Reproductive Biology and Phylogeny of Birds PDF

Reproductive Biology and
Phylogeny of Birds
Phylogeny n Morphology
Hormones n Fertilization
Reproductive Biology and Phylogeny Series
Series Editor: Barrie G. M. Jamieson
Published:
Vol. 1: Reproductive Biology and Phylogeny of Urodela
(Volume Editor: David M. Sever)
Vol. 2: Reproductive Biology and Phylogeny of Anura
(Volume Editor: Barrie G. M. Jamieson)
Vol. 3: Reproductive Biology and Phylogeny of Chondrichthyes
(Volume Editor: William C. Hamlett)
Vol. 4: Reproductive Biology and Phylogeny of Annelida
(Volume Editor: G. Rouse and F. Pleijel)
Vol. 5: Reproductive Biology and Phylogeny of Gymnophiona
(Caecilians)
(Volume Editor: Jean-Marie Exbrayat)
Vol. 6A: Reproductive Biology and Phylogeny of Birds
In press/under preparation:
Vol. 6B: Reproductive Biology and Phylogeny of Birds
Vol. 7: Reproductive Biology and Phylogeny of Cetacea
(Volume Editor: D. Miller)
Reproductive Biology and
Phylogeny of Birds
Part A
Phylogeny n Morphology
Hormones n Fertilization
Volume edited by
BARRIE G.M. JAMIESON
School of Integrative Biology
University of Queensland
St. Lucia, Queensland
Australia
Volume 6A of Series:
Reproductive Biology and Phylogeny
Series edited by
BARRIE G.M. JAMIESON
University of Queensland THE UNIVERSITY
St. Lucia, Queensland OF QUEENSLAND
Australia AUSTRALIA
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ISBN (Vol 6 Pt B) 978-1-57808-444-9
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Preface to the Series
This series was founded by the present series editor, Barrie Jamieson, in
consultation with Science Publishers, in 2001 and bears the title ‘Reproductive
Biology and Phylogeny’, followed in each volume with the name of the
taxonomic group which is the subject of the volume. Each publication has one
or more invited volume editors (sometimes the series editor) and a large
number of authors of international repute. The level of the taxonomic group
which is the subject of each volume varies according, largely, to the amount of
information available on the group, the advice of proposed volume editors,
and the interest expressed by the zoological community in the proposed work.
The order of publication of taxonomic groups reflects these concerns, and the
availability of authors for the various chapters, and it is not proposed to
proceed serially through the animal kingdom in a presumed “ladder of life”
sequence. A second aspect of the series is coverage of the phylogeny and
classification of the group, as a necessary framework for an understanding of
reproductive biology. Evidence for relationships from molecular studies is an
important aspect of the chapter on phylogeny and classification. Other
chapters may or may not have phylogenetic themes, according to the interests
of the authors.
It is not claimed that a single volume can, in fact, cover the entire gamut of
reproductive topics for a given group but it is believed that the series gives an
unsurpassed coverage of reproduction and provides a general text rather than
being a mere collection of research papers on the subject. Coverage in different
volumes varies in terms of topics, though it is clear from the first volumes that
the standard of the contributions by the authors will be uniformly high. The
stress varies from group to group; for instance, modes of external fertilization
or vocalization, important in one group, might be inapplicable in another.
The first five volumes on Urodela, edited by Professor David Sever, Anura,
edited by myself, Chondrichthyes, edited by Professor William Hamlett,
Annelida, edited by Dr. Greg Rouse and Professor Fredrik Pleijel, and
Gymnophiona, edited by Professor Jean-Marie Exbrayat, reflected the above
exacting criteria and the interests of certain research teams. This, the sixth
volume, in two parts, has resulted from my interest in the natural history of
birds, which was stimulated in childhood by Gilbert White’s incomparable
LE Reproductive Biology and Phylogeny of Birds
‘Natural History of Selborne’, and my good fortune in being joined by a most

distinguished group of authors who need no introduction to those familiar
with avian studies. A volume in preparation is on Cetacea (Debra Miller).
My thanks are due to the School of Integrative Biology, University of
Queensland, for facilities, and especially to the Executive Dean, Biological &
Chemical Sciences, Professor Mick McManus, for his continuing
encouragement. I am everlastingly indebted to Sheila Jamieson, who has
supported me indirectly in so many ways in this work. I am grateful to the
publishers for their friendly support and high standards in producing this
series. Sincere thanks must be given to the volume editors and the authors,
who have freely contributed their chapters, in very full schedules. The editors
and publishers are gratified that the enthusiasm and expertise of these
contributors has been reflected by the reception of the series by our readers.
THE UNIVERSITY
OF QUEENSLAND Barrie G.M. Jamieson
AUSTRALIA
21 February 2006 University of Queensland
Classification and Phylogeny LEE
Preface to this Volume
There are almost ten thousand known species of birds of which more than
half are song birds. They are an ideal subject of study as they are one of the
few groups of animals for which almost the total number of species is
estimated to be known, they have been comprehensively catalogued and
illustrated and are readily identified in the field whereas groups such as
annelids (Volume 5) require detailed microscopical work for identification.
Besides these practical qualifications for study and the biological questions
that they pose, they have endeared themselves to humanity not least for their
beauty and their song. This volume, in two parts, attempts to document most
of the important aspects of the reproductive biology of birds and places them
in a setting of phylogenetic relationships. Aspects of reproduction that
comprise this, the first part of volume 6, are classification and phylogeny as
revealed by molecular biology; anatomy of the male reproductive tract and
organs; anatomy and evolution of copulatory structures; development and
anatomy of the female reproductive tract; endocrinology of reproduction;
ovarian dynamics and follicle development; spermatogenesis and testicular
cycles; avian spermatozoa: structure and phylogeny; testis size, sperm size
and sperm competition and, lastly, fertilization. We may here, albeit
superficially, sample some of the many topics treated in the ten chapters.
As more and more homologous DNA sequences and other genetic
characters become available for more and more species, we are gradually
resolving the remaining uncertain nodes of the avian phylogenetic tree, and
this trend will only accelerate as DNA sequencing becomes both cheaper and
more reliable. These are heady times in avian systematics and the present state
of our knowledge is authoritatively reviewed.
The testes of birds are intra-abdominal, and, in contrast with most
mammals, they do not migrate from their site of embryological origin. They
are, thus, closely related, topographically, to the kidneys. The anatomy and
histology of avian testes and their ducts receive a detailed examination.
In birds, unlike most other animal classes, males of some species possess
an intromittent organ, whereas males of other species do not. Thus, in birds at
least, the organ does not seem to be necessary for internal fertilization. This
raises the question whether the avian intromittent organ has evolved as a
LEEE Reproductive Biology and Phylogeny of Birds
primary sexual trait simply for the delivery of sperm or as a secondary sexual
trait. Its absence in so many species is an evolutionary puzzle investigated
here.
Only the left genital primordia develop to functional ovaries in birds except
in a few cases, particularly birds of prey. The reason for the unilateral
development of female genital organs might be to reduce weight for flying. The
question then arises why the falconiforms allow themselves the luxury of two
genital tracts. To reduce weight their hard-shelled eggs are relatively small
and are laid down at an early stage of development. This and other aspects of
the female genital tract are examined. The Müllerian and Wolffian ducts
receive detailed treatment and a valuable analysis of the function of the
oviduct in egg laying is provided.
In an innovative chapter hormonal control systems in birds are
investigated in four major parts: 1) types of environmental signals that
influence reproduction and how they are perceived; 2) the hypothalamus as
an integrator of environmental information from the external and internal
environments, biological clock, etc., that through neuroendocrine and neural
secretions affects all aspects of reproduction; 3) the hypothalamo-pituitary
unit that transduces environmental information processed by higher centers
into endocrine secretions; 4) the functional gonads (ovary and testis)
themselves.
A unique morphological and functional aspect of the reproductively active
avian ovary, as contrasted with the mammalian counterpart, is that follicles at
all stages of development, from resting primordial and primary follicles to the
fully differentiated preovulatory stage, exist simultaneously during egg-
laying. As a consequence, the sequential selection of one undifferentiated
follicle into the final rapid growth stage of development provides for ovulation
of an oocyte from a fully differentiated follicle on an approximate daily basis.
The ovarian follicular hierarchy is a reflection of oviparity, and is a feature
held in common with avian predecessors, the reptiles including, probably,
some dinosaurs. Other aspects of ovarian function are examined.
The seminiferous epithelium in avian testis is made up of germ cells at
varying levels of development, and Sertoli cells. Sertoli cells have multiple
functions, including formation of the blood-testis barrier by means of
junctional complexes, and providing anchorage and nutrition for, as well as
regulation of, germ cells during development. The most primitive or immature
germ cells lie on the basement membrane and the mature germ cells, the
spermatozoa, line the lumen of the seminiferous tubule. Germ cells develop in
close association with one another because as they divide they maintain close
linkage through intercellular bridges which are the result of incomplete
cytoplasmic divisions. A detailed examination of spermatogenesis and factors
affecting it is given from spermatogonia to the mature spermatozoa.
The chapter on sperm structure and phylogeny is the longest of the book, in
keeping with the great amount of information, though often fragmentary,
which is available and the fact that the chapter represents the final scientific
Preface to this Volume EN
contribution of any magnitude by the editor. Former workers recognized the

‘sauropsid’ features of non-passerine spermatozoa. However, the ratite and
lower non-passerine spermatozoon, especially the former, are shown to more
appropriately be termed crocodiloid. Features of Ostrich sperm which are
similar to those of crocodiles are described. These features are also seen in
turtles and are basic (symplesiomorphic) to amniotes, only the fibrous sheath
and the long distal centriole being amniote synapomorphies. The sole
spermatozoal synapomorphy of crocodiles and birds is the dense sheath
investing the two central singlets within the elongate distal centriole. In birds
this sheath is known only in ratites (Ostrich), galliforms and anseriforms. The
literature is reviewed in a phylogenetic context.
Almost since the moment that animal semen was first viewed under the
microscope, in the seventeenth century, it was clear that the shape and size of
spermatozoa varied from one species to the next. Early comparative biologists
similarly noted striking differences in the size of the testis across different
species. Although the general features of the male reproductive system are
rather conservative across extant species of birds, recent work has confirmed
large interspecific variation in the sizes of both testes and sperm. The chapter
addresses the question why should testes mass and sperm size differ so much
from one species to the next?
Until recently, the cellular and molecular events comprising fertilization in
birds were poorly understood. However, several recent studies in mammals as
well as in birds have considerably contributed to our knowledge of the
fertilization process. In mammals only a single spermatozoon enters the egg.
Monospermy is ensured by a variety of mechanisms, which is collectively
known as the block to polyspermy. Unlike mammals, the principal feature of
fertilization in birds is physiological polyspermy, penetration of the ovum by
many sperm. It has been suggested that polyspermy occurs in animals that
produce large yolky (megalecithal) ova and this theme is developed. The
structure of the mature ovum and the events of fertilization are discussed.
There is an extensive section on assisted reproductive technologies. Many
new illustrations are provided throughout the volume.
The generosity of authors and publishers who have allowed illustrations to
be reproduced is most gratefully acknowledged. The kind collaboration of all
the authors, who have borne uncomplainingly the requests of an editor
overseeing the gestation of this work, has been greatly appreciated. Finally,
the courteous and efficient participation of the publishers was indispensable
to production of this volume.
THE UNIVERSITY
OF QUEENSLAND Barrie G.M. Jamieson
AUSTRALIA School of Integrative Biology
21 February 2006 University of Queensland
n n
Contents
Preface to the Series—Barrie G. M. Jamieson v

Preface to this Volume—Barrie G. M. Jamieson vii
1. Classification and Phylogeny of Birds 1

John Harshman
2. Anatomy of the Testis and Male Reproductive Tract 37
Tom A. Aire
3. Anatomy and Evolution of Copulatory Structures 115
Robert Montgomerie and James Briskie
4. Developmental Anatomy of the Female Reproductive Tract 149
Monika Jacob and Murray R. Bakst
5. Endocrinology of Reproduction 181
G. E. Bentley, K. Tsutsui and J. C. Wingfield
6. Ovarian Dynamics and Follicle Development 243
A. L. Johnson and Dori C. Woods
7. Spermatogenesis and Testicular Cycles 279
Tom A. Aire
8. Avian Spermatozoa: Structure and Phylogeny 349
Barrie G. M. Jamieson
9. Testis Size, Sperm Size and Sperm Competition 513
James V. Briskie and Robert Montgomerie
10. Fertilization 553
Urszula Stepinska and Murray R. Bakst
Index 589
n n
About the Series
This series bears the title ‘Reproductive Biology and Phylogeny’ followed by
the name of the taxonomic group which is the subject of the volume. B. G. M.
Jamieson is the founding series editor and each publication has one or more
invited volume editors (if not the series editor) and a large number of authors
of international repute. The series gives a unique coverage of reproductive
biology. The level of the taxonomic group which is the subject of each volume
varies according, largely, to the amount of information available, the advice of
proposed volume editors, and the interest expressed by the zoological
community in the proposed work. Phylogeny and classification is covered as
a necessary framework for an understanding of reproductive biology.
Evidence for relationships from molecular studies is an important aspect of
the phylogenetic section.
About the Volume

There are almost ten thousand known species of birds of which more than
half are song birds. They are an ideal subject of study as they are one of the
few groups of animals for which almost the total number of species is
estimated to be known, they have been comprehensively catalogued and
illustrated and are readily identified in the field. Besides these practical
qualifications for study and the biological questions that they pose, they have
endeared themselves to humanity not least for their beauty and their song.
This volume, in two parts, attempts to document most of the important aspects
of the reproductive biology of birds and places them in a setting of
phylogenetic relationships. Aspects of reproduction that comprise this, the
first part of volume 6, are classification and phylogeny as revealed by
molecular biology; anatomy of the male reproductive tract and organs;
anatomy and evolution of copulatory structures; development and anatomy of
the female reproductive tract; endocrinology of reproduction; ovarian
dynamics and follicle development; spermatogenesis and testicular cycles;
avian spermatozoa: structure and phylogeny; testis size, sperm size and
sperm competition and, lastly, fertilization. Many new illustrations are
included.
About the Series and Volume Editor

Dr. Barrie Jamieson is Emeritus Professor of Zoology in the School of
Integrative Biology, University of Queensland. He holds a Ph.D. from the
University of Bristol, England, a D.Sc. from the University of Queensland, and
is a former Visiting Fellow of, and member of the Association of Corpus
Christi College, Cambridge. In 1990 he was awarded the Clarke Medal for
Research in Natural Sciences, early recipient of which were Thomas Henry
Huxley and Richard Owen. His chief field of research is spermatozoal
ultrastructure and its relevance to phylogeny but he is also an authority on
taxonomy of earthworms and has published on bioluminescence, trematode
taxonomy and life cycles, and DNA-based phylogenetics. He has published
nearly 200 scientific papers and is the author, coauthor or editor of fourteen
books.
n n
CHAPTER
1
Classification and
Phylogeny of Birds
John Harshman
1.1 INTRODUCTION
Avian phylogenetics is currently in its infancy, and paradoxically it is also
nearing maturity. By infancy I mean that we do not currently know much
about the relationships among birds, and by maturity I mean that within a
few years we will know most of what there is to know. The reason for this
apparent paradox lies in the rapidly increasing ease of gathering and
analyzing molecular data. Our ignorance is largely a matter of our not yet
having gathered enough DNA sequences or other molecular data for enough
species, and that condition is being remedied at an increasing rate. If I had
written this chapter a year or two from now, I would have been able to cite
studies incorporating perhaps twice the data so far in published form—this
purely from yet-unpublished studies of whose existence I am now aware—
and I would have been able to say much more about the structure of the avian
tree. But the editor was not anxious to follow that schedule, and so we press
on with what is currently available.
During more than two thousand years of avian systematics—counting from
Aristotle—researchers were able to divide birds into a number of robust
groups based on distinctive morphologies: the non-passerine families. (We
will get to passerines in a moment.) This process was largely completed by the
beginning of the last century. For reviews see Sibley and Ahlquist (1990) and
Cracraft et al. (2004). Progress since then, on relationships between and within
families, has been highly incremental, so much so that at least one prominent
avian systematist (Stresemann 1959) announced that no further progress was
possible. The cladistic revolution did little to help, though at least it focused
our attention on the right questions; apparently it’s very difficult to find
reliable morphological characters that will build a robust tree of birds. And
the first 30 years of the molecular revolution have illuminated only a few of
4869 Pepperwood Way, San Jose, CA 95124; E-mail: jharshman@pacbell.net

Reproductive Biology and Phylogeny of Birds
those questions (Sheldon and Bledsoe 1993), leading another pair of

systematists, once again, to wonder if there would be no further progress (Poe
and Chubb 2004). However, recent discoveries suggest that there is more
reason for optimism.
This chapter will concentrate on what we do know confidently about avian
relationships. Necessarily, it must treat mostly relationships between larger
groups, since the nearly 10,000 living species are too many to be covered in
detail. It is also convenient to divide the birds into two groups, only one of
them monophyletic: passerines and non-passerines. One reason for separate
treatment is that it splits the species roughly in half, but the main reason is
that our state of knowledge differs radically between the groups. For non-
passerines, most traditional families are clearly supported as monophyletic,
and we can summarize relationships as being between these entities. (The
same is not true of traditional orders, as we will see.) I have used the
classification of del Hoyo et al. (1992-2002) as representative of traditional
familial and ordinal assignments. For passerines, however, most traditional
families that have been adequately sampled are not monophyletic, and we
must either speak in extremely general terms or choose individual species as
our units of discussion. In both groups, genera have not fared well, and many
have proven not to be monophyletic when examined closely.
Non-monophyly should come as no surprise. Traditional taxonomy did not
take monophyly into account. What mattered was distinctness, and whether
that distinctness arose from apomorphy or plesiomorphy was not considered
important. Thus if some subset of a group possessed a prominent apomorphy,
that served to characterize two subgroups: one by its presence, and another by
its absence. This practice has continued even to the present, e.g. the vigorous
defense by Short and Horne (2002) of separating Ramphastidae from
Capitonidae.
One caveat should be kept in mind when assessing the results of phyloge-
netic analyses: they can discern the relationships only among those species
actually included in the analysis. This rule may seem obvious, but it has been
violated frequently in the literature. If an analysis of gruiform phylogeny
includes only gruiforms and a single outgroup, the monophyly of gruiforms
cannot be tested. If an analysis of bird phylogeny includes only one represen-
tative from each order, the monophyly of orders cannot be tested. If an attempt
to find the sister group of hoatzins does not include the actual sister group
(whatever it may be), that relationship will not be found. It would thus seem
that every phylogenetic analysis, in order to be valid, must include every
species of bird (and that further assumes that birds are monophyletic, and that
each individual species is also), which is clearly impossible. Fortunately,
chaos is not total, and uncertainties are of limited taxonomic scope: families
whose ordinal relationships are unclear have themselves so far proven to be
monophyletic; all members of non-monophyletic families can be constrained
within single orders, and all members of non-monophyletic genera within
single families. (The latter does not apply to passerines, however.) Some
Classification and Phylogeny of Birds !
groups do have clear morphological synapomorphies, and these can be easy

to assay across species. Genetic distances offer some comfort too. If we find all
species of Anas to be genetically similar, it’s unlikely that any of them is really
a bustard, even if we do not include them all in an analysis with a bustard.
But we must still be careful not to overinterpret any results.
Because of their importance to avian systematics, I must discuss the DNA
hybridization studies of Sibley and Ahlquist (1990) and the classification
based on them (Sibley and Monroe 1990). Their final product, the “Tapestry”,
is a comprehensive and nearly fully resolved tree of over 1100 species (Sibley
and Ahlquist 1990, figs. 357-385). However important, this tree was severely
flawed as a representation of their data, and the data themselves were
insufficient to resolve objectively many of the relationships asserted by the
Tapestry (Lanyon 1992; Harshman 1994). A later attempt to correct some of
the flaws of the Tapestry by using rigorous methods of analysis on subsets of
the data (Sibley and Ahlquist 1990, figs. 325-352)—since they used the Fitch-
Margoliash (1967) method of distance analysis, we may call them “Fitch
trees”—is an improvement but suffers from the absence of an evaluation of
strength of support, which Harshman (1994) attempted to repair by re-
analyzing the data. These latter analyses should be preferred to the Tapestry
in the several cases of conflict. Sibley and Ahlquist’s studies contain some
exciting, corroborated insights, e.g., the sister relationship of mockingbirds
(Mimidae) and starlings (Sturnidae), but also some major mistakes, e.g., the
placement of storks (Ciconiidae) and New World vultures (Cathartidae) as
sister taxa, and even some decisions unjustified by any data, e.g., the
placement of hoatzin (Opisthocomidae) with cuckoos (Cuculiformes). In the
end, without corroboration, it is difficult to separate the Tapestry’s good
estimates from the bad, even with access to their original data (courtesy of the
late C. G. Sibley). Below, I will mention Sibley and Ahlquist’s findings only
when they have been independently corroborated or if their conclusions are
clearly supported by their data, properly analyzed.
1.2 NON-PASSERINES
Relationships among the non-passerine families are not well resolved at
present. If we consider a tree of relationships among the 103 non-passerine
families recognized by del Hoyo et al. (1992-2002) plus the order
Passeriformes, that tree, if fully resolved, will have 103 internal nodes
(counting the root position). We can be fairly confident of only 60 of those
nodes (Figs. 1.1 and 1.2). In contrast to many other taxa, the most basal
relationships are among the best known, but most other resolved nodes unite
small numbers of closely related families, such as Dromaiidae (emu) and
Casuariidae (cassowaries).
1.2.1 Basal Relationships

Modern birds—Aves or Neornithes depending on one’s taste in terminology
(Gauthier et al. 2001)—are divided basally into two clades, Palaeognathae and
" Reproductive Biology and Phylogeny of Birds
Fig. 1.1 Relationships among non-passerine families, so far as they can be

confidently asserted at present according to my perhaps biased estimate. This tree
does not derive from a formal analysis, either of a combined supermatrix or by a
rigorous supertree method. Branches in gray represent paraphyletic groups. This
figure shows Paleognathae through Metaves. For Coronaves see Fig. 1.2. For
reasons behind the topology chosen, see text.
Neognathae. Palaeognathae includes the ratites and tinamous. Neognathae is

divided into Galloanserae, consisting of the sister orders Anseriformes and
Galliformes, and Neoaves, consisting of all other birds. Recent analyses, both
molecular and morphological, have been nearly unanimous on these four
basal clades (Prager and Wilson 1978; Caspers et al. 1997; Groth and
Barrowclough 1999; García-Moreno and Mindell 2000; van Tuinen et al. 2000;
Paton et al. 2002; Cracraft and Clarke 2001; García-Moreno et al. 2003; Mayr
and Clarke 2003; Cracraft et al. 2004). Some other studies lacked an outgroup
root but are consistent with these clades (Sibley and Ahlquist 1990; Chubb
2004a; Fain and Houde 2004).
A few studies using whole mitochondrial genomes or large fractions thereof
have found a different topology, in which passerines are basal to all other
Classification and Phylogeny of Birds #
Fig. 1.2 Relationships among non-passerine families, continued. Coronaves only.

For reasons behind the topology chosen, see text.
$ Reproductive Biology and Phylogeny of Birds
birds (Mindell et al. 1997; Härlid and Arnason 1999; Mindell et al. 1999). But
these have been shown to be the result of long branch attraction (Braun and
Kimball 2002; Paton et al. 2002; García-Moreno et al. 2003). Also see Ericson et
al. (2001), which failed to find a monophyletic Galloanserae.
A note on non-monophyly: there are two forms of non-monophyly,
paraphyly and polyphyly. Strictly speaking, the two cannot be distinguished
on a phylogenetic tree, since the definitions require either explicit assignment
of ancestral nodes to groups or optimization of diagnostic characters. In this
chapter, I will define paraphyly artificially: if we code group membership as a
presence/absence character, a group is paraphyletic if that state can be
optimized as gained only once on the tree and lost one or more times. It is
polyphyletic if group membership must be optimized as being gained at least
twice. This favors paraphyly in ambiguous cases. Results may differ from
those under other definitions, but at least the condition can be determined
from the tree itself.
1.2.2 Palaeognathae
Almost all studies using non-avian outgroups have found paleognaths to be
monophyletic (García-Moreno and Mindell 2000; van Tuinen et al. 2000; Paton
et al. 2002; Cracraft et al. 2004; but see Chapter 8). Under that assumption, we
can add studies using neognath outgroups, and these have found the two
paleognath orders, Struthioniformes (ratites) and Tinamiformes (tinamous)
also to be monophyletic (Sibley and Ahlquist 1990; Lee et al. 1997; van Tuinen
et al. 1998; Cooper et al. 2001; Cracraft and Clarke 2001; Haddrath and Baker
2001). Within ratites, however, there is considerable contention. It is
universally agreed in all these studies that Dromaiidae (emu) and Casuariidae
(cassowaries) are sister taxa. Molecular studies commonly make Apterygidae
(kiwis) the sister of these two, forming an Australasian clade (Sibley and
Ahlquist 1990; Lee et al. 1997; van Tuinen et al. 1998; Cooper et al. 2001;
Haddrath and Baker 2001). The question of whether the Rheidae (rheas) or
Struthionidae (ostrich) is the sister of all other ratites is less clear; the majority
of studies have found the rhea basal (Cooper et al. 1992; Lee et al. 1997; Cooper
et al. 2001; Haddrath and Baker 2001), but others have found the ostrich basal
(Sibley and Ahlquist 1990; van Tuinen et al. 1998).
The sole morphological study has the same unrooted topology as the
molecular studies, but the root attaches to the kiwi, making the rooted
topology quite different (Lee et al. 1997).
Phylogenetic relations within tinamous have been little studied. There is
only one published morphological phylogeny (Bertelli et al. 2002) and no
molecular studies.
1.2.3 Galloanserae
Monophyly of Galloanserae is affirmed by a large number of studies (Sibley
and Ahlquist 1990; Caspers et al. 1997; Livezey 1997; Groth and
Barrowclough 1999; García-Moreno and Mindell 2000; van Tuinen et al. 2000;
Classification and Phylogeny of Birds %
Zusi and Livezey 2000; Cracraft and Clarke 2001; Paton et al. 2002; García-
Moreno et al. 2003; Mayr and Clarke 2003; Sorenson et al. 2003; Cracraft et al.
2004). But see Ericson (1997) and Ericson et al. (2001).
There is also strong support for monophyly of both Galliformes and
Anseriformes (Livezey 1986; 1997a; Cracraft and Clarke 2001; Zusi and
Livezey 2000; Ericson et al. 2001; Sorenson et al. 2003; Chubb 2004a; Cracraft et
al. 2004), though molecular studies with large taxon samples of both orders
have not been published. Sibley and Ahlquist (1990) found strong support for
monophyly of Galliformes, but monophyly of Anseriformes could not be
confirmed (Harshman 1994), and Chubb (2004a) also could not confirm
anseriform monophyly.
1.2.3.1 Relationships within Galliformes
All families are monophyletic except Phasianidae (pheasants), which
includes the two traditional families Meleagrididae (turkeys) and Tetraonidae
(grouse) (Dimcheff et al. 2002). The monophyly of Numididae (guineafowl) is
not supported by morphological characters (Dyke et al. 2003), but at least two
of the four genera have been shown to be closely related by DNA
hybridization (Sibley and Ahlquist 1990). Basal relationships are clear:
Megapodiidae (megapodes) and Cracidae (curassows, guans, and
chachalacas) are successive sister groups to the rest (Sibley and Ahlquist
1990—the Fitch tree, contradicting the Tapestry; Ericson et al. 2001; Dimcheff
et al. 2002; Dyke et al. 2003; Sorenson et al. 2003; Mayr and Weidig 2004;
Pereira and Baker 2006). The major puzzle is whether the sister group of
Phasianidae is Odontophoridae (New World quail) (Armstrong et al. 2001;
Dimcheff et al. 2002) or Numididae (Sibley and Ahlquist 1990; Kornegay et al.
1993; Kimball et al. 1999; Armstrong et al. 2001; Dimcheff et al. 2002; Pereira
and Baker 2006). Some studies support both possibilities in different analyses.
There are several studies of relationships within families: megapodes (Birks
and Edwards 2002), cracids (Pereira et al. 2002), and phasianids (Kimball et al.
1999; Dimcheff et al. 2002).
The phasianid genus Francolinus is polyphyletic (Bloomer and Crow 1998),
and the tetraonid genera Bonasa and Falcipennis are paraphyletic (Ellsworth et
al. 1996; Dimcheff et al. 2002; Drovetski 2002).
1.2.3.2 Relationships within Anseriformes
Monophyly of Anhimidae (screamers) is easily confirmed, as the three species
are quite closely related (Livezey 1986; Sibley and Ahlquist 1990). Sibley and
Ahlquist’s (1990) Tapestry makes Anseranas (magpie goose, sometimes given
its own monotypic family Anseranatidae) the sister group of Anhimidae, thus
making Anatidae (ducks) paraphyletic, but other analyses of the same data
(Sibley and Ahlquist 1990’s Fitch tree; Harshman 1994) restore duck
monophyly. In all other analyses, Anatidae is monophyletic (Livezey 1986;
1997a; Ericson 1997; Ericson et al. 2001; Cracraft et al. 2004).
Relationships within Anatidae are contentious. Though the basal relation-
ships are well established (Anseranas and Dendrocygninae as successive
& Reproductive Biology and Phylogeny of Birds
sister groups to the remaining anatids), the remaining relationships differ

greatly between morphological (Livezey 1997b and references therein) and
molecular (Madsen et al. 1988; Sraml et al. 1996; Donne-Goussé et al. 2002;
Callaghan and Harshman 2005 and references therein) analyses.
Only one genus has been shown to be paraphyletic. As might be expected,
this is the largest, Anas, which includes the steamer ducks (Tachyeres) as well
as three monotypic genera that are sometimes merged with Anas (Amazonetta,
Speculanas, Lophonetta) (Johnson and Sorenson 1999).
1.2.4 Neoaves
Monophyly of Neoaves, also called Plethornithes by Groth and Barrowclough
(1999), has been confirmed in all recent studies (Groth and Barrowclough
1999; Sorenson et al. 2003; Cracraft et al. 2004; Fain and Houde 2004). It
encompasses the great majority of all birds, including Passeriformes.
1.2.4.1 Monophyly of neoavian orders
There are 23 non-passerine orders within Neoaves. Of these, 9 consist of just
a single family, and their monophyly is trivial. Two more orders,
Psittaciformes (parrots) and Strigiformes (owls) each consist of just two closely
related families, the psittaciform pair often merged into a single family,
Psittacidae. Thus there are 12 orders for which monophyly is a serious ques-
tion. Of these, we have good evidence of monophyly for four, good evidence
against monophyly for five, and no strong evidence either way for three.
The single-family orders are Sphenisciformes (penguins), Gaviiformes
(loons/divers), Podicipediformes (grebes), Phoenicopteriformes (flamingos),
Opisthocomiformes (hoatzin), Pterocliformes (sandgrouse), Columbiformes
(pigeons), Coliiformes (colies), and Trogoniformes (trogons).
The clearly monophyletic orders (other than those with only one family) are
Procellariiformes (tubenoses), Apodiformes (swifts and hummingbirds),
Galbuliformes (puffbirds and jacamars), and Piciformes (woodpeckers,
barbets, and honeyguides).
The monophyly of both Pelecaniformes and Ciconiiformes is falsified by the
close relationship among the pelecaniform family Pelecanidae (pelicans) and
the two ciconiiform families Scopidae (hamerkop) and Balaenicipitidae
(shoebill) (Hedges and Sibley 1994; Siegel-Causey 1997; van Tuinen et al. 2001;
Cracraft et al. 2004; Fain and Houde 2004; but see Mayr 2003). There remains
a group that we might call “core pelecaniforms” except that it fails to include
pelicans, consisting of Sulidae (boobies), Phalacrocoracidae (cormorants),
Anhingidae (darters), and Fregatidae (frigatebirds) (Sibley and Ahlquist 1990;
Harshman 1994; Cracraft et al. 2004). Relationships of the remaining
traditional pelecaniform family, Phaethontidae (tropicbirds), are unclear, as
are relationships of the remaining traditional ciconiiforms.
Caprimulgiformes is at least paraphyletic, since one caprimulgiform family,
Aegothelidae (owlet-nightjars) is the sister of Apodiformes (Mayr 2002a;
Cracraft et al. 2004); whether the order would be monophyletic if apodiforms
were included is unclear.
Classification and Phylogeny of Birds '
The monophyly of both Gruiformes and Charadriiformes is falsified by the

discovery that one gruiform family, Turnicidae (buttonquails) is deeply nested
within charadriiforms (Paton et al. 2003). However, charadriiforms are
monophyletic with the addition of turnicids (Paton et al. 2003). But there is no
evidence for monophyly of gruiforms minus turnicids, and some evidence
against it (Fain and Houde 2004). There is a group we might call “core
gruiforms”, consisting of Gruidae (cranes), Aramidae (limpkin), Psophiidae
(trumpeters), Rallidae (rails), and Heliornithidae (finfoots) (Houde et al. 1997;
Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004). For another view, see
Livezey (1998), in which both Turnicidae and Pedionomidae (plains
wanderer) are included in a monophyletic Gruiformes.
There is some morphological evidence for monophyly of Falconiformes
(Griffiths 1994), but no molecular analyses have so far succeeded in putting
all families together. Nor, however, is there strong evidence for any
falconiform family’s relationship to any non-falconiform family. The
hypothesized relationship between Cathartidae (New World vultures) and
Ciconiidae (storks) (e.g., Sibley and Ahlquist 1990) has not survived rigorous
analysis.
There is no evidence for monophyly of Coraciiformes, though there is some
support for a “core coraciiforms” including Alcedinidae (kingfishers),
Todidae (todies), Momotidae (motmots), Meropidae (bee-eaters), Coraciidae
(rollers), and Brachypteracidae (ground-rollers) (Johansson and Ericson 2003;
Cracraft et al. 2004). There are so far only two studies that have included
Leptosomidae (cuckoo-roller), and there was no support for its inclusion in or
exclusion from Coraciiformes (Kirchman et al. 2001; Mayr et al. 2003). The
position shown on Sibley and Ahlquist’s (1990) Tapestry is not based on valid
data (C. G. Sibley, unpublished data). The remaining three coraciiform
families, Upupidae (hoopoe), Phoeniculidae (woodhoopoes), and Bucerotidae
(hornbills) also form a clade, but their relationship to core coraciiforms is
unsupported (Cracraft et al. 2004).
There is no strong evidence for monophyly of Cuculiformes, consisting of
Cuculidae (cuckoos) and Musophagidae (turacos) (Sorenson et al. 2003).
1.2.4.2 Relationships between neoavian orders
(or their monophyletic fragments)
Confirmed relationships between orders are few. Perhaps the most interesting
published hypothesis is that of Fain and Houde (2004), who divided Neoaves
into two basal clades: Metaves, consisting of Caprimulgiformes, Apodiformes,
Podicipedidae, Phaethontidae, Phoenicopteridae, Opisthocomidae,
Mesitornithidae, Rhynochetidae, Eurypygidae, Pteroclidae, and Columbidae;
and Coronaves, consisting of the remaining Neoaves. So far, these clades are
supported entirely by analyses of beta-fibrinogen, intron 7, but it is
encouraging that both sequence characters and indel characters, which evolve
largely by independent processes, support the same clades. In addition, no
rigorous studies contradict this hypothesis. After some internal debate, I have
added these groups to Figs. 1.1 and 1.2. Note that, if accepted, these two clades
make Pelecaniformes polyphyletic (through the inclusion of Phaethontidae in
Metaves and the remaining families in Coronaves) and Gruiformes highly
polyphyletic.
One amply confirmed though surprising relationship is that between
Phoenicopteridae (flamingos) and Podicipedidae (grebes). First discovered by
van Tuinen et al. (2001), it has since been confirmed by many authors (Chubb
2004a; Cracraft et al. 2004; Mayr 2004).
Another well confirmed relationship is that between Aegothelidae (owlet-
nightjars) and Apodiformes (Mayr 2002a; Mayr et al. 2003; Cracraft et al. 2004).
The traditional relationship of Galbuliformes and Piciformes has recently
been questioned strongly enough that the two orders, generally united as
Piciformes, were separated in the classification used by del Hoyo et al. (1992-
2002). However, that relationship has subsequently been conclusively
confirmed (Johansson and Ericson 2003; Mayr et al. 2003; Cracraft et al. 2004).
The long-standing hypothesis that Procellariiformes and Sphenisciformes
are sister groups has received some support in molecular analyses (van
Tuinen et al. 2001); but see Mayr (2005) for a placement of penguins within
“core pelecaniforms”.
One more possible grouping, though it has ambiguous support and its
boundaries are not clearly defined, is sometimes called “water birds”. It
includes most of the traditional members of Pelecaniformes (except
Phaethontidae), Ciconiiformes, and Procellariiformes, plus Spheniscidae
(penguins) and Gaviidae (loons) (van Tuinen et al. 2001; Cracraft et al. 2004).
Support in any individual analysis is not strong, however, and some analyses
include additional groups within the clade (e.g., Cuculiformes, “core
gruiforms”), while other analyses do not sample all groups. Because of the
ambiguity surrounding this potential group, I have omitted it from the tree
(Fig. 1.2).
One prominent hypothesis that has no support is a clade made up of any
combination of Opisthocomidae, Musophagidae, and Cuculidae. Groups
uniting different pairs or all three have been proposed several times (Sibley
and Ahlquist 1990; Hedges et al. 1995; Hughes 1996, 2000; Hughes and Baker
1999), but they have been plagued by data problems (Sorenson et al. 2003), and
as of now no resolution is possible. Note that in the analysis by Fain and
Houde (2004), the hoatzin belongs to Metaves, while turacos and cuckoos are
Coronaves.
There are a few other hypotheses with some support from morphological
data that either contradict or are unconfirmed by molecular data. Mayr
(2004b) proposed a sister group relationship between Mesitornithidae
(mesites) and Cuculidae (cuckoos). Mayr (2003b) proposed a sister group
relationship between Trogonidae (trogons) and Steatornithidae (oilbirds).
Both relationships contradict Fain and Houde’s (2004) Metaves/Coronaves
split, though they do not contradict any strongly supported clades in any
other analyses.
Classification and Phylogeny of Birds
1.2.4.3 Relationships within neoavian orders

(or their monophyletic fragments)
Procellariiformes. Pelecanoididae (diving-petrels) are probably nested within
Procellariidae (petrels and shearwaters) (Cracraft et al. 2004), so I show them
as sister taxa in Fig. 1.2. This is the only resolved node in my reanalysis
(Harshman 1994) of Sibley and Ahlquist’s (1990) data. Hydrobatidae may be
polyphyletic (see below) and this prevents any further resolution among
families.
Pelecaniformes. This order is divided into three pieces. Phaethontidae
(tropicbirds) is apparently not closely related to any of the other families. The
relationships of Pelecanidae (pelicans) have been noted above. This leaves a
clade I will call “core pelecaniforms” despite the absence of pelicans from the
group, composed of Fregatidae (frigatebirds), Sulidae (boobies),
Phalacrocoracidae (cormorants), and Anhingidae (darters). The only resolved
node in my reanalysis (Harshman 1994) of Sibley and Ahlquist’s (1990) data
unites these four families. Siegel-Causey (1997) and Cracraft et al. (2004, fig.
27.4) also find this grouping. In other studies (van Tuinen et al. 2001; Cracraft
et al. 2004, fig. 27.7) only the last three are united or Fregatidae is only weakly
attached to the others (Cracraft et al. 2004, fig. 27.6). Relationships among
Sulidae, Phalacrocoracidae, and Anhingidae are ambiguous, with either
phalacrocoracids (van Tuinen et al. 2001; Fain and Houde 2004) or sulids
(Siegel-Causey 1997; Cracraft et al. 2004) sister to the other two.
Falconiformes. Though the order cannot be confidently asserted as
monophyletic, relationships are well established among families of the
suborder Accipitres, with Sagittariidae (secretary bird) the sister of
Pandionidae (osprey) and Accipitridae (hawks) (Cracraft et al. 2004; Fain and
Houde 2004); but see Griffiths (1994).
Gruiformes. The relationships of Otididae (bustards), Cariamidae (seriemas),
and Mesitornithidae (mesites) to any other families (gruiform or otherwise)
cannot be determined at present. Turnicidae (buttonquail) belongs to
Charadriiformes (see above). Eurypygidae (sunbittern) and Rhynochetidae
(kagu) are sister taxa (Houde et al. 1997; Livezey 1998; Cracraft et al. 2004; Fain
and Houde 2004), but their relationships to other families cannot be
determined. This leaves a “core gruiforms” consisting of Gruidae (cranes),
Aramidae (limpkin), Psophiidae (trumpeters), Rallidae (rails), and
Heliornithidae (finfoots), for which there is strong support (Houde et al. 1997;
Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004). There is strong
support for a sister group relationship between Gruidae and Aramidae and
between Rallidae and Heliornithidae (Houde 1994; Houde et al. 1997; Livezey
1998; Cracraft et al. 2004; Fain and Houde 2004). The placement of Psophiidae
is disputed, but the only strong support is for a position as sister of Gruidae
plus Aramidae (Livezey 1998; Fain and Houde 2004).
Charadriiformes. All molecular analyses (Sibley and Ahlquist 1990; Ericson
et al. 2003; Paton et al. 2003; Fain and Houde 2004) are agreed on the
relationships of families within Charadriiformes. Paton et al. (2003) discuss

incongruence between their tree and that of Sibley and Ahlquist (1990), but
they are referring there to the Tapestry, while I refer here to the more rigorous
Fitch tree (Sibley and Ahlquist 1990; Harshman 1994). The order is
traditionally divided into three suborders, Charadrii, Lari, and Alcae (del
Hoyo et al. 1996), but Alcae is nested within Lari, and the traditional Charadrii
is polyphyletic. The three basal clades do consist of plover-like birds, gull-like
birds, and sandpiper-like birds, which might be given the names Charadrii,
Lari, and Scolopaci, respectively, with the first being sister to the other two.
Ibidorhynchidae (ibisbill) has never been sampled and cannot be placed.
Lacking any better notion, I have placed it at the basal node of
Charadriiformes.
Caprimulgiformes. There is no study strongly supporting the relationship of
any caprimulgiform family to any other (Harshman 1994; Mariaux and Braun
1996; Fidler et al. 2004). The only supported relationship is that of
Aegothelidae to Apodiformes, as discussed above.
Apodiformes. Remarkably few studies have included representatives of all
three families of apodiforms, but those that did have unsurprisingly found
Apodidae (swifts) and Hemiprocnidae (treeswifts) to be sister taxa (Johansson
et al. 2001; Mayr 2002a; Mayr et al. 2003; Chubb 2004a).
Coraciiformes. Relationships within “core coraciiforms” are partially
resolved. One clade is composed of Alcedinidae, Todidae, and Momotidae,
though relationships among these families are contradictory (Sibley and
Ahlquist 1990; Harshman 1994; Espinosa de los Monteros 2000; Johansson et
al. 2001; Johansson and Ericson 2003), and another clade of Coraciidae and
Brachypteracidae (Johansson et al. 2001; Kirchman et al. 2001; Johansson and
Ericson 2003). The position of Meropidae is unresolved, though there is some
evidence that it is sister to the first clade (Johansson and Ericson 2003). There
is another clade of traditional coraciiforms whose relationships to core
coraciiforms are unresolved; it consists of Bucerotidae, Upupidae, and
Phoeniculidae, in which the last two are sister taxa (Sibley and Ahlquist 1990;
Harshman 1994; Johansson et al. 2001; Cracraft et al. 2004). Leptosomidae
certainly does not belong to the roller clade and probably does not belong to
the core coraciiforms (Kirchman et al. 2001).
Piciformes. There is a basal split between Capitonidae (which includes
Ramphastidae) and the other two families, Indicatoridae and Picidae (Sibley
and Ahlquist 1990; Harshman 1994; Johansson and Ericson 2003; Cracraft et
al. 2004).
1.2.4.4 Monophyly of neoavian families
Most non-passerine, neoavian families have clear morphological
synapomorphies, and the monophyly of many has been supported by
molecular analyses. But there are a few exceptions. Charadriidae (plovers) is
both paraphyletic, since it includes the two families Recurvirostridae (stilts
and avocets) and Haematopodidae (oystercatchers) (Ericson et al. 2003; Fain

and Houde 2004) and polyphyletic, since Pluvianellus (Magellanic plover) is
not a close relative of the other plovers, but is instead sister to Chionidae
(sheathbills) (Paton et al. 2003). Glareolidae (coursers and pratincoles) is
polyphyletic, since Pluvianus (Egyptian plover), as its common name might
suggest, is not a member of the family; its actual position is not clear (Fain and
Houde 2004).
In parrots, Psittacidae is paraphyletic to Cacatuidae (de Kloet and de Kloet
2005). There is some evidence to suggest that the procellariiform family
Hydrobatidae (storm petrels) is paraphyletic (Nunn and Stanley 1998) to the
rest of the order, or even polyphyletic (Cracraft et al. 2004), but topologies are
contradictory. I have represented this in Fig. 1.2 by making Hydrobatidae
paraphyletic and its relationships to other families unresolved. There is also
strong evidence that Procellariidae (petrels and shearwaters) is paraphyletic
to Pelecanoididae (diving-petrels) (Nunn and Stanley 1998, Cracraft et al.
2004). And Capitonidae (barbets) is paraphyletic to Ramphastidae (toucans)
(Sibley and Ahlquist 1990; Lanyon and Hall 1994; Barker and Lanyon 2000;
Johansson and Ericson 2003; Cracraft et al. 2004; Moyle 2004).
There may be other exceptions that we are unaware of and that will be
exposed by increased taxon sampling, but I do not expect many.
1.2.4.5 Relationships within neoavian families
Space doesn’t permit a lengthy discussion of intrafamilial relationships.
Instead I have provided a list of references (Table 1.1) that investigate such
relationships with a large enough taxon sample to be useful.
Table 1.1 References to studies of phylogeny within individual neoavian families
Family Reference
Spheniscidae Bertelli and Giannini 2005
Diomedeidae Nunn and Stanley 1998
Procellariidae Nunn and Stanley 1998
Hydrobatidae Nunn and Stanley 1998
Phaethontidae Kennedy and Spencer 2004
Fregatidae Kennedy and Spencer 2004
Ardeidae Sheldon 1987
Sheldon et al. 2000
Ciconiidae Slikas 1997
Accipitridae Griffiths 1999
Falconidae Griffiths 1999
Griffiths et al. 2004
Cathartidae Wink 1995
Gruidae Krajewski and King 1996
Rallidae Livezey 1998
Otididae Pitra et al. 2002
Broders et al. 2003
Table 1.1 Contd. ...

Family Reference
Jacanidae Whittingham et al. 2000
Scolopacidae Ericson et al. 2003
Paton et al. 2003
Thomas et al. 2004
Stercorariidae Braun and Brumfield 1998
Thomas et al. 2004
Laridae Crochet et al. 2000
Thomas et al. 2004
Pons et al. 2005
Alcidae Friesen et al. 1996
Thomas et al. 2004
Columbidae Johnson and Clayton 2000
Psittacidae/ Cacatuidae Miyaki et al. 1998
Brown and Toft 1999
de Kloet and de Kloet 2005
Musophagidae Hughes and Baker 1999
Veron and Winney 2000
Cuculidae Aragón et al. 1999
Hughes and Baker 1999
Hughes 1996, 2000
Johnson et al. 2000
Sorenson et al. 2003
Nyctibiidae Mariaux and Braun 1996
Trochilidae Bleiweiss et al. 1997
Chubb 2004b
Trogonidae Espinosa de los Monteros 2000
Johansson and Ericson 2004
Moyle 2005
Alcedinidae Sibley and Ahlquist 1990
Johansson and Ericson 2003
Brachypteracidae Kirchman et al. 2001
Capitonidae/ Barker and Lanyon 2000
Ramphastidae Johansson and Ericson 2003
Nahum et al. 2003
Moyle 2004
Picidae Prychitko and Moore 1997
Webb and Moore 2005
1.2.4.6 Monophyly of neoavian genera

There are a limited number of families in which taxon sampling has been
dense enough to determine the monophyly of genera. But in those families,
paraphyly of genera has been shown to be frequent. The sparse data so far
suggest that the larger a genus, and the more genera within the family, the
more likely it is that non-monophyly will be found. Most often, as with
families, paraphyly results from aberrant species being assigned their own
genera. But there are additional cases without such clear causes.
The greatest prevalence so far is in the most heavily sampled family with
large genera, Picidae, in which at least six genera are non-monophyletic:
Colaptes, Piculus, Picoides, Dendropicos, Veniliornis, and Picus (Weibel and Moore
2002; Webb and Moore 2005). Other examples include Eupodotis and Ardeotis
in Otididae (Pitra et al. 2002; Broders et al. 2003), Columba and Streptopelia in
Columbidae (Johnson and Clayton 1999; Johnson et al. 2001), Larus in Laridae
(Pons et al. 2005), Tauraco in Musophagidae (Veron and Winney 2000), and
Stercorarius in Stercorariidae (Braun and Brumfield 1998).
We can expect many more non-monophyletic genera to come to light as
species sampling increases.
1.2.5 Classification of Non-passerines

It would be premature to offer a new classification of the non-passerines,
given our great current uncertainty and the hope that it will soon be repaired,
but I can at least make a few recommendations for modification of the
classification I have been using, from del Hoyo et al. (1992-2002).
Paraphyletic and polyphyletic groups should be reconciled so as to make
them monophyletic, and though Linnean ranks are arbitrary, we may as well
avoid leaving traditional family-rank names nested within other family-rank
names. Therefore sevaral families should be merged into the families that
enclose them: Tetraonidae and Meleagrididae into Phasianidae,
Pelecanoididae into Procellariidae, Haematopodidae and Recurvirostridae
into Charadriidae, Cacatuidae into Psittacidae, and Ramphastidae into
Capitonidae. It may be that Hydrobatidae will need to be split into two
families, but more study is needed.
Turnicidae should be moved from Gruiformes to Charadriiformes.
Coraciiformes should be limited to the “core coraciiforms”, and a new order
established for Bucerotidae, Upupidae, and Phoeniculidae, for which
Bucerotiformes is a reasonable name. Likewise, Gruiformes should be limited
to the “core gruiforms”. We should probably wait a while before redefining
Pelecaniformes; perhaps a clade can be found that includes both pelicans and
“core pelecaniforms”, though it will surely include other families too.
Galbuliformes should be returned to its traditional state as a suborder of
Piciformes.
1.3 PASSERINES
The order Passeriformes contains more than half the species of birds (Sibley
and Monroe 1990). In contrast to the difficulties with non-passerines,
molecular techniques have proven to be highly successful in determining
relationships at all levels. The major difficulties have been the sheer number
of species and the unreliability of previous taxonomies as guides to
phylogeny. Thus, single species cannot be used as handy proxies for entire
families, or even genera. The rules of thumb used for non-passerines—in
which genera did not commonly change families, and non-monophyletic
genera at least had their separate parts all within one family, etc.—cannot be
counted on here. What that means for this review is that any summary of
relationships will be difficult, as the species assigned to families and
superfamilies are in flux, and new surprises are expected as more species are
sampled. Further, it becomes even more important to consider the exact nature
of the taxon sampling in any given study and its comparability to sampling in
other studies. I will attempt only an impressionistic summary tree of higher
taxa (Figs. 1.3 and 1.4), for whose suggested species membership the reader
must consult the references provided. One monotypic traditional family,
Hypocoliidae, has never been included in any molecular analysis or any
rigorous, cladistic analysis of morphology, and it has been omitted from
further discussion here.
Many of the analyses used in assembling the passerine tree have used
Bayesian methods (Huelsenbeck and Ronquist 2001). Recently it has become
apparent that Bayesian support values can be highly inflated (Suzuki et al.
2002; Yang and Rannala 2005). Very small differences in data or choice of
evolutionary model can produce contradictory but strongly supported clades
(pers.obs.), and there are many contradictory clades in the sources used in this
review that have high Bayesian support. Consequently, I have not accepted
any relationships based solely upon Bayesian analysis of any single data set.
Either there must be some other measure of support used (e.g., parsimony or
likelihood bootstrap), or different data sets (including indel data if any) must
agree on the topology. Neither has any contradiction in relationships
supported by Bayesian analyses alone caused me to remove resolution from
any clade.
1.3.1 Monophyly of Passeriformes

Passeriform monophyly is well supported by morphological apomorphies
(Raikow 1982). There have been several molecular analyses with moderately
large taxon samples of passerines and other orders, and passerine monophyly
has always been found (Johansson et al. 2001; Johansson and Ericson 2003;
Sorenson et al. 2003; Cracraft et al. 2004; Fain and Houde 2004).
1.3.2 Monophyly of Passerine Families and Genera

One reason that it’s difficult to summarize passerine relationships in the same
way I did for non-passerines is that there are no convenient, easily recognized
subtaxa to use in constructing a summary tree. Sibley and Ahlquist (1990)
showed that many of the traditional passerine families were not
monophyletic, and others since then have found that many of the groups with
which Sibley and Ahlquist (1990) and Sibley and Monroe (1990) replaced
those traditional groups are themselves not monophyletic (see below). Further,
misplaced taxa are not limited to small movements, but may span almost the
range of the passerine tree—“almost” because so far no putative suboscine
has been shown to be oscine, or vice versa. However, species have moved
between superfamilies. Pseudopodoces (Hume’s ground jay), supposed to be a
member of Corvidae and thus of Corvoidea, is instead a member of Paridae (in

fact within the genus Parus, which thereby becomes paraphyletic) and thus of
Sylvioidea (James et al. 2003; Gill et al. 2005). Yuhina (Erpornis) zantholeuca
(white-bellied yuhina) is not a yuhina (Sylvioidea) but a vireo (Corvoidea)
(Barker et al. 2004). Macgregoria (Macgregor’s bird of paradise) is not a member
of Paradiseidae (Corvoidea) but of Meliphagidae (Meliphagoidea) (Cracraft
and Feinstein 2000). Sapayoa (broadbilled sapayoa) is not a New World
suboscine but an Old World suboscine, in terms of phylogeny if not
geography (Fjeldså et al. 2003; Chesser 2004). There is no reason to suppose
that these examples exhaust the list.
Traditional or recently defined (Sibley and Ahlquist 1990; Sibley and
Monroe 1990) families that have proven non-monophyletic include
Eurylaimidae (e.g., Prum 1993), Formicariidae (traditional and Sibley and
Ahlquist versions), Rhinocryptidae, Furnariidae, Tyrannidae, Cotingidae, and
Pipridae, in fact the large majority of suboscine families (Sibley and Ahlquist
1990; Irestedt et al. 2002; Chesser 2004). Oscines are similar in their proportion
of non-monophyletic families, though I will not list them here (e.g. Sibley and
Ahlquist 1990; Cibois and Cracraft 2004; Voelker and Spellman 2004;
Beresford et al. 2005; Alström et al. 2006).
Genera of passerines are also not reliably monophyletic. While this is true
also of non-passerines, there are certainly many more visible examples within
passerines, and genera are more likely to be split into more widely separated
pieces. In an informal poll of bird systematists I conducted at a recent meeting,
I asked them to guess what percentage of passerine genera were non-
monophyletic (excluding monotypic genera). The modal answer was “about
half”. Of course the majority of genera have not been rigorously investigated
so far, which requires sampling most or all the species in the genus plus many
species outside it. A few examples should suffice. In addition to finding New
World warblers polyphyletic, Lovette and Bermingham (2002) found six out of
seven investigated genera with two or more sampled species to be non-
monophyletic; Driskell and Christidis (2003) found three non-monophyletic
genera within Meliphagidae out of sixteen examined; Irestedt et al. (2004a)
found three of four sampled genera of Thamnophilidae non-monophyletic,
and monophyly of the fourth was not confirmed; and Irestedt et al. (2004b)
found two of six sampled genera of Dendrocolaptidae non-monophyletic.
1.3.3 Basal Relationships within Passerines

Basal relationships are clear (Fig. 1.3). The family Acanthisittidae (New
Zealand wrens), with only two genera and three extant species, is the sister
group of all other passerines (Lovette and Bermingham 2000; Barker et al.
2002, 2004; Ericson et al. 2002a; Chesser 2004; Beresford et al. 2005). The
remaining passerines are split into two clades, Suboscines and Oscines
(Sibley and Ahlquist 1990; Lovette and Bermingham 2000; Barker et al. 2002,
2004; Ericson et al. 2002a; Chesser 2004; Beresford et al. 2005).
Fig. 1.3 Relationships among passerine families and other groups, so far as they
can be confidently asserted at present according to my perhaps biased estimate.
This tree does not derive from a formal analysis, either of a combined supermatrix
or by a rigorous supertree method. Branches in gray represent paraphyletic
groups. For Passerida see Fig. 1.4. For reasons behind the topology chosen, see
text.
Within the suboscines basal relationships are also easy to describe, as there
are New World and Old World clades, Tyrannides and Eurylaimides (Sibley
and Ahlquist 1990; Irestedt et al. 2001; Barker et al. 2002, 2004; Chesser 2004;
Beresford et al. 2005). (Recall that Eurylaimides has a single New World
representative, Sapayoa, as mentioned above.) Old World suboscines are also
simple: Pittidae (pittas) is either sister to or paraphyletic to Sapayoa, with a
larger taxon sample necessary to determine which (Fjeldså et al. 2003; Chesser
2004), and Eurylaimidae (broadbills) is paraphyletic to Philepittidae (asities)
(Prum 1993; Irestedt et al. 2001; Barker et al. 2002, 2004; Beresford et al. 2005).
New World suboscines can be divided into two clades, Tyranni and Furnarii
(Lovette and Bermingham 2000; Irestedt et al. 2001; Barker et al. 2002, 2004;
Chesser 2004; note that Sibley and Ahlquist 1990 gave the name Tyranni to a
different group),
In the oscines, Sibley and Ahlquist (1990) erected two groups Corvida and
Passerida. Corvida is however paraphyletic to a (mostly) monophyletic
Passerida (Barker et al. 2002). Within the corvidan grade, there are three
superfamilies: Menuroidea, Meliphagoidea, and Corvoidea, each very roughly
corresponding to Sibley and Ahlquist’s (1990) groupings, plus a number of
additional taxa unincluded in any superfamily (Barker et al. 2002, 2004;
Beresford et al. 2005). Within Passerida, three superfamilies, Sylvioidea,
Muscicapoidea, and Passeroidea, are also roughly similar to Sibley and
Ahlquist’s (1990) groups (Sheldon and Gill 1996; Barker et al. 2002, 2004;
Ericson and Johansson 2003; Beresford et al. 2005). It has however been
necessary to add a fourth, Certhioidea (Barker et al. 2004), and there are also a
number of groups that do not fit securely into any superfamily (Sheldon and
Gill 1996; Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al.
2005; Fuchs et al. 2006).
Ignoring for simplicity groups that do not fit into a superfamily, the
relationships among those superfamilies can be resolved: Menuroidea,
Meliphagoidea, and Corvoidea are successive sister groups to all other oscines
(Barker et al. 2002, 2004; Beresford et al. 2005). Sylvioidea is sister to all other
passeridans, and Muscicapoidea is sister to Certhioidea (Sheldon and Gill
1996; Barker et al. 2002, 2004; Beresford et al. 2005). Relationships are
described in more detail below.
1.3.4 Relationships within New World Suboscines

1.3.4.1 Furnarii
There is a basal polytomy among three clades: Melanopareia (crescent-chests),
a genus traditionally assigned to Rhinocryptidae (Irestedt et al. 2002; Chesser
2004), Thamnophilidae (typical antbirds) plus Conopophagidae (gnateaters),
and remaining Furnarii (Irestedt et al. 2001, 2002; Chesser 2004; but see Barker
et al. 2002, 2004 for a different placement of conopophagids). Formicariidae
(ground antbirds) is divided into two parts; one of them, ant-pittas, is the sister
group of Rhinocryptidae (tapaculos) while the other, ant-thrushes, is sister to
Furnariidae (ovenbirds) and Dendrocolaptidae (woodcreepers). Furnariidae is
paraphyletic to Dendrocolaptidae (Irestedt et al. 2002; Chesser 2004).
1.3.4.2 Tyranni
Several clades within Tyranni are clearly established: Pipridae (manakins),
Tityridae (tityras), Cotingidae (cotingas), Oxyruncus (sharpbill), and
Tyrannidae (tyrant flycatchers). But relationships among them are
contradictory in different analyses, and no resolution is strongly supported in
any analysis (Barker et al. 2002, 2004; Johansson et al. 2002; Chesser 2004).
Relationships within Tyrannidae are likewise contentious, and no molecular
study has so far accumulated a large taxon sample.
1.3.5 Relationships within the Corvidan Grade

1.3.5.1 Menuroidea and other near-basal groups
Sibley and Ahlquist’s (1990) Menuroidea included Menuridae (lyrebirds),
Atrichornithidae (scrub-birds), Climacteridae (Australasian treecreepers), and
Ptilonorhynchidae (bowerbirds). More recent studies show that the latter two
are sister taxa and are more closely related to other oscines than to Menuridae
(Barker et al. 2002, 2004; Ericson et al. 2002b; Beresford et al. 2005; but see
Ericson et al. 2002a). There is so far no sequence data available for
Atrichornithidae, and I have retained it within a reduced Menuroidea (Sibley
and Ahlquist 1990). The unnamed group including Climacteridae and
Ptilonorhynchidae is sister to all oscines other than Menuroidea (Barker et al.
2002, 2004; Ericson et al. 2002b; Beresford et al. 2005; but see Ericson et al.
2002a).
1.3.5.2 Meliphagoidea and other groups outside Corvoidea
Meliphagoidea consists of Maluridae (fairy wrens), Meliphagidae
(Honeyeaters), Pardalotidae (pardalotes), Acanthizidae (scrubwrens,
thornbills), and Dasyornis (bristleheads) (Cracraft and Feinstein 2000; Barker
et al. 2002, 2004) and is sister to the remaining oscines (Barker et al. 2002, 2004;
Beresford et al. 2005). Relationships within Meliphagidae and Acanthizidae
have been investigated by Driskell and Christidis (2004). Two more families,
Pomatostomidae (Australian babblers) and Orthonychidae (logrunners) are
successively more closely related to Corvoidea and Passerida (Barker et al.
2002, 2004).
Three more clades form a polytomy with Corvoidea and Passerida:
Callaeatidae (New Zealand wattlebirds), Cnemophilinae (traditionally
supposed to be birds of paradise, belonging to the corvoid Paradiseidae), and
Melanocharitidae (most berrypeckers—one genus, Paramythia, is corvoid)
(Cracraft and Feinstein 2000; Barker et al. 2002, 2004; Beresford et al. 2005).
1.3.5.3 Corvoidea
This large clade contains most of the member of Sibley and Ahlquist’s (1990)
Corvoidea (Barker et al. 2004, 2004; Beresford et al. 2005). It consists of two
large, resolved subclades, a few pairs of taxa, and a large, basal polytomy. One
large subclade is a collection of mostly shrike-like birds including Cracticidae
(butcherbirds), Gymnorhina (Australian magpie), Strepera (currawongs),
Artamidae (wood-swallows), Aegithinidae (ioras), Malaconotinae (bush-
Classification and Phylogeny of Birds
shrikes), Dryoscopus (puffbacks), Batis (batises), Lanioturdus (chatshrike),

Vangidae (vangas), and Prionopidae (helmet shrikes) (Barker et al. 2002, 2004;
Beresford et al. 2005; Fuchs et al. 2006). The other group consists of taxa close
to Corvidae, including Laniidae (shrikes), Paradiseidae (birds of paradise),
Monarchidae (monarch flycatchers), Struthidea (apostlebird), Corcorax (white-
winged chough), Grallina (mud-nest builders), Melampitta (melampittas),
Dicruridae (drongos), and Rhipiduridae (fantails) (Barker 2002, 2004;
Beresford et al. 2005).
1.3.6 Relationships within Passerida

These are shown in Fig. 1.4.
1.3.6.1 Groups not within any superfamily
Picathartidae (rockfowl) and Petroicidae (Australian robins) are basal
members of Passerida (Barker et al. 2002, 2004; Beresford et al. 2005). Regulidae
(kinglets) and Hyliota are closer to other Passerida than the previous two
families, but cannot so far be linked to any of the superfamilies (Barker et al.
2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005; Fuchs et al.
2006).
1.3.6.2 Sylvioidea
The most difficult problem in establishing sylvioid monophyly has been the
inclusion of Paridae (titmice) and its relatives within the superfamily. Though
no study has supported that conclusion strongly, several have supported it
and none have strongly contradicted it (Sheldon and Gill 1996; Barker et al.
2002, 2004: Beresford et al. 2005; Alström et al. 2006).
In addition to its traditional allies, Remizidae (penduline tits), the clade
around Paridae has recently been shown to include a clade of refugees from
other families, termed Stenostiridae by Beresford et al. (2005). This clade
includes Stenostira (fairy warbler, previously considered a sylviid), Culicicapa
(canary-flycatchers, usually considered muscicapid, but placed among
petroicids by Sibley and Ahlquist 1990), and Elminia (blue-flycatchers,
previously considered monarchids) (Barker et al. 2002, 2004; Beresford et al.
2005; Fuchs et al. 2006).
Monophyly of the remainder of Sylvioidea is strongly supported, though
this group differs from that of Sibley and Ahlquist (1990) by inclusion of
Alaudidae (larks) (Sheldon and Gill 1996; Barker et al. 2002, 2004; Ericson and
Johansson 2003; Alström et al. 2006; Fuchs et al. 2006). Alaudidae, with its
unexpected sister taxon Panurus (bearded tit) (Ericson and Johansson 2003;
Alström et al. 2006; Fuchs et al. 2006) is strongly supported as the sister group
of all remaining sylvioids (Sheldon and Gill 1996, Barker et al. 2002, 2004;
Ericson and Johansson 2003; Beresford et al. 2005; Alström et al. 2006; Fuchs et
al. 2006).
In the rest of Sylvioidea there is a large polytomy consisting of several
genera, a few families, and one large group of families. There are still a great
many sylvioid (or supposed sylvioid) genera and species yet to be sampled;
Fig. 1.4 Relationships among passerine families, continued. Passerida only. For
reasons behind the topology chosen, see text.
more sampling of taxa and genes may resolve this polytomy and illuminate
unknown clades. Beresford (2005) proposed a “Sphenoeacus group” consisting
of Sphenoeacus, Sylvietta, Achaetops, Bradypterus victorini (the genus being
polyphyletic), and Macrosphenus; but this was supported solely by Bayesian
analysis. Other studies, with only partly overlapping taxon samples, have
produced contradictory results (Alström et al. 2006; Fuchs et al. 2006). Alström
et al. (2006) found Macrosphenus to belong to Pycnonotidae (bulbuls) with
strong support; however, the two studies sequenced different species, and it’s
quite possible that both are right and the genus is polyphyletic.
The sylvioid polytomy includes four large clades (Alström et al. 2006). Two
are the families Cisticolidae (cisticolas) and Timaliidae (babblers). Note that
Timaliidae as used here includes the genera Sylvia and Zosterops; there are no
families Sylviidae or Zosteropidae, following the terminology of Alström et al.
(in press). A third clade includes the families Megaluridae (grassbirds) and
Acrocephalidae (acrocephaline warblers) plus the monotypic Donacobius, once
considered a troglodytid (Barker 2004; Alström et al. 2006). The final clade
consists of a number of families: Hirundinidae (swallows), Pycnonotidae
(bulbuls), Phylloscopidae (leaf warblers), Cettiidae (bush warblers), and
Aegithalidae (long-tailed tits), plus the genus Hylia (Alström et al. 2006), and
the last three form a subclade (Beresford et al. 2005).
1.3.6.3 Certhioidea
This superfamily was erected by Cracraft et al. (2004) to cover a clade of four
families removed from Sibley and Ahlquist’s (1990) Sylvioidea. It includes
Troglodytidae (wrens), Polioptilidae (gnatcatchers), Certhiidae (treecreepers),
and Sittidae (nuthatches) (Barker et al. 2002, 2004; Ericson and Johansson
2003; Beresford et al. 2005). Within this clade, Sittidae is basal and
Troglodytidae and Polioptilidae are sister taxa (Sibley and Ahlquist 1990;
Harshman 1994; Sheldon and Gill 1996; Ericson and Johansson 2003; Barker
2004; Alström et al. 2006; but see Barker et al. 2002, 2004).
1.3.6.4 Muscicapoidea
The most difficult question is whether Bombycillidae (waxwings, silky
flycatchers, palmchat) belongs to this superfamily. No analysis has been
conclusive, but several have given weak support (Sibley and Ahlquist 1990;
Barker et al. 2002, 2004; Cibois and Cracraft 2004) and I consider the
combination to offer strong support. Monophyly of the rest of Muscicapoidea
is clear (Sibley and Ahlquist 1990; Harshman 1994; Barker et al. 2002, 2004;
Ericson and Johansson 2003; Cibois and Cracraft 2004; Voelker and Spellman
2004; Alström et al. 2006).
Relationships among the five families Sturnidae (starlings), Mimidae
(mimic thrushes) Cinclidae (dippers), Turdidae (thrushes), and Muscicapidae
(sensu stricto—Old World flycatchers) are also contentious, but a strong case
can be made for the topology I have chosen, in which Sturnidae and Mimidae
are sisters, Turdidae and Muscicapidae are sisters, and Cinclidae is sister to
the turdid-muscicapid clade (Sibley and Ahlquist 1990; Barker et al. 2002,
2004; Cibois and Cracraft 2004; Beresford et al. 2005; Fuchs et al. 2006). An
alternative arrangement in which Cinclidae is sister to the sturnid-mimid
clade is supported only by Bayesian analysis, though the relationship
between Turdidae and Muscicapidae is also supported by parsimony
jackknifing (Ericson and Johansson 2003). Voelker and Spellman (2004) show
a substantially different topology, in which Muscicapidae is basal, but
contradictory nodes again have weak support.
Two additional genera form a polytomy with Sturnidae and Mimidae
(Cibois and Cracraft 2004). Buphagus (oxpeckers) is traditionally considered
part of Sturnidae. Rhabdornis is a genus of previously uncertain relationships,
sometimes placed in Certhiidae or Timaliidae (Sibley and Monroe 1990).
1.3.6.5 Passeroidea
It may be that Promeropidae (sugarbirds) is the basal family in Passeroidea,
but that conclusion is supported by Bayesian analyses alone, and only by
different versions of one data set (Barker et al. 2002, 2004; Beresford et al. 2005).
What may be partial confirmation is offered by Fuchs et al. (2006). The two
genera Modulatrix (spot-throat) and Arcanator (dapple-throat, sometimes
merged into Modulatrix) are strongly supported as the sister group of
Promerops (Barker et al. 2002, 2004; Beresford et al. 2005). Fuchs et al. (2006),
again supported only by Bayesian analysis, show Modulatrix and Arcanator as
the sister group of Passeroidea, but Promerops appears in a polytomy at the
base of Passerida, and in light of the ambiguity of this information, that is
where I have left the entire family. However, there does seem to be good
evidence for inclusion of those two species, whose relationships have
previously been highly uncertain (Sibley and Monroe 1990), as sister taxa
within Promeropidae.
The remainder of Passeroidea is clearly monophyletic (Barker et al. 2002,
2004; Ericson and Johansson 2003; Beresford et al. 2005). Nectariniidae
(sunbirds) and Dicaeidae (flowerpeckers) are sisters (Barker et al. 2002, 2004;
Ericson and Johansson 2003; Beresford et al. 2005), and form a trichotomy
with Irenidae (fairy bluebirds) and remaining passeroids (Barker et al. 2002,
2004; Beresford et al. 2005). Peucedramus (olive warbler), previously thought to
be either a parulid or basal within 9-primaried oscines (Sibley and Monroe
1990) is instead sister to Prunellidae (accentors) (Ericson and Johansson
2003), and these are sister to the remaining passeroids (Barker et al. 2002,
2004; Ericson and Johansson 2003; Beresford et al. 2005). Passeridae (Old
World sparrows) and Motacillidae (pipits and wagtails) are successive sister
groups to the nine-primaried oscines (Barker et al. 2002, 2004; Ericson and
Johansson 2003). Though this is weakly supported in each analysis,
agreement between analyses and a three-codon insertion in the c-myc
sequences (Ericson et al. 2000) are conclusive.
The nine-primaried oscines form a strongly supported group (Klicka et al.
2000; Ericson and Johansson 2003; Barker et al. 2004), including the families
Fringillidae (finches), Emberizidae (buntings and New World sparrows),

Icteridae (New World blackbirds), Parulidae (New World warblers),
Thraupidae (tanagers), and Cardinalidae (grosbeaks). Fringillidae is the sister
of the others (Klicka et al. 2000; Yuri and Mindell 2002; Ericson and Johansson
2003; Barker 2004). Two genera previously considered emberizids, Calcarius
(longspurs) and Plectrophenax (snow buntings) are sister taxa of each other
and together the sister of the remaining nine-primaried oscines (Klicka et al.
2000; Yuri and Mindell 2002; Ericson and Johansson 2003). Relationships
among the remaining five families are contentious. Of analyses with
representatives of all five families, most unite Cardinalidae and Thraupidae
(Bledsoe 1988; Barker et al. 2002, 2004; Yuri and Mindell 2002), though two
(Sibley and Ahlquist 1990; Harshman 1994; Klicka et al. 2000) do not. Again,
most unite Icteridae and Parulidae (Bledsoe 1988; Barker et al. 2002, 2004),
though two (Klicka et al. 2000; Yuri and Mindell 2002) do not. But support for
the minority arrangements are weak. The position of Emberizidae is unclear;
Barker et al. (2002, 2004) have strong support for relationship to the cardinalid-
thraupid clade; Bledsoe (1988) found a relationship to the parulid-icterid
clade; Yuri and Mindell (2002) and Klicka et al. (2000), though failing to find
the parulid-icterid clade, did find a clade consisting of Emberizidae, Icteridae,
and Parulidae. For the present, relationships of Emberizidae are best treated
as unresolved.
1.4 CONCLUSION
Progress in avian systematics during the past few years has finally, after a
long period of frustration, become worthy of optimism. As more and more
homologous DNA sequences and other genetic characters become available
for more and more species, We will gradually chip away at the remaining
uncertain nodes of the tree, and this trend will only accelerate as DNA
sequencing becomes both cheaper and more reliable. These are heady times in
avian systematics.
1.5 ACKNOWLEDGMENTS
I would like to thank Keith Barker and Fred Sheldon for their comments on the
manuscript, and the other members of the Early Bird project, particularly
Kathy Miglia, for sending me copies of many of the referenced articles.
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enigmatic cranial features in galliform and anseriform birds. Annals of Carnegie
Museum 69: 157-193.
n n
CHAPTER
2
Anatomy of the Testis and
Male Reproductive Tract
Tom A. Aire
2.1 INTRODUCTION
The testes of birds are intra-abdominal, and, unlike in most mammals, they do
not migrate from their site of embryological origin. They are, thus, closely
related, topographically, to the kidneys. As in mammals, birds have two testes,
one on either side of the midline, bordering the aorta and caudal vena cava,
laterally. They are attached from their dorso-medial borders to the dorsal
abdominal wall by a short mesorchium, and as the kidneys with which they
are related embryologically, they are largely retroperitoneal, and ventral to the
vertebral column. Topographically, the cranial poles (extremitates craniales) of
the testes lie close to the ventral border of the lungs, while their caudal poles
(extremitates caudales) lie cranio-ventral to the cranial divisions of the kidneys
(Nickel et al. 1977). The dorsomedial aspect of the testis attaches to the
relatively small epididymis. The ductus deferens runs distally from the caudal
border of the epididymis, toward the cloaca, into which it opens. There are no
known accessory sex organs or glands in birds that are either homologous or
analogous to those found in mammals. Mammalian terminologies have often
been erroneously applied to certain structural modifications in birds, such as
seminal vesicle in passerine birds, and the ampulla of the ductus deferens.
These have to be understood for what they are, structurally and functionally,
as segmental convolutions or enlargements, respectively, of the ductus
deferens.
The testis is surrounded by the cranial and caudal thoracic as well as
abdominal air sacs, but contrary to the opinion of Cowles and Nordstrom
(1946) that this relationship helps to cool the testes, as the mammalian
scrotum does, Williams (1958) has observed no differences in the temperature
Department of Anatomy and Physiology, Faculty of Veterinary Science, University of
Pretoria, Onderstepoort, Republic of South Africa. E-mail: tom.aire@up.ac.za
!& Reproductive Biology and Phylogeny of Birds
in the area of the viscera and at the testicular surface in the domestic fowl.
Herrin et al. (1960) have also disproved the assertion by Cowles and
Nordstrom (1946), that adjacent air sacs cool the testis. Testicular color is
nearly white in sexually mature and active birds, but it may be grey or black,
or a mixture of black and white patches or may be entirely greyish black due
to the presence of melanin pigments in melanoblasts, in the testicular
connective tissues. The testes of seasonally resting birds are very small, being
functionally atrophic. Organs of sexually immature or resting birds have
yellowish white to black or grey colour, being quite black in regressed testes
that already contain melanoblasts, as a result of the concentration of these
pigment cells in the connective tissues of much smaller, regressed organs.
2.1.1 Testis Shape, Asymmetry and Size

The avian testes are usually bean- or oval-shaped (Fig. 2.1), but they are
normally vermiform in Cypseloides spp., swifts (Marshall 1961). Although both
left and right testes are symmetrically situated on either side of the median
plane, they are, however, often dissimilar in size. The left testis is often larger
than the right one in most species of birds. Riddle (1918, 1925) observed that
the left testes were larger than the right in pigeons (Columba species), but in 36
of 39 in the dove (Streptopelia risorsia), the right was larger than the left. In
rooster (Gallus domesticus), the left testis is larger than the right in 57% (Mimura
1928) and 65.3% (Marvan 1969) of the birds studied. The right was larger in
only 26.5% while both testes were equal in 8.2% of the birds (Marvan 1969). In
169 genera of birds he studied, Friedmann (1927) concluded that the right
testis was not larger than the left in any genus, but in 104 genera, both testes
were of the same size and the left was larger than the right in 60 genera. Five
genera were inconstant, varying between the first two positions. The left organ
is, also, larger than the right organ in the ratite, Emu (Dromaius
novaehollandiae), although each gram of testis tissue contributes equally to the
production of androgen and, possibly, also, spermatozoa (Malecki et al., 1998).
It seems that the right testis diminishes in size with age, as the left testis was
larger than the right in 90.9% of day-old Turkey toms (Meleagris gallopavo),
94.3% in toms 53-55 wk old and 100% in toms 63-67 wk of age. Care is needed
in removing and identifying each testis especially when the carcass of the bird
is placed on dorsal recumbency, with the left side of the specimen being on
the right of the operator. That is probably why Law and Kosin (1958) found
that all Turkey toms they investigated had larger right testes than the left,
whereas Burke (1973) reported that the left testis surpassed the right testis in
size, with increasing age in their own Turkey toms. The reason for the
generally observed difference in testis size, in favor of the left, is not clearly
understood, especially given the variations that have been reported by
different authors. However, the embryological study by Witschi (1935) shows
that the cortex of the right indifferent gonad loses its chemotactic attraction for
primordial germ cells, in favor of the cortex of the left gonad, from the
beginning of the 3rd day of embryogenesis. Nevertheless, there are no
CMYK
Anatomy of the Testis and Male Reproductive Tract !'

CMYK
CMYK
Fig. 2.1 The topography of the reproductive organs of the quail (Coturnix japonica),
from a ventral view. T, testis; E, epididymis; D, ductus deferens; P, pars recta ductus
deferentis; R, receptacle of the ductus deferens; L, lung; K, kidney. Original.
CMYK
differences in sizes between the left and right testes in the Marsh hawk (Circus
cyaneus) (Witschi 1935; Stanley and Witschi 1940).
The size of the avian testis, except that of several breeds of Domestic Fowl
and Turkey developed for agricultural production, varies considerably
between phases of the reproductive cycle. Thus, the testis reaches maximum
size for the species or breed during the peak of the breeding season, and
becomes considerably smaller, following regression, during the sexually
inactive or resting period of the reproductive cycle, in most wild birds. This
variation in size may be as much as 400- to 500-fold in seasonally breeding
species (Lofts and Murton 1973). During the active breeding season, testis size
is positively related to sperm production rate (Møller 1991), as has been
observed in mammals (Møller 1989). Testis sizes and body weights of 247
species of birds, from 152 genera, 37 families, and 16 orders, have been
compiled by Møller (1991). Testis size in birds is further discussed in Chapter
9.
2.1.2 Body Temperature and Testis Function in Birds

Whereas in most mammals the testes migrate from the site of their
embryogenesis distally and ventrally into the scrotum, those of birds retain
their position of origin, close to the kidneys. Mammalian male germ cells
degenerate at the body’s internal temperature of 37∞C, and even slight and
temporary elevations of scrotal temperature may cause male infertility (Plöen
1972, 1973a,b). On the contrary, spermatogenesis occurs normally at core body
temperature (of 40-41∞C) in birds (Béaupre et al. 1997). Mezquita et al. (1998)
recently demonstrated a number of apparently genetically controlled
biochemical effects that may contribute to ensure the synthesis of essential
proteins which achieve thermotolerance during avian, but not mammalian,
spermatogenesis.
2.1.3 Consistency and Structure of the Testis

The testis of sexually active birds is soft to touch and quite fragile compared to
the mammalian testis. The main exception is the Ostrich (Struthio camelus),
whose testicular substance is enclosed in a firm testicular capsule which is
relatively very thick (vide infra); in other investigated birds it is thin. An
incision through the testicular capsule into the testis causes a mild protrusion
of the testicular substance from which there is a dripping of a relatively
copious amount of milky fluid, even in the Emu whose testis is very dark in
color. Sexually active avian testes have a considerable amount of fluid content
(Lake 1957; Aire 1979a; Clulow and Jones 1988).
2.1.4 The Excurrent Ducts of the Testis

The avian epididymis is a small spindle-shaped structure that is attached
intimately to the medial border of the testis (Fig. 2.1). A thin cranial extension
of this structure is incorporated into the adrenal gland capsule, as the appendix
paradidymidis (Gray 1937; Budras and Sauer 1975a; Budras and Meier 1981).
Anatomy of the Testis and Male Reproductive Tract "
The caudal end of the epididymis also thins out, and is continued by the
ductus deferens. The epididymis of the rooster is 3-5 mm thick at its base, by
which it is attached to the testis (Hodges 1974; Pers. obs.). The epididymis is
relatively large in the ostrich, being between 21 and 31 g in weight, 12 and
15.4 cm in length and 3-7 cm in height, at its highest point at the caudal one-
third of the organ (Pers. obs.). It is about 2.4 cm wide (Soley 1992). The
epididymis, as the testis, is also firm to touch in the Ostrich.
In all birds, the ductus deferens leaves the caudal end of the epididymis in
a slightly wavy manner, but becomes considerably convoluted, increases in
diameter, cranio-caudally, and is situated lateral to the relatively firm and
regular ureter. A short segment of the duct straightens out to form the pars recta
ductus deferentis, just before it enters the cloaca. The ductus deferens terminates
thereafter, in a spindle- or barrel-shaped enlargement, the receptaculum ductus
deferentis, which is embedded in the cloacal wall, but opens into the urodeum
through its protruding and pointed distal end, the papilla ductus deferentis.
Birds do not have accessory sex organs that are to be found in mammals.
2.1.5 Blood Supply to the Reproductive Organs

The blood supply to the male reproductive organs of birds has been studied
fully only in the rooster (Nishida 1964) and Pigeon (Bhaduri et al.1957), and
partly by Siller and Hindle (1969) and Kurihara and Yasuda (1973) in their
reports of the vasculature of the kidney in the rooster. Blood supply to the
male organs in the ostrich has also been studied grossly to complement these
reports (Elias M, Aire, T. A. and Soley, J. T. unpublished results), and the
pattern of arterial supply as well as venous drainage of the male organs is
generally similar in these three species of birds. The following account is
based on these reports.
The avian male reproductive organs are intra-abdominal and retain their
embryological positions, and therefore their blood supply is relatively simpler
than in mammals. A short testicular artery (Arteria testicularis) arises from the
A. cranialis renalis, and runs into the testis, along its dorso-medial border,
supplying the testis, and sending thin branches to the epididymis (Figs. 2.2,
2.3 and 2.4). One or two short accessory testicular arteries (Aa. testiculares
accessoria) may originate directly from the aorta, unilaterally or bilaterally, and
similarly run into the hilus of the testis. The testicular arteries run short
courses into the hilus of the testis from where they divide into intratesticular
loops (Fig. 2.3). Branches ramify in the interstitial tissue, from these loops. The
cranial part of the ductus deferens is supplied by the R. ureterodeferentialis
cranialis that arises from the A. cranialis renalis. The A. renalis communis
originates from A. sciatica in the Ostrich (Elias et al., unpublished observations)
and immediately divides into the A. renalis medius and A. renalis caudalis. The
former sends one or two twigs to the middle part of the ductus deferens. The
A. pudenda sends several branches (Rr. ureterodeferentiales caudales) to the
caudal part of the ductus deferens, and several other branches (Rr. cloacales)
into the cloaca and root of the phallus. The general disposition of the blood
Fig. 2.2 The arterial supply to the reproductive organs of male Gallus domesticus.
Ara, A. renalis cranialis; At, A. testicularis; Ae, Aa. epididymicae; Auda, Aa. uretero-
deferentiales craniales; Audm, Aa. uretero-deferentiales mediae; Audp, Aa.
uretero-deferentiales caudales; Apc, A. pudenda communis. From Nishida, T. 1964
Japanese Journal of Veterinary Science 26: 211-221. Figure 1. Reproduced with
the permission of Japanese Society of Veterinary Medical Science.
Anatomy of the Testis and Male Reproductive Tract "!
Fig. 2.3
Fig. 2.4
Figs. 2.3 and 2.4 A semi-diagrammatic illustration of blood vascular supply in the
right testis of Gallus domesticus. Aoa, aorta; At, A. testicularis; Ata, A. testicularis
accesorius; Vic, V. iliaca communis; Vcp, V. cava caudalis. From Nishida, T. (1964)
Japanese Journal of Veterinary Science 26: 211-221. Figure 4. Reproduced with
the permission of Japanese Society of Veterinary Medical Science.
"" Reproductive Biology and Phylogeny of Birds
vessels in the rooster, pigeon and ostrich are similar, with only minor intra-
and inter-species variations. The veins draining the testicular substance are
tributaries of larger veins which run peripherally into the testicular capsule
(Figs. 2.3 and 2.4). The latter are large, wide and clearly visible as they run
radially toward the hilus from which they emerge and open into the caudal
vena cava as numerous short veins (Nishida 1964). The veins draining the
ductus deferens are mostly satellites of the arteries that supply the duct
(Nishida 1964).
2.2 THE TESTIS

2.2.1 The Testicular Capsule
The avian testicular capsule is generally very thin (Lake 1971). In a
preliminary study, figures for testicular capsule thickness in certain species of
birds are, on the average, 578.1 mm in the ostrich, 81.5 mm in the rooster, 91.7
mm in the Japanese quail (Coturnix japonica) and 91.8 mm in the drake (Anas
platyrhynchos) (Pers. obs.). However, at the interface between the testis and
epididymis, the capsule is much thicker, and has been found to be, on the
average, 1215.9 mm thick in the ostrich, 515.0 mm in the rooster, 255.4 mm in the
Japanese quail, and 233.7 mm in the drake. There are no readily available data
on testicular thickness in mammals, but Davis et al. (1970) indicate that the
thickness of the testicular capsule varies from one testicular region to another.
The tunica albuginea in man is approximately 1000 mm thick, at 75 years of
age (Yoshimura and Fukunishi 1965, cited by Davis et al. 1970).
The testicular capsule in the bird divides in the epididymal region, and
sends a thick branch around the epididymis. The other branch, devoid of the
tunica serosa, runs on the orchido-epididymal border, and is pierced at a
number of points, in most birds, by the intracapsular portion of the rete testis
(RT) ductules, to enter the epididymis.
Histologically, the testicular capsule is composed of three main tissue
layers: an outer, thin tunica serosa, a thick tunica albuginea and the innermost,
very thin tunica vasculosa (Fig. 2.5). A basement membrane separates the thin
mesothelial cells of the t. serosa from the bulky connective tissue of the t.
albuginea. The latter forms the bulk of the capsule, and is composed of
collagen, elastic fibers and abundant fibroblasts (Hodges 1974). Blood vessels,
especially veins of varying sizes run within these bundles of fibroblasts and
collagen, and are easily observed running radially on the surface of the testis,
grossly. Smooth muscle actin intermediate filaments have been demonstrated,
immunohistochemically, in the testicular capsule of the Japanese quail, Drake,
Turkey, rooster and Ostrich (Aire, T.A. unpublished observations). This
indicates that there are smooth muscles in the testicular capsule of birds, as
has been demonstrated in some mammals (Davis et al. 1970; Hargrove et al.
1977). Smooth muscle cells in the testis capsule are probably responsible for
the spontaneous and drug-induced contractions of the capsule of mammals.
These contractions may assist in moving immobile testicular spermatozoa
Anatomy of the Testis and Male Reproductive Tract "#
Fig. 2.5 Histological section of testicular capsule of the ostrich (A, C) and drake
(B). A. The capsule is thick and shows the tunica serosa (arrowheads), tunica
adventitia (T) and tunica vasculosa (arrow). V, large intracapsular blood vessels; S,
seminiferous epithelium. B. A thinner capsule, displaying the three tissue layers,
as in A. Nu, an elongated nucleus of a myofibroblasts. C. testicular capsule (T) of
a juvenile ostrich. There are no obvious septa originating from the testicular
capsule, rather, connective tissue containing oval, euchromatic nuclei aggregate
and pass between the seminiferous cords (Sc) of the testis. Bars: All figures =
100 µm. Original.
"$ Reproductive Biology and Phylogeny of Birds
toward the epididymis (Davis et al. 1970). A similar function may be expected
to occur in birds.
There are no septa running from the testicular capsule into the testicular
substance, as occurs in most mammals (Fig. 2.5A, C). Loose connective tissue,
which appears better formed in the ostrich than in other birds, may be seen
conducting blood vessels from the subcapsular region into the testis
substance, and sending slivers of connective tissue between the seminiferous
tubules (Fig. 2.5A, C).
2.2.2 Seminiferous Tubules

The seminiferous tubules of birds are dissimilar to those of mammals by
forming highly and complexly anastomotic, non-blind-ending network of
tubules (Huber 1916; Bailey 1953; Lake 1957; Marvan 1969). Evidence of this
structure is commonly found in histological sections (Fig. 2.6), and is probably
responsible for the lack of connective tissue septa, as well as the non-
lobulation of the avian testis. The seminiferous epithelium is several cells
thick, and contains two cell types: fixed, somatic cells, represented by the
Sertoli cells, and temporary and mobile germ cells, comprising a series of
differentiating cells (spermatogonia, primary and secondary spermatocytes
and spermatids) (Fig. 2.6 B, inset). The basal stem cell is the spermatogonium
that divides repeatedly to form larger, upward-mobile, cells in successive
stages of meiotic division and maturity. Spermatogenesis in birds, as in
mammals, involves a series of divisions of spermatogonia, resulting in
primary spermatocytes and secondary spermatocytes, both of which undergo
meiotic divisions, culminating in the evolution of spermatids. The latter
differentiate to form motile, itinerant spermatozoa. Spermatogenesis is
discussed fully in Chapter 7, but the Sertoli cell is discussed below.
2.2.3 Sertoli Cells

The Sertoli (sustentacular) cell is the fixed or permanent and non-germinal cell
in the epithelium of the seminiferous tubule. It is known as a “nurse” cell
because it supports and provides nutrition for the developing germ cells.
Avian Sertoli cells are generally similar to those described for the mammal
(Cooksey and Rothwell 1973). Sertoli cells are tall, columnar and extend from
the basal lamina to the luminal border of the seminiferous epithelium (Fig.
2.7A, B). Sertoli cells in the rooster have fewer broad base contacts with the
basement membrane than in mammals (Osman et al. 1980). A similar
observation has been made in the Japanese quail (Fig. 2.7A, B). The cytoplasm
of this cell is generally more electron-dense than that of attaching or adjacent
germ cells (Aire, T. A. personal observations), and arborizes between the germ
cells that attach to, or embed in, it in an irregular manner. The nucleus lies at
the level immediately above the spermatogonia, and is relatively large and
euchromatic. It may therefore be relatively of high activity. It is usually
irregular, has a prominent nucleolus, and is situated close to the basal lamina
in most birds except in the ostrich where it lies in the middle part of the cell
Anatomy of the Testis and Male Reproductive Tract "%
Fig. 2.6 Coturnix japonica. A. A survey photomicrograph of the seminiferous

tubules showing complex branching of the seminiferous tubules. B. Higher power
view of seminiferous tubules displaying various germ cells in the epithelium. Inset:
shows Spermatogonia (arrows); Sertoli cell nucleus (arrowhead); round
spermatids (Sr); elongated spermatids (Sel). Bars: A = 100 µm, B, including inset
= 200 µm. Original.
"& Reproductive Biology and Phylogeny of Birds
Fig. 2.7 Coturnix japonica. A. A resin section of seminiferous tubules stained with
toluidine blue, showing positions of concentrated Sertoli cell cytoplasm
(arrowheads). Dark particles and elongated profiles in the Sertoli cell cytoplasm
are sections of embedded elongated spermatids. B. A TEM micrograph of the
basal half of the seminiferous epithelium showing a Sertoli cell (S) with its
characteristic elongated and irregular nucleus (N), electron-dense cytoplasm
(stars), spermatogonium (Sg) and spermatocyte (Sp). T, peritubular and interstitial
tissue. Bar: A = 200 µm, B = 2 µm. Original.
Anatomy of the Testis and Male Reproductive Tract "'
and hence epithelium (Aire, T. A. personal observations). The cytoplasm

contains abundant, small, smooth endoplasmic reticulum (SER), numerous
free ribosomes and polyribosomes, a well-developed Golgi complex and
micropinocytotic vesicles, both basally and between adjacent cells (Fig. 2.8).
Microtubules are conspicuous in the cell body and processes that surround
the germ cells. Lipid droplets are dense and small in size, and are few in the
rooster (Cooksey and Rothwell 1973) unlike in the Budgerigar (Melopsittacus
undulatus) (Humphreys 1975), where they are numerous.
Sertoli cells are absolutely necessary for the development of germ cells, from
spermatogonia to spermatozoa, not only because they provide physical
support and nutrition for the germ cells, but also because they provide the
avenue for substances to pass between the blood and germ cells. In mammals,
there is a close temporal relationship between the development of the secretion
of many Sertoli cell products and the appearance and increase in number of
primary spermatocytes and early spermatids (Jégou 1991). The same is
probably true in birds because Jégou (1991) considers that the interaction
between late spermatids and the Sertoli cell has survived throughout
evolution and therefore constitutes a major aspect of the paracrine regulation
of spermatogenesis. The passage of substances from Sertoli cells to germ cells
is regulated by a peculiar structural feature of Sertoli cells (Mann and Lutwak-
Mann 1981), which involves cell-cell contacts in the seminiferous epithelium.
These contacts are necessary because there is an intimate interdependence of
germ cells and Sertoli cells. Elongating and elongated spermatids are difficult
to isolate from Sertoli cells without their being considerably damaged.
Besides, early spermatids have not survived well in cell culture (Jutte et al.
1981; Le Magueresse and Jégou 1988). Indeed, Jégou (1991) emphasises that
spermatids are major regulators of Sertoli cell function.
Details of the cellular contacts between the Sertoli and germ cells have been
reported in mammals (Fawcett 1973; Dym 1973; Dym and Fawcett 1970;
Fawcett 1975) and birds (Cooksey and Rothwell 1973; Osman et al. 1980;
Bergmann and Schindelmeiser 1987; Sprando and Russel 1987; Pelletier 1990;
Pfeiffer and Vogl 1993). The main cell contacts in the seminiferous epithelium
of the rooster (Osman et al. 1980; Bergmann and Schindelmeiser 1987; Pelletier
1990) are similar to those described in mammals, and are classified as Sertoli-
germ cell junctions and Sertoli-Sertoli cell junctions (Fig. 2.9).
Sertoli-germ cell contacts consist of gap and adhering junctions that have
subsurface condensations of 7 nm filaments that are lined internally by
cisternae of endoplasmic reticulum (Pelletier 1990). Where two germ cells are
apposed, contacts are made by means of gap junctions that are similar to those
of Sertoli-germ cell adhesions (Fig. 2.9). The Sertoli-Sertoli cell junctions
comprise occluding, gap and adhering junctions, with the occluding junction
being most basal, but situated above spermatogonia as well as early primary
spermatocytes (Pelletier 1990). The gap junction separates the occluding
junction from the adhering junction. The inter-Sertoli cell-occluding junction
is the morphological basis of the intra-testicular and extremely effective blood-
# Reproductive Biology and Phylogeny of Birds
Fig. 2.8 Coturnix japonica. High power view of the cytoplasm of the Sertoli cell of
the quail exhibiting an abundance of smooth endoplasmic reticulum (SER), a few
lipid droplets (L), some mitochondria (M), Golgi complex (G), a few microtubules
(upper arrowheads), and a lysosome (Y). Sp, spermatogonium; RER (lower
arrowheads) B, basal lamina. Bar = 1 µm. Original.
Anatomy of the Testis and Male Reproductive Tract #
testis barrier. The avian occluding junction is different from that of mammals
in only a few details, such as the absence, in birds, of subsurface filaments,
disposed of as bundles, that lie between the adjacent tight junctions and the
subsurface cisternae (Fig. 2.9). This structural barrier, as in mammals,
separates the seminiferous epithelium into the basal and adluminal
compartments, and is able to prevent tracer compounds in the basal
compartment from entering the adluminal compartment of the seminiferous
epithelium in the rooster (Osman et al. 1980; Bergmann and Schindelmeiser
1987; Pelletier 1990). The occluding Sertoli-Sertoli cell junctions are present
and relatively constant in position, above the spermatogonia, in both
regressed testes (Pelletier 1990), and active testes (Osman et al. 1980;
Bergmann and Schindelmeiser 1987; Pelletier 1990) of birds. Thus, in the birds
investigated, the Sertoli cell junction is constantly present and active,
irrespective of the phase in the reproductive cycle.
2.2.3.1 Functions of the Sertoli cell
Since avian Sertoli cells are similar to those of mammals, structurally, it is
tempting to assume that the main functions in both classes of animals are
generally similar. The abundance of SER in the mammalian Sertoli cell
indicates a hormone-secreting cell (Fawcett 1975). Sertoli cells are unable to
significantly synthesise steroids de novo, per se, but promote interconversion of
steroids e.g. progesterone and androstenedione to testosterone and reduced
5 a-androgens (Mann and Lutwak-Mann 1981). Whereas the presence of
steroids was demonstrated in extracts of the fowl testis (Delrio et al. 1967), it
was Tingari’s (1973) histochemistry study that demonstrated steroidogenic
activities in the seminiferous epithelium, and particularly in those parts that
are consistent with Sertoli cell locations, in the rooster. Androgen binding
protein (ABP), inhibin and activin are endocrine regulators of follicular-
stimulating hormone (FSH) production and of testicular steroidogenesis and
spermatogenesis, not only in mammals but also in birds (Johnson and Brooks
1996; Lovell et al. 2000; Onagbesan et al. 2004). It is likely that the avian Sertoli
cell functions as its mammalian counterpart, in secreting ABP, inhibin,
activin, and a part of the testicular fluid (Hagenäs et al. 1975) that have the
same types of functions, as in mammals. The phagocytic ability of the Sertoli
cell is well known in both mammals and birds. These cells have been observed
to remove residual bodies in the rooster and Japanese quail (Cooksey and
Rothwell 1973; Sprando and Russel 1987; Lin and Jones 1992) and India ink
particles (Fig. 2.10) that were introduced into the seminiferous tubular lumen
in the rooster (Pers. obs.).
Perhaps one of the most important functions of the Sertoli cell is the
establishment of the most durable component of the blood-testis barrier, which
creates two fluid compartments within the seminiferous epithelium. This
barrier, already described above, is similar to the mammalian barrier, in major
particulars. It is present and functional in both sexually mature and active as
well as regressed testes in birds, by preventing tracers from entering the
adluminal compartment, from the basal compartment (Osman et al. 1980;
Fig. 2.9. A. A diagrammatic representation of Sertoli-germ cell and Sertoli-Sertoli

cell ectoplasmic specializations and junctions in an active testis. Sert, Sertoli cell;
Fig. 2.9 Contd. ...

Anatomy of the Testis and Male Reproductive Tract #!
Pelletier 1990). It has an important protective role in safeguarding the germ

line from noxious influences originating both from within and external to the
individual, “…including both isolation of sperm-specific autoantibodies and
autoimmunocytes from the tubule in the event that sensitization does occur”
(Neaves 1977).
In addition to these functions, there are peritubular-Sertoli cell interactions.
Peritubular cells constitute an important extra-tubular part of the blood-testis
barrier, and they also have important regulatory interactions with Sertoli cells
in the seminiferous tubule, via the production of paracrine factors that
enhance Sertoli cell functions, including the production of ABP and
transferrin (see Skinner et al. 1991). Sertoli cell-Leydig cell interactions also
exist, in which both cells influence each other with regard to steroidogenic
activities, as well as inhibin and activin secretions (see Skinner et al. 1991).
2.2.4 Interstitial Tissue of the Testis

The interstitial tissue of the testis consists of two main components. The first
is that compact layer of myofibroblasts and connective tissue which closely
surrounds the seminiferous tubule, and known as the boundary tissue. The
other is the loose connective tissue, the interstitium, which lies between
seminiferous tubules, with full expression in the angular areas or wedges
between 3 or more adjacent seminiferous tubules (Fig. 2.11). The boundary or
peritubular tissue consists of subepithelial tissue and layers of alternating
elongated cells and their interconnecting amorphous tissue. The interstitium,
on the other hand, consists of single, or groups of a few, Leydig cells (Cellulae
interstitiales), blood vessels, fibroblasts and cells of the macrophage system,
such as macrophages, lymphocytes and monocytes. The interstitial tissue of
Fig. 2.9 Contd. ...
Sg, spermatogonium; Sp, spermatocyte; Sd, elongating spermatids; B, basal

lamina; L, lumen of seminiferous tubule. Arrowheads indicate a junctional complex
between adjacent Sertoli cells (Sertoli-Sertoli junctional complex) situated above
the spermatogonium, and consisting of a varying number of tight junctions and
parallel-running subsurface cisternae of endoplasmic reticulum (highlighted in
Figure B). Other less frequently encountered inter-Sertoli cell junctions, lying apical
to the tight junctions, are adhering junctions (Ad) (comprising intracytoplasmic
condensation of material, on both sides of the Sertoli cell plasma membranes,
and a line of dense material between the two cell membranes, and gap junctions
(Gp). The former is also found between adjoining Sertoli and germ cells. A special
type of Sertoli cell-elongating spermatid cell junction (arrows) displays a
subsurface condensed material in the Sertoli cell cytoplasm, but none in the
spermatid cytoplasm (hence also known as hemi-ectoplasmic specialization). B. A
higher power representation of tight junctions between adjacent Sertoli cells.
Arrowheads, focal tight junctions; Sert, Sertoli cell; er, cisternae of endoplasmic
reticulum. Unlike in mammals, there are no layers of filaments between the
cisternae and the cell membranes, on either side. Original.
#" Reproductive Biology and Phylogeny of Birds
Fig. 2.10 Coturnix japonica. India ink particles (arrowheads) occur in the apical
half of the Sertoli cytoplasm after 15 m of retrograde infusion of India ink through
the rete testis into the seminiferous tubules. Bar = 50 µm. Original.
the testis has been described in a number of mammals (Christensen 1965;

Fawcett et al. 1969, 1970; Dym 1973; Fawcett et al. 1973; Weaker 1977; Skinner
et al. 1991), but reports on this tissue in birds are rather scanty, and are, in the
main, on domestic species of birds, such as the rooster (Rothwell and Tingari
1973, 1974; Rothwell 1975; Aire 1997), Duck (Marchand 1973; Aire 1997),
Guineafowl (Numida meleagris) and Japanese quail (Aire 1997). Although a
very important component of the interstitium, Leydig cells have been
described in only a few species of birds (Connel 1972; Garnier et al. 1973;
Marchand 1973; Rothwell 1973; Scheib 1973; Aire 1997). The following review
of the testicular interstitial tissue is based largely on these reports, and a
number of unpublished observations from our laboratory.
2.2.4.1 The boundary (peritubular) tissue
Figures 2.11A and B show interstitial tissue located between three
seminiferous tubules in the drake. Figure 2.11C illustrates the relationship
between the seminiferous tubules, the boundary tissue, and the interstitium
containing blood vessels and Leydig cells. The boundary tissue in the rooster
(Rothwell 1975) and Ostrich (Soley, J. T., van Wilpe, E. and Aire, T. A.
unpublished observations) consists of an inner fibrous layer, subjacent to the
seminiferous epithelium, and an outer cellular layer of myofibroblasts. The
inner fibrous layer or lamella exhibits a homogeneous, moderately dense basal
lamina, and an adjacent layer of multi-directional collagen fibrils. The latter
layer, of collagen fibrils, is absent in the birds studied by Aire (1997) (see Fig.
2.12A). It is not known why this structural difference occurs between the bird
Anatomy of the Testis and Male Reproductive Tract ##
Fig. 2.11 Anas platyrhynchos (A, B), Coturnix japonica (C). A. Wedges of interstitial
tissue between three seminiferous tubules shows a peripheral lymphatic vessel
(arrows). V, blood vessels; L, Leydig cells. B. The lymphatic vessel (arrow) in this
histological section appears to be centrally located in the interstitium. L, Leydig cell.
C. An electron micrograph displays a lymphatic vessel (star) situated between the
boundary tissue (B) of a seminiferous tubule (S), a blood vessel (V) and a Leydig
cell (L). The blood vessel and Leydig cell are within the interstitium. Bars: A = 60
µm; B = 60 µm; C = 2 µm. Original.
#$ Reproductive Biology and Phylogeny of Birds
tissues that were fixed by immersion fixation (Rothwell and Tingari 1973;
Rothwell 1975; Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished
observations) and those fixed by vascular perfusion (Aire 1997). The basal
lamina rests on a thin layer of microfibrils and amorphous, moderately
electron-dense material in perfused birds (Aire 1997). However, Humphreys
(1975) reports that the basement membrane of the budgerigar testis exhibits
“irregular collagen content, and that many birds do not show collagen”, at
all. Microfilament rearrangement and re-configuration in the Leydig cells of
the dog has been described in tissues that were re-fixed by immersion in
glutaraldehyde after a previous perfusion fixation (Connel and Christensen
1975). Collagen fibrils have been observed, recently, in unpublished
micrographs, below the basal lamina in a patchy manner, in some, but not all,
of the seminiferous tubules, in intravascularly perfused adult sexually active
Japanese quail, in our laboratory (Fig. 2.12B). This is in accord with the
findings by Humphreys, referred to above. Further studies are necessary,
using various fixatives and buffers, as well as fixation methods in several
species of birds in order to clarify this concern.
The mesenchymal layer of myofibroblast cells varies in thickness, based
upon the number of concentric, alternating or overlapping cells. There are up
to 5, and occasionally, more, cellular lamellae (Aire 1997; Soley, J. T., van
Wilpe, E and Aire, T. A. unpublished observations). The myofibroblasts
contain highly elongated, uniformly granular nuclei displaying a few foci of
marginated chromatin and eccentric nucleoli. Short profiles of moderately
distended RER, a few oval mitochondria, a well-developed Golgi complex and
a number of micropinocytotic vesicles are found in the cytoplasm, which, also,
abounds in typical 5 nm-thick intermediate filaments and associated focal
intracytoplasmic densities. In the rooster and ostrich tissues fixed by
immersion, bundles of collagen fibres lie in the intercellular spaces between
myofibroblast cells (Fig. 2.13). Myofibroblasts that exhibit features which are
found in either fibroblasts or smooth muscle cells are not uncommonly seen.
In the quail and ostrich, the peritubular cells are invested by what appears to
be an incomplete layer of basal lamina (Fig. 3.12B). The endothelium of blood
or lymphatic capillary forms the peripheral limit between the boundary tissue
and the interstitium (Fig. 2.11C). Birds are similar to the rat, chinchilla, guinea
pig and mouse, in possessing a relatively small volume of Leydig cells and in
the location of the lymphatic vessels, but differ from these mammals in lacking
an extensive peritubular lymphatic system (Fawcett et al. 1973). However, the
arrangement of the myofibroblasts is similar to that in human and cat (Burgos
et al. 1970), in being multilayered. The peritubular tissue of birds therefore
varies in certain particulars between species (Rothwell 1975; Aire 1997; Soley,
J. T., van Wilpe, E. and Aire, T. A. unpublished observations), and combines
structural features of the interstitium found variably in mammals.
2.2.4.2 The interstitium
The interstitium is relatively compact in birds (Aire 1997) except in the ostrich
in which it forms a loose and ‘oedematous’ connective tissue (Soley, J. T., van
Anatomy of the Testis and Male Reproductive Tract #%
Fig. 2.12 Anas platyrhynchos (A) and Coturnix japonica (B). A. The basal lamina
(B) of the seminiferous tubule does not rest on an internal lamella of collagen
fibers, but directly on an amorphous material adjacent to myofibroblast cells (M) of
the boundary tissue. Extensions of the basal lamina (arrows) into the seminiferous
epithelium occurs frequently. B. The internal (fibroreticular) lamella of the boundary
tissue displays a patchy presence of collagen fibers (C) below the basal lamina
(B); Arrow indicates a part of the internal lamella devoid of collagen fibers.
Arrowheads, dense material, similar to the lamina densa of the basal lamina,
interposed between myofibroblasts (M); S, seminiferous epithelium. Bars: A = 2
µm; B = 1 µm. Figure A is from Aire, T. A. 1997 Onderstepoort Journal of Veterinary
Research 64: 291-299, with permission of the Editor. Figure B is original.
#& Reproductive Biology and Phylogeny of Birds
Fig. 2.13 Struthio camelus. A. The boundary tissue at the base of the germinal
epithelium (GE) displays a regular basal lamina (arrowheads), the inner fibrous
lamellum (F) and the outer peritubular layer consisting of alternating cellular (1-4)
and acellular lamellae. Collagen fibrils (cf) are the most conspicuous element of
the acellular lamellae. B, Dilated profiles of rough endoplasmic reticulum (RER),
bundles of microfilaments (arrow), focal densities (squat arrow) and coated pit
(arrowhead) are present in the myofibroblasts. Cross-sections of collagen fibers
occur in the acellular lamellae. Basal lamina (double arrowhead). Bars for both
figures = 1 µm. (From Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished
micrographs).
Anatomy of the Testis and Male Reproductive Tract #'
Wilpe, E. and Aire, T. A. unpublished observations). The component tissue

and cell types include a number of centrally situated blood vessels, varying in
size from capillaries to large vessels (Fig. 2.11). Lymphatic vessels are sparse
and usually peripherally, but occasionally, centrally, located in the
interstitium. When peripheral, they meander between the boundary tissue and
the central components of the interstitium (Aire 1997). They are commonly
located centrally in the Ostrich (Soley, J. T., van Wilpe, E. and Aire, T. A.
unpublished observations). Other component cells include very few
macrophages that are closely associated with a few, single or groups of a
small number of Leydig cells. It has not been determined in birds if there are
structural and functional relationships between these two cell types, as in
mammals in which they are morphologically and functionally coupled (Bergh
1985, 1987; Gayton et al. 1994). Other blood-derived cells, such as
lymphocytes, plasma and mast cells may occur in the interstitium. The central
role of the Leydig cell in the secretion of the primary male sex hormone, and
possibly also testicular estrogens in man, the stallion and boar (Akingbemi et
al. 1999) sets this cell apart for special treatment in this review.
2.2.4.3 The Leydig cell
Leydig cells of birds (Connel 1972; Nicholls and Graham 1972; Garnier et al.
1973; Marchand 1973; Rothwell 1975; Aire 1997; Soley, J. T., van Wilpe, E. and
Aire, T. A. unpublished observations) are generally similar, structurally, to
those of mammals (Neaves 1975; Russel 1996; Akingbemi et al. 1999). The
number of Leydig cells that are present in the interstitial wedges between
multiple seminiferous tubules is small (Fig. 2.11C). Leydig cells seem to form
columns of cells in the interstices in the turkey (personal observations).
References to information on the ostrich are taken from unpublished results of
studies done on the interstitial tissue of this bird by Soley, J. T., van Wilpe, E.
and Aire, T. A. Ultrastructurally, Leydig cells of gonadally active birds are
large, relatively electron-dense, and contain euchromatic, oval, polygonal or
elongated nuclei, depending on location in the interstitial tissue (Fig. 2.14).
The Golgi complex is generally moderately developed in birds (Aire 1997)
except in the ostrich, in which it is well developed and displays numerous
Golgi fields (Fig. 2.15B). Prominent, relatively numerous, oval or elongate
mitochondria contain tubular cristae that are embedded in an electron-dense
matrix (Fig.2.14). The rough endoplasmic reticulum (RER) is poorly developed,
relative to the smooth endoplasmic reticulum (SER) which is more developed
(Fig. 2.14), but not nearly as well as in mammals (Neaves 1975; Aire 1997).
The SER profiles are short, moderately dilated, or, in the ostrich, may form
branched or anastomotic strands. Whorls of SER are present only in the
guineafowl (Aire 1997), and, in the ostrich, there are concentric collections of
thickened membranes which develop within existing cisternae of SER,
resulting in arrays of parallel-oriented, long, rod-shaped structures of greater
electron density than normal strands of SER with which they are continuous
(Fig. 2.15). Their functions are not known. Scattered ribosomes, polyribosomes,
Fig. 2.14 Coturnix japonica. Part of an electron micrograph of a Leydig cell

showing mitochondria (M) with tubular cristae within a dense matrix. Abundant
profiles of smooth endoplasmic reticulum (SER), several lipid droplets (D) and only
a few, short profiles of rough endoplasmic reticulum (arrowhead) occur in the
cytoplasm. The nucleus (N) is oval in shape. F, microfilaments in peritubular
myofibroblasts (P); S, seminiferous tubule. Bar = 1 µm. Original.
Anatomy of the Testis and Male Reproductive Tract $
dense bodies and intermediate filaments of variable prominence and location

within the cell are present in the ostrich. Lipid droplets, often partially
extracted, are fewer and larger in the drake, guineafowl and rooster, than in
the Japanese quail (Fig. 2.14) and ostrich. In several cells, a solitary cilium, of
undetermined axoneme structure, originates deeply in the cytoplasm and,
therefore, traverses an intracytoplasmic canal to reach the cell surface, in the
ostrich.
Non-myelinated nerve cell processes, as well as occasional naked axons are
to be found throughout the interstitium. These nerves are transmitted into the
substance of the testis by septa-like strands of connective tissue that surround
the seminiferous tubules. The boundary tissue, however, does not display any
neural elements, but some nerve fibers seem to lie close to the basal lamina of
the seminiferous tubules in the rooster (Tingari and Lake 1972a). Adrenergic
nerves appear to innervate interstitial cells in Mute swan (Cygnus olor)
(Baumgarten and Holstein 1968).
2.2.4.4 Functions of the intertubular tissue
Peritubular cells are involved not only in environmental interactions, that is
structural interactions, but also regulatory interactions, in which they produce
a paracrine or an autocrine agent to elicit a signal transduction event that
influences cellular functions on a molecular level (Skinner 1987). Considerable
and varying interactions occur between the cells of the testis (Skinner et al.
1991). These include peritubular cell-Sertoli interactions, all of which are vital
to the proper structural and functional roles of the various cells in the testis.
For example, the peritubular cells secrete PModS which influences Sertoli cell
functions that are vital for the maintenance and control of spermatogenesis
(Skinner 1987).
2.3 THE EXCURRENT DUCTS OF THE TESTIS

The excurrent ducts of the testis which comprise various ducts that transport
spermatozoa and fluid produced by the seminiferous epithelium, in birds,
have, surprisingly, been described in only a few species: the rooster (Gray
1937; Lake 1957; Marvan 1969; Tingari 1971, 1972; Budras and Sauer
1975a,b), Turkey (Hess and Thurston 1977; Hess et al. 1976; Aire 2000a; Aire
and Josling 2000), Guineafowl (Aire et al. 1979; Aire 1980, 1982a), Japanese
quail (Aire 1979a, 1980, 1982b, 2000a; Clulow and Jones 1982, 1988), Ostrich
(Budras and Meier 1981; Aire and Soley 2000, 2003), passerine birds (Bailey
1953; Traciuc 1967, 1969; Middleton 1972; Barker and Kendall 1984), Pigeon
(Stefanini et al. 1999), Mute Swan (Mehrotra 1962, 1964) and drake (Marchand
and Gomot 1973; Aire 1982a,b; 2000a).
The excurrent duct system of birds, as in mammals, comprises (a) the rete
testis (RT) unit, (b) the efferent duct unit and (c) the epididymal duct unit.
The epididymal duct unit, in turn, comprises the ductus conjugens or
connecting duct, the ductus epididymidis or epididymal duct and the ductus
deferens or deferent duct. The epididymis of birds is not divided into the
Fig. 2.15 Struthio camelus. A. A Leydig cell displays an array of rod-shaped

structures (R) in longitudinal profiles. These structures, some of which appear
branched (arrows), are more electron dense than the smooth endoplasmic
reticulum (SER) (arrowheads). Only a few profiles of rough endoplasmic reticulum
or sparsely granulated endoplasmic reticulum (squat arrows) occur in the
Fig. 2.15 Contd. ...

Anatomy of the Testis and Male Reproductive Tract $!
customary gross segments of caput, corpus and cauda epididymides, seen in

mammals, but it is a complex structure (Fig. 2.16) consisting of several duct
units (Lake 1957; Tingari 1971; Budras and Sauer 1975; Hess et al. 1976; Aire
1979a; Aire et al. 1979; Budras and Meier 1981). This organ incorporates the
extratesticular portion of the RT, the efferent ducts, the connecting and
epididymal ducts (Fig. 2.17). A description of the histological and
ultrastructural features of the various duct units of the epididymis will be
based, in the main, on the various reports referred to, above.
2.3.1 The Rete Testis

The rete testis (RT) of birds is connected to the seminiferous tubules whose
products it transports to the succeeding segment of the excurrent duct system.
The occurrence of divisions, based on their location, of the RT has been a
controversial subject. Tingari (1971) considers that the rete is located entirely
outside the testis but it is generally agreed that there are intracapsular
(intratunical) and extratesticular portions of this duct unit. In addition, Bailey
(1953), Budras and Meier (1981), Aire (1982a) and Stefanini et al. (1999) have
described an intratesticular portion which is made up of rete channels that
are similar in epithelial lining to those in the intracapsular and extratesticular
segments, or appear in the form of tubuli recti in various species of birds
(Gray, 1937; Mehrotra 1964; Aire 1979a; Aire et al. 1979; Budras and Meier
1981; Barker and Kendall 1984). Osman (1980), however, reports that the
seminiferous tubules of the testis of the domestic fowl are joined to the RT in
three ways: by way of a terminal segment and a tubulus rectus, a terminal
segment only, or by direct opening into the rete lacunae and Lake (1957)
describes a transitional tubule, lined by modified Sertoli cells, at the junction
between the seminiferous tubule and RT. The intratesticular portion of the RT
is rarely observed in birds, but in both birds and mammals it constitutes a
minor part of the duct unit (Amann et al. 1977; Roosen-Runge and Holstein
1978; Budras and Meier 1981). They are more distinctly evident in regressed
testes (Fig. 2.17B). However, in birds, most of the seminiferous tubules
terminate by opening into the RT directly (Aire 1982b).
The main part of the RT of birds is extratesticular, forming an integral part
of the epididymis. Both the intracapsular and extratesticular parts of the RT of
the Ostrich (Budras and Meier 1981) are even more complex in organisation
than in most of the other birds studied. They consist of a series of connected

cytoplasm. The perimeter of the cell is demarcated by a basal lamina-like material
(double arrowheads). B. Rod-shaped structures, as in Fig. 2.15A, are sectioned
transversely. Strands of SER and the rods seem to be continuous or closely
associated (arrowheads). Some SER profiles display thickened membranes in the
lumen (arrows), probably indicating the formation of rod-shaped structures from
normal elements of the SER. An extensive Golgi complex (G), a dense body (D)
and a vesicular nucleus (Nu) are present in the cell. Bars: A = 1 µm; B = 2 µm.
(From Soley J. T., van Wilpe, E. and Aire, T. A. unpublished micrographs)
$" Reproductive Biology and Phylogeny of Birds
Fig. 2.16 Gallus domesticus. A diagrammatic representation of the epididymis

and its duct units, based partly on casts. T, testis; RT, rete testis; P, proximal efferent
ductule; D, distal efferent ductule; CD, connecting ductule; DE, ductus epididymidis;
DD, ductus deferens. From Nasu, T., Nakai, M., Murakami, T., Saito, I., and
Takahara, H. 1985 Japanese Journal of Zootechnical Science 56: 81-85. Fig. 2.1-7.
Reproduced with the kind permission of Japanese Society of Animal Science.
longitudinal cisterns and ducts that open ultimately into the efferent duct unit
(Budras and Meier 1981). The distribution of the RT in mammals also varies
between species, being septal and mediastinal in the rat, Rattus norvegicus
(Roosen-Runge 1961; Dym 1976; Hermo and Dworkin 1988), Guinea pig,
Cavia porcellus (Fawcett and Dym 1974), Goat, Capra hircus (Ezeasor 1986;
Goyal et al. 1992), bull, Bos taurus (Hees et al. 1987) and man, Homo sapiens
(Roosen-Runge and Holstein 1978). An extratesticular portion appears to be
found in only a few mammalian species, e.g. in man (Roosen-Runge and
Holstein 1978) and goat (Goyal et al. 1992).
In birds, the RT is the smallest duct unit, by volume, in both the epididymis
(Table 2.1), and the entire excurrent duct system, constituting only 2.3% of the
extra-testicular ducts in the Japanese quail (Clulow and Jones 1988). But, the
ratio of surface area of luminal border:luminal volume for this duct unit
(43.4:1) is quite large in the Japanese quail (Clulow and Jones 1988).
2.3.1.1 Surface features of the rete testis tubules/lacunae
Scanning electron microscopical features of the RT of birds, have been
reported in the drake (Aire 1982a), Ostrich (Aire and Soley 2000) and certain
domestic galliform birds—Turkey, rooster, Guineafowl and Japanese quail
(Bakst 1980; Aire and Josling 2000). The RT spaces are, not infrequently,
broken up into interconnecting cavernous spaces by lamellae or sheets of
connective tissue which are, themselves, lined by the rete epithelium. Narrow
cylindrical struts of connective tissue or chordae retis, lined by rete epithelium,
Anatomy of the Testis and Male Reproductive Tract $#
Fig. 2.17 Coturnix japonica. A. Low power histological view of the epididymis
showing profiles of the rete testis (RT) opening into ductuli efferentes proximales
(PED). DED, ductuli efferentes distales; DC, ductus conjugens and DE, ductus
epididymidis. B. A histological section of the para-epididymal region of an
involuting testis showing exaggerated profiles of the intratesticular duct system
(asterisks), which are portions of the rete testis. TC, testicular capsule; IR, capsular
portion of the rete testis. Bar: A = 200 µm, B = 100 µm. Original.
$$ Reproductive Biology and Phylogeny of Birds
Table 2.1 Species variation in volumetric proportions (%) of epididymal ducts and structures
Species and number of birds

Structures Chicken (4) Japanese quail (5) Guinea-fowl (3) Turkey (3) Ostrich (4)
Rete testis 13.3 ± 1.5* 9.9 ± 0.7 10.7 ± 1.8 14.0 ± 6.8 2.4 ± 1.8
Proximal 27.6 ± 3.9 40.8 ± 3.5 45.7 ± 4.2 28.1 ± 3.9 78 11.8 ± 1.8
6.2 ± 2.8 9
efferent duct
Distal efferent duct 7.7 ± 1.3 15.2 ± 2.5 16.2 ± 2.1
5.2 ± 2.2 7
8 26.1 ± 4.1
Connecting 2.3 ± 0.4 1.7 ± 0.4 0.7 ± 0.0
3.3 ± 1.6 9
duct
Ductus epididymidis 7.6 ± 0.4 2.4 ± 0.6 1.8 ± 0.2
Connective tissue 38.7 ± 3.7 27.3 ± 5.4 22.6 ± 1.5 38.8 ± 4.1 58.2 ± 4.9
Blood vessels 2.5 ± 0.4 2.7 ± 0.5 2.3 ± 0.3 4.4 ±1.6 1.8 ± 1.4
Aberrant ducts 0.3 ± 0.0 – – – –
* ± Standard error. Adapted from Aire, T.A. 1979. Journal of Anatomy 129: 703 – 706, Table 1, with
permission of Blackwell Publishing Ltd.
may be seen to traverse the lacuna, linking opposite walls. Chordae retis,
named by Roosen-Runge and Holstein (1978), may be a common feature of the
RT in many species, and probably acts as transluminal coupling device for
the contractile system of myofibroblasts (Hees et al. 1989), as do the trabeculae
septomarginales of the heart.
The surface of the epithelial lining of the RT ductules, except for a few
minor details, is generally regular, but may bear a few shallow grooves in the
ostrich (Aire and Soley 2000). The apical cell outlines are elongated or
polygonal in shape. The cell surfaces extend into short, stubby regular
microvilli which vary from very few and sparsely distributed to very
numerous and evenly distributed (Fig. 2.18). A single, central cilium projects
from most cells into the duct lumen in all birds studied (Aire 1982a,b; Aire
and Soley 2000; Aire and Josling 2000). The solitary cilium, that exhibits the
9+2 axonemal structure in the bull (Hees et al. 1989), and is probably sensory
in function, appears to be a common feature of the rete cells in animals, as it
has also been reported in other mammals, including man (Dym 1976; Roosen-
Runge and Holstein 1978; Goto 1981; Hees et al. 1989). The solitary cilia of the
non-ciliated cells of the human oviduct, which are similar to those described
in the male reproductive organs, have been shown, using phase- or video-
microscopy, to have vortical or funnel-like movement (Odor and Blandau
1985; Nonaka et al. 1998), contrary to Ghadially’s (1997) assertion that they
are immotile. In most birds studied, especially in the rooster, macrophages are
seen in the RT lumen, resting on the epithelium (Aire and Malmqvist 1979a;
Osman 1980; Budras and Meier 1981; Aire 1982b; Aire and Josling 2000). Only
a few spermatozoa and earlier germ cell series occur in the lumen, and each
spermatozoon in the RT of the ostrich bears a single, spindle-shaped, distal
cytoplasmic droplet (Aire and Soley 2000), a phenomenon that appears to be
unique to this bird, and perhaps other ratites. The motility and fertilizing
ability of the rete spermatozoa, and, indeed, of spermatozoa in other segments
of the excurrent ducts in the ostrich need to be investigated, as has been done
Anatomy of the Testis and Male Reproductive Tract $%
Fig. 2.18 Coturnix japonica (A), Gallus domesticus (B, C). Surface morphology of
the rete testis epithelium. A. Short, stubby microvilli are concentrated centrally and
around the edge of the cell in the quail, or B. are evenly and sparsely distributed
throughout the surface. In C. the microvilli are restricted to the edges of the cell
surfaces. In B. and C., solitary cilia project from most cells. Bars: A = 10 µm; B and
C = 2 µm. Original.
$& Reproductive Biology and Phylogeny of Birds
for some other birds (Munro 1938; Bedford 1979; Howarth 1983, 1995), for
purposes of comparability.
2.3.1.2 The histology and ultrastructure of the rete testis cells
The rete epithelium varies from simple, low cuboidal to squamous. Because
the apical portions of some of the rete cells overlap sections of adjacent cells,
the histological sections of the epithelium often display a pseudostratified
appearance (Fig. 2.19). This appears to be a common feature in all birds except
the ostrich in which the epithelium is almost always simple (Aire and Soley
2003). In mammals, the rete epithelium is simple squamous to low columnar
(Leeson 1962; Dym 1976; Bustos-Obregon and Holstein 1976; Osman 1978;
Hees et al. 1989). The rete epithelium contains only one, non-ciliated cell type.
Other cell types that may be found in the epithelium are intraepithelial
lymphocytes (Fig. 2.19A) and an occasional ciliated cell. Ciliated cells have
been described by Barker and Kendall (1984) as being a usual component of
the epithelium in some wild birds, but Aire (2002a) is of the opinion that a few
scattered ciliated cells occur in the rete epithelium of gonadally-resting birds.
Most of these cells, obviously, are lost during recrudescence, preparatory to
resumption of active gonadal function.
The surfaces of the rete cells display short, straight and regular microvilli,
in well-fixed tissue. Adjacent lateral cell membranes, or parts thereof, are
either straight and regular (Tingari 1972) or form complex interdigitations
(Aire 1982b; Aire and Soley 2003) that may extend to the basal part of the cell.
Extensive, intricate cell membrane interdigitation is probably associated with
active transport of substances (Morales et al. 1984) but there is little net fluid
reabsorption in the RT of the Japanese quail (Clulow and Jones 1982). The
apical tight junctional complexes between the rete cells contain only one or
two punctate fusions which, however, do not permit tracer compounds to
reach the duct lumen via the paracellular space (Nakai and Nasu 1991). This
type of junctional complex has also been reported in the mammalian rete
epithelium (Dym 1976) but Claude and Goodenough (1973) regard it as a
‘leaky’ junctional complex. Desmosomes are also present, both in the apical
and basal zones of the plasma membrane (Barker and Kendall 1984). In the
ostrich, a unique lateral cell membrane modification, similar, in some respects,
Fig. 2.19 Coturnix japonica (A) and Anas platyrhynchos (B). Transmission elec-
tron micrographs of rete testis epithelial cells. A. The epithelium often appears
pseudostratified. The nuclei (N) are deeply indented, euchromatic and irregular in
shape. The cytoplasm contains sparse organelles. L, an intraepithelial lymphocyte;
P, periductal tissue. B. Part of the rete cell exhibiting short, stubby
microvilli (arrowheads), a number of multivesicular bodies (arrows), a small Golgi
complex (G), a few subapical vesicles (V) and round/oval profiles of mitochondria
(M). Bars: A = 20 µm; B = 1 µm. A is adapted from Aire, T. A. 2002 Anatomy Histol-
ogy Embryology 31: 113-118, Fig. 2, with permission of Blackwell Publishing Ltd.,
and B is adapted from Aire, T. A. 1982 Journal of Anatomy 135:
97-110, Fig. 15, with permission of Blackwell Publishing Ltd.
Anatomy of the Testis and Male Reproductive Tract $'
Fig. 2.19
% Reproductive Biology and Phylogeny of Birds
to a hemi-desmosome, occurs frequently along the length of this membrane. Its

function is unknown (Aire and Soley 2003). Rete cell nuclei are typically
irregular in outline, being often deeply indented and exhibiting marginated
chromatin as well as a single, central nucleolus (Fig. 2.19A). However, in the
ostrich, the nuclei have regular, vertically elongated profiles, and are more
euchromatic than in other birds. Oval to elongate mitochondria occur above
and below the nucleus in moderate numbers, and are more numerous in the
ostrich rete cells (Aire and Soley 2003) than has been reported for other birds
(Tingari 1972; Aire 1982b; Barker and Kendall 1984). The Golgi complex is
moderately large and usually lies in the perinuclear region (Fig. 2.19B). Other
organelles worthy of note are multivesicular bodies and strands of rough
endoplasmic reticulum, both of which may be numerous in the drake (Aire
1982b). A few, small, lipid droplets are a constant feature of this cell. However,
in the ostrich, a solitary, large heterogeneous lipid droplet is a consistent
feature in the immediate supranuclear region (Aire and Soley 2003).
Intermediate filaments commonly surround the nuclei and support the basal
part of the cell, especially in the ostrich (Aire 1982b; Aire and Soley 2003). The
chordae retis is lined by an epithelium that is similar to that of the rest of the
RT, but its core substance is composed of loose spongy connective tissue, as
has also been observed in the bull (Hees et al. 1989). Phagocytised sperm
fragments are found occasionally in the rete cell of the rooster (Tingari 1972),
and in certain mammalian species (Sinowatz et al. 1979; Holstein 1978).
Luminal macrophages contain ingested fragments of spermatozoa only
occasionally (Aire and Malmqvist 1979a; Aire and Josling 2000), apparently,
removing by ingestion, only damaged or abnormal spermatozoa, but they
become avidly spermiophagic in vasectomized or vasoligated birds (Tingari
and Lake 1972b; Aire and Heath 1977; Nakai et al. 1989b; Aire 2002a).
The subepithelial tissue is composed of dense connective tissue and
myofibroblasts or smooth muscle cells (Tingari 1972; Aire 1982b; Aire and
Soley 2003). This tissue is richly innervated (Tingari 1972), but poorly
vascularized (Barker and Kendall 1984; Nakai et al. 1988) by a non-fenestrated
capillary network that is irregularly arranged (Fig. 2.20) (Nakai et al. 1988).
Aire (1982b) has observed a rich lymphatic drainage system in the
subepithelial tissue in the vicinity of the RT in the drake.
Intraepithelial lymphocytes, exhibiting irregular outlines, light-staining
cytoplasm and considerably heterochromatic nuclei (Fig. 2.19A), are not
uncommon components of the rete epithelium in birds (Aire and Malmqvist
1979b; Aire 1982b; Osman 1980; Stefanini et al. 1999) and mammals (Dym and
Romrell 1975; Hees et al. 1989). They have sparse organelles, as in mammals,
and there are no junctional complexes between them and epithelial cells. They
occur at all levels in the epithelium. These cells probably monitor the
epithelium with a view to sequestering germ cell antigens and preventing their
escape into the blood or periductal connective tissue (Dym and Romrell 1975;
Osman and Plöen 1978).
Anatomy of the Testis and Male Reproductive Tract %
Fig. 2.20 Gallus domesticus. Three types of microvasculature of the rete testis
ductule (a), efferent ductule (b) and epididymal duct and ductus deferens (c) are
shown diagrammatically. The rete testis has a sparse and irregular capillary
network, while both the efferent ductule and epididymal duct unit have a dense
capillary network. The meshwork of capillaries is polygonal in (b) and elongated in
(c). From Nakai, M., Hashimoto, Y., Kitagawa, H., Kon, Y. and Kudo, N. 1988
Japanese Journal of Veterinary Science, with the kind permission of Japanese
Society of Veterinary Medical Science.
2.3.1.3 Functions of the rete testis

The functions of the avian RT are still quite conjectural. The RT constitutes
only 2.4% in a preliminary study in the ostrich, and between 10% and 13% of
the entire volume of the epididymis in galliform birds (see Table 2.1). This does
not take into account the small intratesticular and intracapsular portions of
this duct unit. The RT is, understandably, the smallest component (2.3%) of
the units of the extratesticular genital ducts of the Japanese quail (Clulow and
Jones 1988). The histology and ultrastructure of the RT epithelium in birds
(Tingari 1971 1972; Budras and Sauer 1975; Hess and Thurston 1977; Hess et
al.1976; Aire 1979a, 1982b; Budras and Meier 1981; Barker and Kendall 1984;
Aire and Soley 2003) is similar, in most respects, to those of mammals (Dym
1976; Roosen-Runge and Holstein 1978; Goto 1981; Hees et al. 1989), and
because of the ‘leaky type’ apicolateral junctional complex between the
epithelial cells (Claude and Goodenough 1973; Lopez et al. 1997), this segment
of the excurrent duct system may be a particularly weak one, with regard to
the blood-epididymal barrier. In mammals, the RT epithelium secretes about
65% of total testicular fluid (Tuck et al. 1970), but the fluid entering the rete
testis is the primary secretion of the seminiferous tubules in the quail (Clulow
and Jones 2004). However, even though there is a net reabsorption of
testicular fluid by all of the epididymal ducts in the Japanese quail, there is
little net fluid transport across the RT epithelium (Clulow and Jones 1988).
Spermatozoa spend only 25 sec in traversing the RT in the Japanese quail

(Clulow and Jones 1988), and therefore the through-flow that enters the
proximal efferent duct is highly diluted fluid which contains only a few germ
cells (Aire 1979a, 1982b).
The RT cells are capable of endocytic activity by both fluid-(bulk)-phase
and adsorptive endocytosis in the rat (Morales et al. 1984). Tracers interiorised
from the lumen by rete cells are disposed of by the lysosomal system in a
similar fashion, irrespective of mode of endocytosis. Morales et al. (1984) have
concluded that the RT cells may play a role in determining the composition of
the RT fluid. These cells in the Japanese quail are also capable of interiorizing
intraluminally-introduced India ink particles which are then conveyed to the
lysosomal system of the cells (Aire T.A. unpublished observations). However,
RT cells fail to phagocytize intraluminally-introduced avirulent strain of
Salmonella gallinarium organisms in the Japanese quail (Aire et al. 2004).
The myofibroblast layer surrounding the RT lacunae is well developed, and
probably acts to move testicular fluid forward into the efferent ducts. The
relaxation of the contractile layer, including the chordae retis, could suck fluid
from the seminiferous tubules, by means of vis-a-fronte forces, into the
cavernous spaces of the RT. Actin, desmin and vimentin filament staining are
strongly positive in the myofibroblast layer in the Japanese quail and drake
but only slightly positive in the ostrich (Pers. obs.).
2.3.2 The Efferent Ducts (Ductuli Efferentes)

In birds, the RT lacunae are continued by the efferent duct unit (Bailey 1953;
Lake 1957; Traciuc 1969; Tingari 1971; Budras and Sauer 1975a; Hess et al.
1976; Aire 1979a, 1980; Aire et al. 1979; Budras and Meier 1981; Bellamy and
Kendall 1985), as in mammals (Reid and Cleland 1957; Ladman and Young
1958; Ilio and Hess 1994). The volume proportion of the efferent ducts in the
epididymis varies between 35% and 62% in domestic Galliform species of
birds and 12% in the ostrich (Table 2.1), and about 19% of the total volume of
the genital ducts in the Japanese quail (Clulow and Jones 1988). Two different
but serially arranged segments of the efferent duct occur in birds, viz.,
proximal efferent duct [ductuli efferentes proximalis] (PED) and the distal
efferent duct [ductuli efferentes distalis] (DED). The latter is the distal
continuation of the PED, and opens into the connecting duct [ductulus
conjugens] (Budras and Sauer 1975a; Aire 1979a). The ductuli efferentes,
according to Ilio and Hess (1994), are unique in being the only duct unit of the
male reproductive tract that is lined by a ciliated epithelium. Whereas in birds
there is a distinct histological division of the efferent ducts into the two
segments, PED and DED, such a division is not clearly marked in mammals,
among which are species-based differences in the non-ciliated cell types
present along the length of the duct. Thus, in the laboratory animals, there is
a single type of non-ciliated (NC) cell (Hamilton 1975; Robaire and Hermo
1988) and three types in the cane rat, Threonomys swinderianus (Aire and van
der Merwe 2003), man, goat and bull (Goyal et al.1992; Morita 1966; Goyal and
Hrudka 1981; Gray et al. 1983; Goyal and Williams 1988). Ciliated (C) cells in
Anatomy of the Testis and Male Reproductive Tract %!
birds, as in mammals, do not vary structurally along the efferent duct, but the
non-ciliated (NC) cell types (types I and II, in the PED and DED, respectively)
exhibit different and characteristic cytological features (Aire et al. 1979; Aire
1980). Tingari (1971,1972), Marchand and Gomot (1973), Hess et al. (1976),
Hess and Thurston (1977) regarded the distal efferent ductule to be the
connecting duct, in error, and, consequently, described only a single non-
ciliated cell type in the efferent duct system.
2.3.2.1 Surface morphology of the efferent ducts
The surface features of the efferent ducts have been described, using scanning
electron microscopy, in the rooster and Turkey (Bakst 1980; Aire and Josling
2000), drake (Aire 1982a), rooster, drake and Japanese quail (Aire and Josling
2000) and Ostrich (Aire and Soley 2000). The following account is derived
from these reports.
Proximal Efferent Ducts (PED). The epithelial lining, and, in some instances,
the mucosa of the PED is highly folded, presenting an irregular, festoon
appearance (Fig. 2.21A,B). The folds project prominently into the ductal lumen
and are lined by cells whose apical surfaces extend into a lush brush border
of microvilli (non-ciliated cells) or numerous cilia (ciliated cells). In all
investigated birds, the non-ciliated cells (NC) are more numerous than the
ciliated (C) cells, and their microvilli are closely packed, long and regularly
cylindrical in shape. The microvilli of the C cells are fewer, shorter and
thinner than those of the NC cells, and are scattered between the cilia which
usually overshadow adjacent NC cells. The microvilli of the NC type I cell of
the PED are shorter in the Turkey and very much so in the ostrich than in the
rooster and Japanese quail (Aire and Josling, 2000; Aire and Soley 2000). A
single cilium projects into the duct lumen from the central region of most NC
cells (Aire 1982b; Aire and Soley 2000; Aire and Josling 2000). In vascularly
perfused birds (Aire 1982a; Aire and Soley 2000; Aire and Josling 2000), the
NC cells do not exhibit apical blebbing, as reported by Bakst (1980), whose
specimens were fixed by immersion fixation. Aire (1980; 2000a) regards apical
blebbing in this cell type to be a fixation artifact.
Distal Efferent Ducts (DED). The epithelium of this round or oval duct is
regular and exhibits no folds that are very prominent in the PED. The cilia of
the predominant cell type, C cell, overhang the fewer NC type II cells whose
apical surface features are similar to those of the NC type I cell of the PED
(Fig. 2.21C).
2.3.2.2 Histology and ultrastructure of the efferent duct epithelia
The histology of the efferent ducts in birds has been reported in only a small
number of species: in the rooster (Lake 1957; Tingari 1971; Budras and Sauer
1975a, b), turkey (Hess et al. 1976), Japanese quail (Aire 1979a), Guineafowl
(Aire et al. 1979), duck (Marchand and Gomot 1973), Pigeon (Stefanini et al.
1999) and the Common Starling, Sturnus vulgaris (Bellamy and Kendall 1985).
Histology of Efferent Ducts. The epithelial lining of the ductuli efferentes
proximalis (proximal efferent duct) (PED) or occasionally, the mucosa, projects
%" Reproductive Biology and Phylogeny of Birds
Fig. 2.21 Gallus domesticus. Scanning electron microscopy of the surface

features of the efferent ducts. A. The duct wall of the proximal efferent duct (PED)
displays variably-sized epithelial and mucosal folds. Note that the epithelial surface
is devoid of apical cytoplasmic blebs. B. A higher power view of the epithelial
surface of PED. C, cilia of ciliated cells; V, microvilli of non-ciliated cells. Cilia of
ciliated cells frequently over-shadow the non-ciliated cells which are more
numerous that the former cells. C shows an overwhelming preponderance of cilia-
bearing ciliated cells over the non-ciliated type II cell of the DED. Bars: A = 50 µm;
B and C = 5 µm. Original.
Anatomy of the Testis and Male Reproductive Tract %#
into the duct lumen as folds of varying length and thickness, thus conferring
a ‘festoon’ appearance on transverse profiles of the PED (Fig. 3.17A). The
epithelium is columnar and pseudostratified because the C cells usually
appear truncated between NC cells, and their nuclei are usually situated in
the apical half of the cells, while those of the NC type I cells are located in the
basal half (Fig. 2.22A). Both cell types make contact with the basement
membrane. The NC cells are predominant over the C cells, and, in the common
starling in the ratio of 5:1 (Bellamy and Kendall 1985). The luminal content is
mainly proteinaceous fluid, in which there is a suspension of sparse
spermatozoa and earlier germ cell series.
The apical surface of the NC cell is extended by long, closely bunched,
regularly cylindrical microvilli that project into the duct lumen. In plastic
sections, the subapical region of the cell displays a few vacuoles of varying
sizes, below which are rows of round dense bodies extending to the level of
the nucleus. The nucleus is round or, more commonly, oval in shape, and
contains one or two nucleoli. The C cell is generally of a lighter stain than the
NC cell. The nuclei of the C cells are also round or oval, but may be irregular
in shape. Even in plastic sections, profiles of organelles in the C cell, other
than the nuclei, are hardly discernible. The epithelium rests on a distinct
basement membrane that is supported by periductal tissue of fibroblasts,
collagen fibers and myofibroblasts.
The ductuli efferentis distalis (DED) has a regular profile, both externally
and internally (Fig. 2.22B). It has a columnar or high cuboidal,
pseudostratified epithelium consisting of NC cells, and a preponderance of C
cells. The apical morphological features of these cells are similar to those of
the PED. In plastic sections, there are no obvious subapical vacuoles or dense
bodies in the NC type II cells of this duct. The C cell is similar, structurally, to
that in the PED.
The microvasculature of the avian epididymis is derived from branches of
testicular artery, including the cranial ureterodeferential ramus, that supply
blood to the epididymis in the rooster (Nishida 1964; Nakai et al. 1988) and
Ostrich (Elias M, Aire T.A. and Soley J.T. unpublished observations). Smaller
branches of these arteries run along the length of the efferent duct and produce
a rich periductal network of fenestrated capillaries (Fig. 2.20B).
Nerves that supply the epididymis of the rooster are derived from the
testicular plexus, and reach the organ by accompanying the testicular arteries
(Nishida 1964; Tingari 1971). Both cholinergic and adrenergic components,
which have a similar distribution, have been demonstrated in the efferent
ducts. These nerve fibers run around the ducts and are associated with the
walls of blood vessels and muscle fibers (Tingari and Lake 1972a).
Ultrastructure of the Efferent Ducts. Detailed reports of the fine structure of
the epithelium in both segments of the efferent ducts (PED and DED) have
been published mainly in Galliformes (Tingari 1972; Budras and Sauer 1975a;
Hess and Thurston 1977; Aire 1980), drake (Aire 1982b, 2002b) and Pigeon
(Stefanini et al. 1999).
%$ Reproductive Biology and Phylogeny of Birds
Fig. 2.22 Gallus domesticus. Histological sections of the PED (A) and DED (B). In
A., the NC type I cell (N) contains numerous supranuclear, dense bodies; C,
ciliated cell. In B., the NC type II cell (N) lacks the dense bodies found in the
corresponding cell type of the PED; C, ciliated cell, and S, spermatozoa in the DED
lumen. Bars: A = 200 µm; B = 10 µm. Original.
Anatomy of the Testis and Male Reproductive Tract %%
The classification of the NC cell into types I and II is based upon important
characteristic organelle differences and disposition between the two non-
ciliated cells lining the PED and DED, respectively (Aire 1980). In mammals,
NC cells have also been known to vary structurally along the length of the
efferent ducts: thus there is only one type in rodents (Hoffer and Greenberg
1978; Ilio and Hess 1994), but there are three types in the Great cane rat,
Threonomys swinderianus (Aire and van der Merwe 2003), and 3 in man, bull,
Goat and Dog, Canis canis (Morita 1966; Goyal and Hrudka 1980, 1981; Gray
et al. 1983; Goyal and Williams 1988). Variations in the disposition and
number of vacuoles and/or granules have been used as criteria for classifying
the non-ciliated cells in the efferent ducts of animals. It is not clearly
understood whether or not these organelle differences influence the functions
of these cells, individually in mammals, but there appears to be an obvious
dichotomy in function(s) between the NC type I and NC type II, in birds (Aire
1979a, 1980, 2000b, 2002b; Aire et al. 2004; Clulow and Jones 1988).
The NC Type I Cell. The apical surface extends into a microvillous brush
border of closely bunched, long, and uniformly cylindrical microvilli (Fig.
2.23). The apical surface may also invaginate as fuzzy-lined, tubular coated
pits into the subapical cytoplasm which is remarkably endowed with an
elaborate endocytic or tubulovacuolar system. The tubular coated pits are
continued by straight or coiled apical tubules, which together with endosomes
made up of large dilated membranous vacuoles, as well as a few
multivesicular bodies (MVBs) occupy the apical one-fourth of the cell (Fig.
2.24). The apical tubules frequently contain an amorphous inspissated
material, probably protein, taken in from the duct lumen. Distal to the
endocytic system there occurs a large number of round, variably-sized
homogeneous or heterogeneous dense bodies. The dense bodies may become
heterogeneous as a result of endocytosis and lysosomal activity by the cell.
This type of dense body is probably a telolysosome. Numerous, elongated or
oval mitochondria occur in both the supranuclear, and to a greater extent, in
the subnuclear regions of the cell. They may measure up to 0.6 mm in breadth
(Aire 2002b). The Golgi complex is moderately developed in the supranuclear
region of the cell. The oval nucleus is situated in the basal half of the cell,
contains a central or eccentric nucleolus, and is generally euchromatic. Lipid
droplets are uncommonly encountered in the cytoplasm. Strands of RER and
short, small profiles of SER are scattered in the cytoplasm.
The apical junctional complexes between the NC cells of the efferent ducts
in G. domesticus show, apico-basally, a series of punctate fusions (zonula
occludentes), adhering or intermediate junctions (zonula adherens), and
desmosomes (macula adherens), in that order, along the complex. A continuous
line of fusion between the outer leaflets of adjacent cell membranes in the
apical junctional complex is also evident (Nakai and Nasu 1991). But Claude
and Goodenough (1973) and Suzuki and Nagano (1978) regard these
junctional complexes as being of the ‘leaky type’, in the rat. However, Nakai
and Nasu (1991) have shown that the tight junctions in both the rete and
%& Reproductive Biology and Phylogeny of Birds
Fig. 2.23 Gallus domesticus. Transmission electron micrograph of epithelial cells

of the PED. N, non-ciliated type I cell; C, ciliated cell. Note the large number of
dense bodies (D) and long mitochondria (arrowheads) in the supranuclear region
of the NC cells. The mitochondria of the C cells (arrow) are thinner than those of
the NC cells. The lush microvilli (m) of the NC cells are regularly cylindrical and
longer than the inter-cilial microvilli of C cells. Bar: 1 µm. Adapted from Aire, T. A.
and Josling, D. 2000 Onderstepoort Journal of Veterinary Research 67: 191-199,
Figure 12, with permission of the Editor.
Anatomy of the Testis and Male Reproductive Tract %'
Fig. 2.24 Anas platyrhynchos (A), Gallus domesticus (B, C, D). Non-ciliated type I
cells of the PED. A. Supranuclear region of NC type I cells showing an elaborate
subapical tubulovacuolar or endocytic system, comprising coated pits
(arrowheads), coated apical tubules (arrows) and vacuole (V). D, dense bodies; N,
nucleus. B. A coated pit (arrowhead) leads into a non-coiled apical tubule (T); M,
microvilli; J, junctional complex. C. Transverse sections of apical tubules contain
inspissated material (arrowhead). D. Heterogeneous dense bodies (H) and
mitochondria in a cell that has experienced phagocytosis. Bars: A = 10 µm; B = 2 µm;
C and D = 1 µm. Adapted from Aire, T. A. 2002 Journal of Morphology 253: 64-75,
Fig. 4., with permission of Wiley-Liss, Inc.
efferent duct epithelia are able to exclude lanthanum nitrate from the duct
lumen in the rooster. It is not known if this complex can exclude compounds
of smaller molecular weights from passing into or out of the lumen. Large and
extensive intercellular spaces occur between adjacent NC cells in the PED in
the Ostrich. These spaces usually occur in the basal two-thirds of the
epithelium, and are frequently seen to extend to the basal lamina in electron
micrographs. Dilated intercellular spaces are usually associated with
enhanced fluid absorption by epithelia (Pudney and Fawcett 1984), and in
this case, paracellular fluid movement, apparently by active solute transport
(Suzuki and Nagano 1978).
The NC Type II cell. The microvillous brush border is similar to that of the
NC type I cell. The type II cell lacks the characteristic, elaborate, subapical
endocytic apparatus and numerous dense bodies in the supranuclear zone of
the type I cell (Fig. 2.25). Instead, only a few, sparsely-distributed, subapical
coated pits and apical tubules, containing inspissated material, are scattered
between the bundles of microfilaments which project from the microvilli into
the subapical cytoplasm. Vacuoles are usually few, small and scattered in the
apical half of the cytoplasm. Mitochondria are fewer than in the NC type I,
and are scattered within the supranuclear, and to a lesser extent, in the
subnuclear cytoplasm. The nuclei are similar to those of NC type I cells in
location, size, shape and configuration. Strands of RER and a few quite small
dense bodies and lysosome-like bodies may be seen in the cytoplasm.
Ciliated Cells. Readily discernible differences are not to be found in the
ultrastructure of the C cells in both segments of the efferent ducts, but the
number of C cells relative to NC cells increases markedly in the DED, with a
ratio of 4:5, respectively, in the Common starling (Bellamy and Kendall 1985)
and 9:1 in the ostrich (Budras and Meier 1981) than in the PED that has a
respective ratio of C to NC cells of 1:5 in the common starling (Bellamy and
Kendall 1985) and 3:7 in the Ostrich (Budras and Meier 1981).
Uniformly-spaced cilia, interspersed with a few, short and thinner
microvilli than in the NC cells, project into the lumen (Figs. 2.23 and 2.25). A
few coated apical pits are also present. Mitochondria are mainly in the
supranuclear region of the cell; they are only about 30% as broad as those of
the NC cells (Aire 2002b). The Golgi complex is of moderate size. Numerous
bundles of microfibrils, possibly assisting in stabilizing the cell whose role
includes movement of the luminal through-flow, may be seen running in
different directions, in the supranuclear and perinuclear zones of the cell (Aire
1980). A euchromatic nucleus, which is generally oval in profile, but is often
indented or invaginated, is situated in the apical half of the cell. Profiles of
RER, polyribosomes, a few small dense bodies and lysosomes may be seen in
the cytoplasm. The C cell is thought to assist in moving the seminal content of
the efferent ducts, in addition to a limited endocytic activity, and, thus, also
influencing the composition of the luminal content.
Intraepithelial lymphocytes are not uncommonly present, in varying
numbers and at various levels, in the epithelium of both segments of the
Anatomy of the Testis and Male Reproductive Tract &
Fig. 2.25 Gallus domesticus. A. A survey transmission electron micrograph of the

DED epithelium. C, ciliated cell; N, non-ciliated type II cell showing one or two
subapical vacuoles; L, intraepithelial lymphocyte. B. High power of the supranuclear
region of ciliated (C) and non-ciliated type II (N) cells. The N cell lacks the
numerous dense bodies found in the supranuclear region of the corresponding
cell in the PED, and only a few apical tubules (arrowheads) are present in the
DED. Arrow, microfibrillar bundles in the supranuclear region of a C cell. Bars: A =
200 µm, B = 2 µm. Original.
efferent ducts (Fig. 2.25A), as in the RT epithelium (Aire and Malmqvist

1979a).
Function of the Efferent Ducts. The structure of the efferent ducts is generally
similar in mammals and birds (Burgos 1960; Ladman 1967; Tingari 1972;
Ramos and Dym 1977; Jones et al. 1979; Aire 1980; Hermo et al. 1988; Robaire
and Hermo 1988). It is therefore very tempting to ascribe similar functions to
the ducts in most, if not all, animals. In birds, however, unlike in mammals,
the proximal segment (PED) of the duct possesses a highly folded epithelium
which increases considerably the surface area that is available and exposed
to the luminal content. Besides, the PED constitutes a greater proportion of the
epididymal volume (about 300% more) than the distal segment (DED), but,
together, both of them constitute between 35% and 62% of the entire
epididymal volume in various species of birds (Aire 1979b). This is significant
because the avian testis has a high fluid content and sperm production, the
latter being also very rapid. The structure of the PED epithelium is consistent
with active uptake of luminal macromolecules and considerable luminal fluid
reabsorption, and transport across the epithelium. The PED must therefore
play a major role in modifying luminal content, and, in general, the
functioning of the avian excurrent duct system, as in mammals. However, little
is known about endocrine regulation of the avian male reproductive tract and
its role in production of fertile spermatozoa (Janssen et al. 1998). The efferent
ducts are probable sites of steroidogenesis in the rooster (Tingari 1973), but
androgens do not prolong sperm viability in the ductus deferens of the rooster
(Munro 1938). Estrogen receptors are strongly expressed in its efferent ducts
(Kwon et al. 1997) and in several mammals (Goyal et al. 1997, 1998; Fisher et al.
1997). Estrogens seem to have a profound effect on the ability of the efferent
ducts in Mouse (Mus musculus) to reabsorb luminal fluid (Hess et al. 1997).
In mammals, micropuncture studies indicate that the efferent ducts
reabsorb most of the testicular fluid entering the excurrent ducts of the testis
(Crabo 1965; Jones 1980; Jones and Clulow 1987; Clulow and Jones 1988;
Clulow et al. 1994; Man et al. 1997). In their excellent studies in the Japanese
quail, Clulow and Jones (1988) show that even though spermatozoa (in their
fluid medium) spend only 3 min traversing the PED, yet about 86% of the fluid
leaving the testis is reabsorbed there, and another 6.5% in the DED. Important
ion transporters, such as sodium-potassium ATPase [Na+, K+-ATPase]
carbonic anhydrase II (CA II) and sodium hydrogen exchanger isoform
3(NHE3) have been immunolocalized in the efferent ducts of the rooster (Bahr et
al. 2006). Transmembrane water channel proteins (aquaporins –2, –3, and –9)
that are responsible for water flow, have similarly been localized in efferent
ducts of the large white turkey (Zamboni et al. 2004). The arrangement of the
efferent duct unit into a large number of narrow ducts in a parallel array
provides a large ratio of luminal surface area:luminal volume in the Japanese
quail (Clulow and Jones 1988). Spermatozoa traverse the entire efferent duct
unit in 8 min in the Japanese quail (Clulow and Jones 1988) in contrast with
about 45 min in the rat (English and Dym 1981). It is clear that the rate of fluid
reabsorption of testicular fluid by the efferent ducts of the testis is much higher
in birds than in mammals (Jones 1998).
The inspissated material in the lumen of apical tubules of the NC type I cell
in birds (Fig. 2.24C) is probably proteinous, and, if so, it indicates that this
type of cell, and therefore the entire efferent duct unit, is capable of absorption
of testicular proteins secreted into the testicular fluid, as has been established
for mammals (Koskimies and Kormano 1975; Olson and Hinton 1985; Jones
and Jurd 1987; Veeramachaneni and Amann 1991; Clulow et al. 1994).
Anatomy of the Testis and Male Reproductive Tract &!
Morales and Hermo (1983) and Hermo and Morales (1984) have demonstrated
that the non-ciliated cells of efferent ducts are capable of internalizing specific
substances from the duct lumen by both adsorptive and fluid-phase
endocytosis in the rat. Nakai et al. (1989a) and Aire (2000a) have also
demonstrated the respective ability of NC cells in the PED of birds to
interiorize testicular proteins and India ink particles (Fig. 2.26). The absorbed
materials are destined for the lysosomal system of the cell. The NC cells in the
PED are able to recognise, by an unknown mechanism, and remove cationic
ferritin, even in bicameral chambers (Janssen et al. 1998), as well as
‘designated’ spermatozoa (Hess et al. 1982; Aire 2000b, 2002a) and
intraluminally introduced avirulent strain of Salmonella gallinarium (Aire et al.
2004) from the luminal through-flow in the rooster and Japanese quail.
In vasectomized birds, desquamated germ cells that are transported from
the seminiferous tubules to the excurrent ducts are sequestered in the PED
where they degenerate and their fragments are removed by phagocytic
acitivities of the NC type I cells (Tingari and Lake 1972b; Aire and Heath 1977;
Nakai et al. 1989b; Aire 2002a). The DED seems to be screened, by an unknown
mechanism, from such germ cell debris by the PED. Remarkably, the NC type
I cells are also capable of proliferating and forming new adluminal sheets of
cells which are highly spermiophagic on both their free surfaces, in the
process of removing an overwhelming accumulation of sperm debris,
following vasectomy in the Japanese quail and rooster (Aire 2000b, 2002a) or
carbendazim exposure in Coturnix (Aire 2004). These new sheets of cells
subsequently sequester small ducts from the original duct lumen, during the
process of microrecanalization in the efferent ducts (specifically the PED
segment) in birds (Aire 2004). Microrecanalization, specifically of the
transected end of the vas deferens, has been described previously only in
vasectomized men, and was probably responsible for unexpected fathering of
babies by such men (Cruickshank et al. 1987; Freund et al. 1989).
2.3.3 The Epididymal Duct Unit

2.3.3.1 General organization and features
From ontogenetical, structural and functional perspectives, the connecting
duct (ductus conjugens), epididymal duct (ductus epididymidis) and deferent duct
(ductus deferens) are essentially similar and constitute a functional unit (Gray
1937; Lake 1957; Tingari 1971, 1972; Budras and Sauer 1975a; Budras and
Meier 1981; Aire 1979a; Aire et al. 1979; Aire 2000a). This is also in accord
with the observations made by Tingari (1971, 1972) and Aire et al. (1979), that
the NC type III cell is identical in the epithelia of these ducts, which shows
that the avian ductus epididymidis and ductus deferens are merely different
segments of the same organ, an organ equivalent to, but not grossly structured
as, the epididymis in mammals. This functional unit may therefore be referred
to, for convenience, as the “epididymal duct unit”. This duct unit forms the
greater proportion (about 74%) of the entire excurrent duct volume than all
other duct units put together, in the Japanese quail (Clulow and Jones 1988).
&" Reproductive Biology and Phylogeny of Birds
Fig. 2.26 Coturnix japonica. A. Rete testis-infused India ink particles are present
in the non-ciliated, but not the ciliated cells of the PED epithelium, and are absent
in the epithelial cells of the DED and ductus epididymidis (DE). B. India ink particle-
filled lysosomes are present in transverse sections of non-ciliated (N) cells but not
ciliated (C) cells of the PED. Bars: A = 200 µm; B = 1 µm. Original.
Anatomy of the Testis and Male Reproductive Tract &#
Similarly, the epididymis and vas deferens in mammals, together, constitute, a

priori, the greatest proportion by volume of the excurrent duct system.
The efferent ducts are continued distally by the epididymal duct unit that
is derived from the Wolffian duct (Budras and Sauer 1975a). This duct is lined
by a non-ciliated epithelium. The first portion of this unit is the short
connecting duct (CD) that joins the efferent duct, specifically, the DED segment
of the efferent duct unit, to the epididymal duct (ED). The CD opens into the
ED which is a slightly wavy duct (Fig. 2.16), but may be complexly tangled in
the Common Tern (Sterna hirundo) and Jackdaw (Corvus monedula) (Traciuc
1967, 1969). It is situated longitudinally on the dorsomedial border of the
epididymis, and runs caudally into the ductus deferens (DD). Neither the ED
nor DD is therefore similar to those of mammals in length, gross features or
configuration.
2.3.3.2 The ductus deferens and its modifications
In non-passerine birds, the ductus deferens is highly wavy and increases in
diameter cranio-caudally, in sexually active birds (Fig. 2.1). Close to the cloaca,
the ductus deferens straightens out for a variable length, depending on the
species and size of bird, to form the pars recta ductus deferentis (Marvan 1969;
Lake 1971; Marchand and Gomot 1973). This part subsequently enlarges into
a thick-walled, barrel-or spindle-shaped structure, the receptaculum ductus
deferentis that pierces the wall of the cloaca to open into the urodeum of the
cloaca by an opening in its pointed end, the papilla ductus deferentis.
In passerine birds, the ductus deferens is complexly thrown into coils that
form a compact ball, the seminal glomus or sac, caudally (Bailey 1953) (Fig.
2.27). The seminal glomus is a tubular and highly coiled structure that is
encapsulated in loose connective tissue (Bailey 1953; Salt 1954 and Wolfson
1954). It lies beneath the skin, dorso-lateral to the cloaca (Salt 1954), where its
coiled ducts may be visible through the skin of the cloacal protuberance
(Mulder and Cockburn 1993). The latter, akin to the scrotum in scrotal
mammals, is a swelling of the skin, that accommodates the seminal glomus.
The seminal glomus is a characteristic feature of passerine birds (though also
reported for the psittacid Melopsittacus undulatus, see Chapter 8), and is
regarded as an anatomical adaptation that ensures a storage and probable
maturation site for spermatozoa (Lake 1981) that are necessary for sperm
competition (Birkhead et al. 1993). It is tempting to regard the lower
temperature in the seminal glomus as beneficial to spermatozoa in passerine
birds, as it is in scrotal mammals. Wolfson (1954) believes that the lower
temperature is necessary for sperm maturation. But, avian sperm do not
require maturation and capacitation in order to be fertile (Munro 1938;
Bedford 1979; Howarth 1983). The seminal glomus may therefore serve as a
daily sperm store since, it appears, sperm production in the testis as well as
sperm transport to the glomus seminalis occurs more rapidly at night, while
its sperm content declines significantly during the day (Birkhead et al. 1994).
The seminal glomus may, in addition to acting as a storage area, provide a
&$ Reproductive Biology and Phylogeny of Birds
Fig. 2.27 A diagrammatic representation of a ventral view of the reproductive

organs and tract of a sexually mature and active passerine bird, Passer domesticus
(the House sparrow). T, testis; E, epididymis; D, ductus deferens; SG, seminal
glomus; CL, cloaca; R, rectum; V, vent; Arrowhead, skin of the cloacal protuberance.
Not drawn to scale. Original.
lower temperature for spermatozoa, in order to conserve their energy or

perhaps permit a biochemical or physiological reaction or adjustment in the
spermatozoa, before insemination into the female tract. The precise function
of the seminal glomus remains speculative. This structure is neither
homologous nor analogous to the seminal vesicle of mammals.
The cloacal protuberance is a very useful structure for sexing passerine
birds, and in determining the prevailing phase of their reproductive cycle.
The size of the protuberance is strongly and positively correlated both with
the mass of the seminal glomus and its sperm content (Birkhead et al. 1993)
Anatomy of the Testis and Male Reproductive Tract &%
and, therefore, a guide to the sperm production capacity of the bird under
examination. In the Superb Fairy-Wren (Malurus cyaneus), a pointed anterior
‘tip’ of the cloacal protuberance, not yet described in other passerine birds,
probably facilitates effective sperm transfer, during short periods of
copulation (Mulder and Cockburn 1993).
2.3.3.3 Surface features of the epididymal duct unit
The epididymal epithelium, at low power magnification, appears smooth and
presents no folds except at sharp angulations and at the entry of the CD into
the ED where several longitudinal ridges and grooves occur (Aire and Soley
2000). A number of irregularly distributed invaginations or ‘craters’ (Fig. 2.28)
occur on the epithelial surface in the drake (Aire 1982b). In all birds studied
(the ostrich by Aire and Soley 2000; Turkey, rooster and Japanese quail by Aire
and Josling 2000), except the drake (Aire 1982b), the luminal surfaces of the
CD, ED and DD appear cobbled, with distinct intercellular grooves. Close-up
views show numerous, evenly distributed, regular microvillar extensions of
the apical surfaces of the principal epithelial cells (Fig. 2.28B). A single, central
cilium projects from several cell surfaces into the duct lumen.
2.3.3.4 Histology of the epididymal duct unit
In non-passerine birds, the histological features of the epididymal duct unit
have been reported in several species. Discrepancies or errors in the
nomenclature of the ducts, as well as the interpretation of normal structure,
are to be found in the literature (Gray 1937; Lake 1957; Tingari 1971, 1972;
Budras and Sauer 1975a; Hess et al. 1976; Aire 1979a, 2000a). These will be
highlighted in this review. The CD, ED and DD are lined by cuboidal to
columnar, non-ciliated epithelium, simple or pseudostratified, depending on
the angle of section (Fig. 2.28). The epithelium consists of the non-ciliated type
III cell (Aire et al. 1979) which is distinctly different, ultrastructurally, from the
non-ciliated types I and II cells of the PED and DED, respectively. Basal cells,
not present in the more proximal duct units, are wedged between the NC type
III cells, and their long axes typically lie parallel to the basal membrane. Basal
cells increase in number, cranio-caudally, i.e., they are least numerous in the
CD and ED, but quite numerous and virtually form a distinct layer of cells in
the caudal part of the DD. The ductal lumen is regular in outline and oval in
cross-section, save at the entry of the CD into the ED where epithelial ridges
and grooves occur (Aire and Soley 2000). A short microvillous brush border,
varying in height from 0.7 mm in the drake to 1.6 mm in the Japanese quail
(Aire 2000a) projects from the NC type III cells into the lumen. Nuclei of the
NC type II cells are round or oval in shape, and display single, central
nucleoli. Intraepithelial lymphocytes are also present in the epithelium (Aire
and Malmqvist 1979b). The epithelium rests on a compact, richly vascular
peritubular boundary tissue composed of fibroblasts, collagen fibers and
several layers of smooth muscle cells (Aire 2000a).
In passerine birds, not much has been reported on the normal structure of
the excurrent ducts of the testis, even though passerines constitute a
&& Reproductive Biology and Phylogeny of Birds
Fig. 2.28 Anas platyrhynchos (A), Meleagris gallopavo (B) and Struthio camelus (C).
In A., A SEM view of the surface epithelium of the epididymal duct, showing a
regular surface interspersed with crater-like depressions. Several solitary cilia
Anatomy of the Testis and Male Reproductive Tract &'
significantly large proportion of all birds. In fringillids, the epithelium of the

epididymal duct is reported to be columnar and ciliated (Bailey 1953) but that
of the ductus deferens is columnar or pseudostratified and non-ciliated.
Bedford (1979) considers that the DD has a ciliated epithelium. The apical
cytoplasm of NC cells blebs into the duct lumen (Bailey 1953), in a manner
that is reminiscent of apocrine secretion. Care must be taken in regarding
these blebs as representing apocrine secretion, because histological sections of
this tissue, fixed by immersion, but not by vacular perfusion, in non-passerine
birds, display such apical blebs that are regarded as artifacts of fixation (Aire
1980, 1982b, 1997, 2000a). Basal cells that lie on the basement membrane are
present between non-ciliated cells.
The epithelial lining of the seminal glomus is cuboidal to low columnar,
and is, also, composed by non-ciliated and basal cells (Bailey 1953; Salt 1954;
Wolfson 1954; Middleton 1972). However, Bullough (1942) and Bhat and
Maiti (2000) have described a ciliated columnar epithelium. Middleton (1972)
considers that the epithelium contains secretory cells whose product is PAS-
positive, and probably glycogen. The duct is filled with spermatozoa the
heads of which are pointed toward, and make contact with, the epithelium,
which in some cases is penetrated and eroded by the spermatozoa, thus
creating the impression of holocrine secretion by this epithelium. The
epithelium is invested by a well-defined fibromuscular layer of boundary
tissue. There is a great deal of discrepancy in the nomenclature of the various
ducts of the epididymis in these reports. Further and more detailed studies of
the structure of the epithelial lining of the various excurrent ducts of the testis
of passerine birds is necessary in order to minimise the confusion that exists
in the literature.
The Receptaculum Ductus Deferentis. This structure displays a large
number of epithelial and mucosal folds that are not obliterated, even when
full of spermatozoa. The folds are longer and more complex toward the papilla
of the receptacle. The epithelium is columnar, may be pseudostratified and
comprises non-ciliated cells and numerous basal cells (Fig. 2.29). Several deep
grooves or crypts, and their cross-sections occur in histological sections. In
the ostrich, the cross-sections of the crypts, especially toward the papilla,

(arrowheads) project from most cells. S, spermatozoon. B. Higher power view of
the surface epithelium of the epididymal duct, showing uniformly distributed,
regular microvilli and solitary cilia projecting from several cells. C. A histological
section of the epididymal duct of the ostrich, exhibiting a columnar profile; basal
cells (arrowheads) are present, and increase considerably in number cranio-
caudally. Bars: A = 200µm; B = 2 µm; C = 200 µm. Figure A is adapted from Aire, T.
A. 2000 Anatomy Histology Embryology 29: 179-191, Figure 2. Reproduced with
permission; Figure B is from Aire, T. A. and Josling, D. 2000 Onderstepoort Journal
of Veterinary Research 69: 191-199, reproduced by permission of the Editor. Figure
C is original.
' Reproductive Biology and Phylogeny of Birds
Fig. 2.29 Struthio camelus. Histological sections of the receptacle of the ductus
deferens. A. The epithelium projects finger-like processes into the duct lumen.
Eosinophilic and PAS-positive, but not glycogen, secretory material (arrowheads)
occurs within the epithelium, particularly in the region of the crypts between
epithelial folds. B. Basal cells (arrows) are extremely numerous, and line the basal
part of the epithelium. Bar: A = 100 µm, B = 200 µm. Original.
often display spermatozoa and/or an eosinophilic content that is PAS-

positive, non-glycogen polysaccharide material (Fig. 2.29), of which the mode
of secretion and function are unknown (pers. obs.). The epithelium of the
receptaculum ductus deferentis therefore appears to be secretory and capable of
influencing or modulating sperm viablility or nutrition. Its sperm content is
highly concentrated, and therefore can be rapidly regulated (Clulow and
Jones 1988). In the drake, the epithelial grooves contain only spermatozoa, if
any (Aire et al. 1979). The epithelium is enclosed by a very thick coat of
fibromuscular tissue. In the rooster, this tissue contains subepithelial sinuses
and tortuous arteries, akin to the situation in the mammalian penis (Lake
1957), but the function and mechanism of action of this structural peculiarity,
with regard to ejaculation in birds, is unknown. Nevertheless, the receptacle
appears to be a temporary storage compartment for a relatively large number
of spermatozoa, close the point of their emission.
2.3.3.5 Ultrastructure of the epididymal duct unit
Reports of ultrastructural studies on the avian epididymal duct unit are
scarce, and are mainly on the rooster (Tingari 1972; Aire 2000a), turkey (Hess
and Thurston 1977; Aire 2000a), drake and Japanese quail (Aire 2000a).
Non-ciliated Type III Cells. The apical surface of the type III cells extend into
a microvillous brush border (Figs. 2.30 and 2.31), which is only about 65% as
Anatomy of the Testis and Male Reproductive Tract '
Fig. 2.30 Anas platyrhynchos. A survey electron micrograph of epithelial cells

lining the epididymal duct unit, taken from the ductus epididymidis. Short microvilli,
a well developed Golgi complex (G), complexly folded lateral plasmalemma (open
arrows); nucleus (N) and an investing layer of intermediate filaments (IF).
Numerous mitochondria occupy the supranuclear region of the cell. Bar = 1 µm.
Original.
Fig. 2.31 Anas platyrhynchos. Magnified portions of the non-ciliated type III cell of
the epididymal duct unit. A. The microvilli may branch (small arrowhead), the Golgi
Anatomy of the Testis and Male Reproductive Tract '!
long as those of NC types I and II cells in efferent ducts of the Japanese quail
(Aire 2000b). The microvilli are regularly cylindrical in profile, and blebbing
of the apical cytoplasm is rarely seen in tissues that are well fixed, either by
very good immersion fixation or, more importantly, by good intravascular
perfusion fixation. Apical blebbing in NC types I and III cells has been
described by various authors (Budras and Sauer 1975a; Hess and Thurston
1977; Bakst 1980; Stefanini et al. 1999). However, Aire (1979a, 1980, 1982b),
Aire and Josling (2000) and Aire et al. (1979) have found that these blebs are
absent in very well fixed tissues. Nicander (1970) and Hamilton (1975) have
also regarded these structures, in mammalian epididymal tissues, as artifacts
of fixation. Ericsson (1964) has made similar observations in poorly fixed
homologous cells of the kidney.
Adjacent lateral plasma membranes are intricately folded (Fig. 2.30),
especially in the basal two-thirds of the cell length. These foldings may serve
a mechanical purpose of stabilizing intercellular attachments, or in water
transport (Pease 1956). Apicolateral junctional complexes are well formed
(Friend and Gilula 1972) and both the tight junction (zonula occludens) and
adhering intermediate junction (zonula adherens) are composed of multiple
punctate fusions which exclude tracer compounds from the duct lumen, in the
rooster (Nakai and Nasu 1991). Desmosomes on the lateral membranes and
hemi-desmosomes in the basal plasma membrane are also present.
The nucleus of the NC type III cell is round or oval in shape in the drake
(Aire 2000a) and Ostrich (pers. obs.) or vertically elongated in the rooster,
Turkey and Japanese quail (Tingari 1972; Hess and Thurston 1977; Aire
2000a). It is moderately heterochromatic, contains a usually eccentric
nucleolus, and is basally situated in the cell. Bundles of intermediate filaments
(IFs), up to 640 nm wide, may surround the nucleus, partially or wholly
(Fig. 2.30), particularly in the drake (Aire 2000a) and may be seen to attach to
other organelles as well as the plasma membrane. The function of this distinct
assemblage of IFs in the drake is not clearly understood, but IFs are known to
form the cytoskeleton component that connects different parts of the cell into
an organic network, act as organizers that control the distribution of different
subcellular structures, and as integrators of the cellular space (Lazarides
1980; Geiger 1987 and Zhu et al. 1997). A moderately abundant endoplasmic
reticulum occurs in the cell, and are mainly of the sparsely granulated (SGER)
type in the drake, in particular, while in other birds, it is represented mainly
complex (G) is well developed but consists of only a few saccules; the cytoplasm
contains abundant, distended profiles of SER or sparsely granulated endoplasmic
reticulum (SGER) (large arrowheads); the subapical region shows numerous
secretory vesicles with a dense content (arrows). B. Apical half of part of the NC
type III cell displaying profiles of microtubules (arrowheads) extending to the apical
membrane of the cell. Arrows, clathrin-coated vesicles. Bars for both A and B =
2 µm. Original.
'" Reproductive Biology and Phylogeny of Birds
by profiles of SER and RER. In Turkey, a whorl of RER occurs invariably in

the immediate supranuclear zone (Aire 2000a). Abundant free ribosomes or
rossettes of ribosomes occur in the cells.
Lipid droplets are uncommon in all birds (Tingari 1972; Aire 2000a) except
the turkey in which they are quite abundant, as lipid aggregates, in the
supranuclear, and to a lesser extent, infranuclear regions of the NC type III
cell (Hess and Thurston 1977; Aire 2000a). The lipid content in the cells,
particularly in the Turkey, may be related to some form of steroidogenesis, as
Tingari (1973) has shown that 3b-and 17b-hydroxysteroid dehydrogenases
are moderately localized in the epididymal duct of the rooster. Mitochondria
are dispersed evenly in the cytoplasm, and are each usually surrounded by a
strand of RER. A well developed, supranuclear Golgi apparatus occurs in the
drake and Japanese quail. Numerous smooth-surfaced vesicles and clathrin-
coated vesicles are observed in the region of the Golgi complex. The Golgi
complex, in this duct unit, is very well developed (Fig. 2.31), but is not as
elaborate as in the epididymal duct of mammals (Nicander and Glover 1973;
Ramos and Dym 1977; Hermo et al. 1991; Stoffel and Friess 1994). Its function
in birds appears quite significant (Aire 2000a). Noteworthy features include
numerous secretory vesicles, with increasing condensation of their content, as
they move from the peri- and supra-nuclear zone toward the apical
plasmalemma (Fig. 2.31B), in the rooster (Tingari 1972) and drake (Aire
2000a). Microtubules extend from the region of the Golgi complex to the apical
zone in the Japanese quail (Aire 2000a) and drake. Movement of secretions by
means of vesicles and/or microtubules is well established in, possibly, all
eukaryotic cells (Palade 1975; Cooper et al. 1990; Darnell et al. 1990). The
secretory vesicles contain dense, amorphous material, which is probably
discharged in a merocrine manner of secretion into the duct lumen (Tingari
1972; Aire 2000a). The nature of this secretion is unknown. However, about
four Wolffian duct proteins bind to spermatozoa as they pass through the
epididymis and the ductus deferens in the rooster (Esponda and Bedford
1985; Morris et al. 1987). Only one androgen-dependent protein of 17kDa has
been identified in the Japanese quail (Kidd 1982, cited by Jones 1998). These
proteins seem to have a specificity that is confined to the same order of birds,
in this case, Galliformes (Esponda and Bedford 1985; Morris et al. 1987).
Coated pits invaginate into the subapical cytoplasm, and lead to only a
few, scattered subapical coated vesicles. These indicate that the NC type III
cell is capable of micropinocytosis, probably of luminal proteins, along with a
very small quantity of fluid, because Clulow and Jones (1988) have shown
that the epididymal duct unit (CD, ED and DD) reabsorbs only about 0.8% of
the total testicular plasma output in the Japanese quail. The NC type III cells
appear to be incapable of endocytosis of India ink particles infused into the
excurrent duct system through the RT (Fig. 2.26A), whereas numerous rete
cells and almost all of efferent duct NC type I cells, but not NC type II or
ciliated cells, avidly take up the ink particles (Aire 2000a, and unpublished
observations). Similarly, the epididymal duct unit fails to interiorize
Anatomy of the Testis and Male Reproductive Tract '#
horseradish peroxidase, while both the RT cells and NC type I contain large
amounts of the substance (Nakai et al. 1989b). On the whole, the NC type III
cell, in birds, appears to be similar, functionally, to the principal cell of the rat
epididymis, in being involved primarily, but not exclusively, in secretion,
while the clear cells, not described in birds, are primarily involved in
absorption (Hamilton 1975; Robaire and Hermo 1988; Hermo et al. 1988).
Basal Cells. Basal cells are found between the bases of NC type III cells only
in the epididymal duct unit, and they increase in frequency, cranio-caudally
(Figs. 2.26, 2.28 and 2.29C). They are cuboidal or pyramidal in shape,
containing nuclei of varying shape, from elongated to triangular or irregular
(Fig. 2.32). The nucleus contains a central nucleolus and heterochromatin
aggregations attached to the inner nuclear membrane. The organelle content
of the cell is sparse, and includes a small Golgi apparatus, a few
mitochondria and strands of RER (Fig. 2.32). The nucleus is encircled by
bundles of fibrillar material that are best developed in the rooster and
Japanese quail, but are also present, to a lesser extent, in the drake and Turkey
(Aire 2000a). Tingari (1972) therefore speculates that basal cells subserve a
similar function as myoepithelial cells, in assisting the contraction of the
muscular coat, during ejaculation. Croisille et al. (1978) suspect that basal
cells serve as stem cells for the regeneration of the periodically exfoliating
epithelial lining of the epididymal duct unit. Intraepithelial lymphocytes are
also seen in the epithelium of the epididymal duct unit, as in other duct units,
described above.
Periductal Tissue. One or two layers of fibroblasts and up to ten concentric
layers of smooth muscle cells constitute the periductal tissue which
progressively thickens, cranio-caudally. The smooth muscle cells contain
highly elongated, euchromatic, regular nuclei, surrounded by longitudinally
orientated filaments, and organelles that are typical of smooth muscle cells
(Fig. 2.33). This tissue, in the epididymal duct unit, is richly vascularized,
being penetrated by a dense peritubular blood capillary network (Nakai et al.
1988; Aire 2000a), contrary to Tingari’s (1971) observation. The blood
capillaries are fenestrated and close to the epithelium in the domestic fowl
(Nakai et al. 1988) but not as close to the epithelium as in the mammalian
epididymis (Abe et al. 1984). Encapsulated nerve endings are found between
the blood capillaries and the ductal epithelium (Nakai et al. 1988), and both
cholinergic and adrenergic nerve components have been demonstrated
(Tingari, and Lake 1972a). The adrenergic nerve component is particularly
closely associated with the epithelium, and ramifies abundantly in the walls
of the ducts as well as the receptacle and papilla of the ductus deferens. The
abundance of fine intrinsic nerves in the epididymal duct unit may be related
to the higher level of development of smooth muscle cells in the boundary
tissue of this duct unit than in the more cranial duct units, particularly the RT
and efferent ducts. Therefore this unit may have a greater contractile force for
onward movement of spermatozoa toward the cloaca than the more cranial
duct units (Tingari and Lake 1972a).
'$ Reproductive Biology and Phylogeny of Birds
Fig. 2.32 Gallus domesticus. A basal cell rests on the basal lamina of the
epithelium of the epididymal duct, and its nucleus (N) is oval in shape and
surrounded by bundles of microfilaments (arrows) which may attach to the cell
membrane (arrowheads). Organelles are sparse. Bar: 1 µm. Adapted from Aire, T.
A. 2000 Anatomy Histology Embryology 29: 179-191, Figure 15. Reproduced with
permission of Blackwell Publishing Ltd.
2.4 APPENDIX EPIDIDYMIDIS

The appendix epididymidis is made up of blind-ending tubules in the rooster
(Budras and Sauer 1975a) and lies close to the adrenal gland, into which its
cranial portion may be incorporated, for which reason, the appendix
epididymidis is usually separated from the epididymis, on the removal of the
testis from the body cavity. In the ostrich, the appendix epididymidis is very
large and forms the cranial 40% of the entire length of the epididymis in the
Anatomy of the Testis and Male Reproductive Tract '%
Fig. 2.33 Gallus domesticus. The epithelium of the ductus epididymidis rests on a
relatively thick periductal layer of smooth muscle cells (S), displaying typical
organelle features. Bar: 2 µm. Adapted from Aire, T. A. 2000 Anatomy Histology
Embryology 29: 179-191, Figure 6. Reproduced with the permission of Blackwell
Publishing Ltd.
sexually mature and active bird (Budras and Meir 1981). It is attached to the
dorsal body wall by the mesepididymis, to the testis by the epiorchium and to
the adrenal gland, cranially, by connective tissue as well as by some of its
ductules and duct, whose free ends embed in the adrenal gland.
The appendix epididymidis contains two vestigial duct components: (i) the
ductus aberrans, that represents the straight, cranial blind end of the ductus
epididymidis (Wolffian duct), and into which open (ii) the ductuli aberrantes.
The latter are vestiges of the nephrons of the mesonephros that are farther
away than others from the testis, and, instead, lie close to the adrenal gland.
Ontogenetically, the ductuli aberrantes fail to make contact with the RT ducts
because of the distance between them. They have lost their Bowman’s
capsules, and become blind-ended (Budras and Sauer 1975b). The ductulus
aberrans therefore constitutes the distal end of the nephron that normally gives
rise to the DED and CD, developmentally (Budras and Sauer 1975b; Croissile
et al. 1978). Other categories of ductules, are ductuli aberrantes which are
connected to the RT but not to the epididymal duct, as well as tubuli
paradidymidis, which are blind at both ends, and are seldom seen in
histological sections along the length of the epididymis (Budras and Sauer
1975b).
'& Reproductive Biology and Phylogeny of Birds
Transverse sections of these ducts exhibit regular outlines, simple cuboidal

or low columnar epithelium, and a homogeneous, non-cellular luminal
content (Tingari 1971), or small clumps of cellular debris in the lumen
(Fig. 2.34). Budras and Sauer (1975b) have demonstrated, by histochemical
and ultrastructural methods, moderate steroid hormone synthesis in the
ductuli efferentes, ductus conjugens, and ductus epididymidis, and, in particular,
high synthesis in the ductuli efferentes proximales segment of the efferent
ductule, ductus aberrans and ductuli aberrantes in the rooster. The noduli
epididymidis, in the Ostrich, is situated within the substance of the appendix
epididymidis rostrally, and is derived from the swollen ends of the ductuli
aberrantes. It is involved in steroidogenesis (Budras and Meier 1981).
2.5 THE PHALLUS

In most birds there is no organ that is homologous to the mammalian penis
structurally. Closely related folds of tissue, derived from the ventral wall of
the proctodeum of male birds, collectively form the phallus. The phallus is
analogous to the penis in mammals because it is the organ of transfer of
ejaculated spermatozoa from the male into the female reproductive tract. The
phallus in most birds, for example, the domestic fowl, turkey and quail, is
small and not intromittent. An engorgement of the vascular folds by lymph in
the phallus causes an ‘erection’ that permits contact between this sperm-
bearing organ and the slightly everted vagina of the female, during copulation.
Fig. 2.34 Coturnix japonica. A histological section of the rostral part of the
epididymis, containing profiles of proximal efferent ductules (PED), ductus
epididymidis (ED) and ductuli aberrantes (DA) which are round/oval and display
cuboidal, ciliated epithelium and relatively empty lumina. Bar: 100 µm. Original.
Anatomy of the Testis and Male Reproductive Tract ''
On the other hand, in Anseriforms and Ratites, the phallus is long, may be
coiled, and intromittent (see King 1981 for an excellent review of the structure
of avian phallus). This better developed phallus consists of paired fibrous
bodies that constitute the bulk of the organ, a phallic sulcus that bears and
transports the ejaculated semen, and an elastic vascular body which probably
inclines the phallus cranioventrally, during erection. The avian phallus is in
the subject of Chapter 3.
2.6 HISTOCHEMISTRY OF THE MALE REPRODUCTIVE ORGANS

A number of enzymes and other chemical compounds have been studied in
specific regions of the male genital tract in mammals (Nicander 1954, 1957;
Dawson and Rowlands 1959; Allen and Slater 1961; White et al. 1961; Mann
1964; Risley and Skrepetos 1964a,b; Stallcup and Roussel 1965; Blackshaw
and Samisoni 1967). In order to ascertain homologies between parts of the
male reproductive tract in mammals and birds, and to relate structure to
function, it is necessary to evaluate a number of relevant enzymes and
compounds in the male reproductive tract of birds.
Glycoproteins, but not glycogen, are present in small amounts in the
epithelium of the reproductive tract of the rooster (Tingari 1972), and Esponda
and Bedford (1985) have shown that most of the sperm-binding secretions in
the excurrent ducts of the rooster, save for one glycoprotein, are proteins. Four
Wolffian duct proteins have thus been identified in the ducts of the rooster
(Morris et al. 1987). Efferent duct non-ciliated cells seem capable of many
metabolic functions, including the probable production of glutamic acid by
mitochondrial-rich NC type I cells. Most of the amino acid content of the
semen of the rooster is glutamate (Lake and McIndoe 1959). Lactic
dehydrogenase (LDH) exhibits strong activity in the seminiferous tubules, but
lipid reactivity and GDH, SDH and G-6-DH activities are very weakly
expressed in the seminiferous tubules and RT (Tingari and Lake 1972c).
The more distal excurrent ducts, however, have shown the presence of lipids
(phopholipids, neutral lipids and free fatty acids) in both the epithelial cells
and lumen (Tingari and Lake 1972c). Of all of the excurrent ducts, the PED
exhibits the highest level of lipid content specifically in the NC type I cells. The
function of the lipids may be related to estrogen production and/or utilization
by efferent ducts of the rooster (Hess et al. 1997). Tingari (1973) concludes rightly
that there exists a mechanism for the metabolism of steroids in the male fowl
tract. The presence of both 3b- and 17b-hydroxysteroid dehydrogenases, and 3-
b-ol-steroid dehydrogenase (Budras and Sauer 1975b) in the various epithelia,
and the presence of estrogen receptors (ER) in the germ cells and efferent ducts
of the rooster (Kwon et al. 1995, 1997) confirm this assumption. Similar findings
have been reported in the mouse (Janulis et al. 1996). It has been established that
the absence of estrogen receptors in the efferent ducts of mice compromises
considerably the indispensable fluid-resorptive ability of these ducts (Hess et al.
1997). The non-ciliated cells in efferent ducts appear to have a common function
in all species studied (Hess 2000). Thus, it is assumed that estrogen receptors in
the homologous avian ducts have a similar function, perhaps, among others, of
regulating fluid reabsorption in the efferent ducts of these animals, as in
mammals. Strong cholinesterase activity is expressed in the epididymal duct,
and this may be related to ionic movements, as found in homologous
mammalian ducts, but it is absent in the seminiferous tubules, RT and efferent
ducts (Tingari 1972). Recent reports show that the efferent ductule epithelium
immunohistochemically expressed sodium-potassium ATPase (Na+, K+ -
ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform
3 (NHE3), and that the connecting ductule and epididymal duct epithelia
immunoexpressed Na+, K+ -ATPase and CA II (Bahr et al. 2006). Similarly,
Zamboni et al. (2004) have shown that transmembrane water channel proteins
(aquaporins –2, –3, and –9), that are responsible for water flow, are present in
the epithelia of efferent ducts, collecting ducts and ductus epididymidis.
Acid phosphatase activity is present only in the luminal macrophages in
the RT, as well as in the dense bodies in NC type I cells of the PED, indicating
that these bodies are lysosomal (Nakai et al. 1989a). The microvillous border,
as also the lateral plasma membrane of the epididymal duct unit, shows
intense acid phosphatase activity. Alkaline phosphatase activity is present
only on the outer covering of the microvilli of the efferent ducts, and is
completely absent in the epididymal duct unit. The functions of these enzymes
in the male tract are only speculative. However, according to Aitken (1971), a
significant concentration of acid phosphatase is characteristic of all sperm-
storage areas although its exact significance in storage is not clearly
understood. Sperm are stored in the ductus deferens in birds, albeit for only
short periods of time.
2.7 ACKNOWLEDGMENTS
The University of Pretoria kindly provided library support for this review, as
well as research grants for new material contained in the text. I also
acknowledge the assistance of Mrs. Wilma Olivier who made all the line
diagrams. The assistance of both Dr. Peter Ozegbe and Dr. Wahab Kimaro in
computerization and composition of the text and figures is gratefully
acknowledged.

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n n
CHAPTER
3
Anatomy and Evolution of
Copulatory Structures
Robert Montgomerie1 and
James Briskie2
3.1 INTRODUCTION
Unlike most other animal Classes, Aves (birds) is a taxon in which males of
some species possess an intromittent organ (IO), whereas males of other
species do not (King 1981a). Indeed, birds are virtually unique among internal
fertilizers that most species lack an IO. Thus, in birds at least, the IO is not
necessary for internal fertilization. This raises the question, then, whether the
avian IO has evolved as a primary sexual trait simply for the delivery of
sperm, as is sometimes assumed for other taxa, or as a secondary sexual trait
(Eberhard 1990; Briskie and Montgomerie 1997). Most of the scant literature
on avian IOs has focused on the seemingly odd presence of IOs in a few
orders, but it is in fact their absence in so many species that is the evolutionary
puzzle.
Despite this interesting question about the absence of IOs in birds,
relatively little is known about the anatomy, physiology, and evolution of
structures that facilitate copulation in this taxon. There was quite a bit of
interest in the phallus of ratites in the 19th century with J. Müller’s (1836)
anatomical study being the most comprehensive—King (1981a) provides a
useful review of this early work. Later, Eckhard (1876), R. Müller (1908) and
Liebe (1914) showed that the Mallard (Anas platyrhynchos) phallus is erected
by a lymphatic, rather than blood-vascular mechanism as had previously been
thought. Gerhardt (1933) and other early workers also noted that there were
two types of true phalluses in birds—those with and without a blind cavity—
and there was a smattering of other studies on the phalluses of wild birds in
the early part of the 20th century. Beyond that early work, only the non-
1
Department of Biology, Queen’s University, Kingston, ON K7L 3N6, Canada
2
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
intromittent phalluses of the domestic chicken (Gallus gallus) and turkey

(Meleagris gallopavo) received much anatomical or physiological study (King
1981a). However, during the past decade or so, there has been renewed
interest in the avian IO, with new hypotheses to explain the evolutionary
patterns of occurrence (Briskie and Montgomerie 1997; Wesolowski 1999;
Briskie and Montgomerie 2001; Wesolowski 2001), comparative analyses of
anatomical structure (Coker et al. 2002), detailed anatomical work using
modern techniques (Oliveria and Mahecha 2000; Oliveria et al. 2003, 2004),
and some excellent natural history on the most bizarre copulatory structures
yet discovered in birds (Birkhead et al. 1993; Mulder and Cockburn 1993;
Birkhead and Hoi 1994; Wilkinson and Birkhead 1995; Winterbottom et al.
1999, 2001; McCracken 2000; McCracken et al. 2001).
In this chapter we present an overview on what is known about the
anatomy, histology, and physiology of the avian phallus—both intromittent
(phallus protrudens) and non-intromittent (phallus nonprotrudens) forms—as
well as other copulatory structures involved directly in the transfer of sperm
from male to female. King’s (1981a) comprehensive review is our point of
departure for this material and should be referred to by the reader needing
more detail on work done up to 1980. We also discuss the evolutionary history
of IOs in birds in relation both to phylogeny and to the various selective
pressures that may have favored the evolutionary loss of the IO and the
appearance of phallus-like copulatory structures in some lineages. Our goal
in this chapter is to provide a framework for understanding the evolution of
all copulatory structures in birds in the context of both the evolutionary
history of birds and the influence of both natural and sexual selection on the
modification and loss of phalluses in some lineages.
3.2 EVOLUTIONARY HISTORY OF COPULATORY STRUCTURES

The various kinds of copulatory structures found in modern birds
(Neornithes) can be mapped onto the most recent avian phylogenies (e.g., Figs.
3.1-3.3) to illustrate their probable evolutionary history. In birds, a true phallus
(both intromittent and non-intromittent) is found only in the orders
Struthioniformes (ostrich, rheas, emu, kiwis and cassowaries), Tinamiformes
(tinamous), Anseriformes (waterfowl and screamers), and Galliformes
(pheasants, grouse, megapodes and currasows). The true phallus is clearly
homologous to the phallus of crocodiles, turtles and tortoises (King 1981a),
their closest living relatives (Fig. 3.1). A phallus-like structure (or
pseudophallus) used in copulation is known from only three other bird
genera (see below); in only one of these is it intromittent, and in none is it
synapomorphic with the crocodile phallus. Male birds in all remaining avian
taxa apparently have no phallus, but instead copulate solely by cloacal
apposition, usually for only a few seconds (see Volume 6B, Chapter 6).
Problems in rooting the avian tree with respect to the Crocodylia has made
placement of the sister taxa Galliformes and Anseriformes controversial with
Anatomy and Evolution of Copulatory Structures %
Fig. 3.1 Phylogenetic relationship of birds to crocodiles, turtles, lizards, snakes,

and some extinct dinosaurs. Modified after Padian, K. and Horner, J. R. 2002.
Trends in Ecology and Evolution 17: 120-124, Fig. 3.
respect to the Palaeognathae and Neognathae (Cracraft et al. 2004). Thus to

provide some context for the discussion of phallus evolution in birds, we have
mapped the avian phallus and other copulatory organs onto two different
phylogenies that have recent support (Figs. 3.2 and 3.3). These two
phylogenetic hypotheses are based on both molecular and morphological data
and are different in many respects from earlier phylogenies based on
morphology alone.
The comprehensive phylogeny (‘Tapestry’; see Chapter 1) of bird evolution
proposed by Sibley and Ahlquist (1990), based on DNA-DNA hybridization,
grouped the ratites (ostrich, rheas, and tinamous) into one clade
(Palaeognathae), and the Galliformes and Anseriformes into a separate clade
Fig. 3.2 Summary of the Sibley-Ahlquist ‘Tapestry’ of bird phylogeny, based on

DNA hybridization, illustrating the evolution of the avian phallus. The Megapodiidae
are marked as equivocal because IOs have been recorded in some species but
not others, based on very sketchy evidence (see text). Modified after Cracraft, J., et
al. 2004. Pp 468-489. In Cracraft, J. and Donoghue, M. J. (eds), Assembling the
Tree of Life. Oxford University Press, New York, Fig. 27.2.
Anatomy and Evolution of Copulatory Structures '
Fig. 3.3 Summary of a recent consensus phylogeny of birds, based on both

molecular and morphological evidence, illustrating the evolution of the avian
phallus. The Megapodiidae are marked as equivocal because IOs have been
recorded in some species but not others, based on very sketchy evidence (see
text). Modified after Cracraft, J., et al. 2004. Pp 468-489. In Cracraft, J. and
Donoghue, M. J. (eds), Assembling the Tree of Life. Oxford University Press, New
York, Fig. 27.10.
(Galloanserae) that is the sister clade to the Palaeognathae (Fig. 3.2).

According to this phylogenetic hypothesis, the clade comprising the
Palaeognathae and the Galloanserae is separate from the clade comprising all
other birds (Neoaves). Though the Sibley-Ahlquist Tapestry was radically
different from accepted phylogenies at the time, it received widespread
support because it was based on DNA analyses, and made sense in many
respects. Problems with both the use of DNA-DNA hybridization and the
interpretation of data, however, encouraged other researchers to employ a
large number of other kinds of molecular and phylogenetic analyses that
continue to refine our understanding of the relationships among and within
the clades that Sibley and Ahlquist (1990) proposed. Probably the best
consensus phylogeny currently available, based on nuclear genes as well as
other traits, agrees with the Sibley-Ahlquist Tapestry in many respects, but does
not isolate the Galloanserae and Palaeognathae in a separate clade (Fig. 3.3).
The evolution of avian phalluses maps onto both of these general molecular
phylogenies in a similar fashion (Figs. 3.2 and 3.3), with the true phallus
being monophyletic in both cases. The intromittent true phallus is clearly the
ancestral type and is shared by all species in the Palaeognathae and several
families of the Galloanserae; the non-intromittent true phallus, on the other
hand, has probably arisen only once, in the ancestor to the clade comprising
the Phasianidae, Odontophoridae, and Numididae, though possibly also in
the Megapodiidae (see below).
Several other copulatory structures that have so far been discovered in
birds are all in the Neoaves, but only in the Psittaciformes (genus Coracopsis)
has a copulatory structure evolved with an intromittent function. In all other
species in which the genitalia of males appear to have been modified for
copulation, none is known to have a structure that is inserted into the female’s
cloaca.
3.3 FORM AND FUNCTION

3.3.1 Ancestral Phalluses
The true phalluses of birds all have traits similar to the phalluses of their
closest living relatives—the crocodiles, turtles, and tortoises (Fig. 3.4)—and
appear to be monophyletic (Figs. 3.1-3.3). Given our current understanding of
the evolutionary relationships among birds and dinosaurs (Fig. 3.1), it seems
quite likely that theropod dinosaurs also possessed an intromittent phallus
(Larson and Frey 1992) not unlike that of crocodiles, chelonians, and the
intromittent true phalluses of birds.
The phalluses of male chelonians (turtles and tortoises) comprise a
thickening along the midline of the ventral wall of the proctodeum (Wood
Jones 1915). In most species, the caudal portion of the phallus is separated
from the wall of the proctodeum so that most of the phallus is free (Fig. 3.4A),
and is inserted into the female during copulation. The phallus is made up of
two fibroelastic bodies (fibrous tissue with large vascular spaces), which in
Anatomy and Evolution of Copulatory Structures
Fig. 3.4 Typical intromittent true phalluses of (A, B) turtles and (C) crocodiles
showing the location of the ejaculatory groove (phallic sulcus). B. Cross section of
A. Not drawn to scale, nor were scale bars shown on the original drawings.
Modified after King, A.S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds),
Form and Function in Birds. Academic Press, London, Fig. 3.2.
most species are fused into a single structure of erectile tissue (Fig. 3.4). The
fibroelastic bodies are separated by a median ejaculatory groove, called the
phallic sulcus, that extends from the opening of the urogenital sinus nearly to
the tip of the phallus. As the phallus becomes erect, the fibroelastic bodies
swell such that this groove becomes a closed channel along which the semen
travels (Wood Jones 1915).
The phalluses of crocodilians are clearly homologous to those of
chelonians, but the free part is longer and projects more prominently from the
proctodeal wall when it is not erect, the shape tends to be more cylindrical
(Fig. 3.4C), and the glans may be more complex (Gerhardt 1933). In addition,
the crocodilian phallus is distinctly curved, particularly when it is erect,
compared to the relatively straight phallus of turtles and tortoises (Fig. 3.4).
In chelonians and crocodilians, the mechanism of erection appears to be
blood vascular, though King (1981a) hints that this needs to be better
documented. In both taxa, females also have a phallus (i.e., clitoris) that is very
similar to that of the male, but is much smaller and cannot be extruded from
the cloaca.
3.3.2 Bird Phalluses

King (1981a) and earlier workers classified true avian phalluses into
intromittent and non-intromittent types based on their gross anatomy, and
further divided the intromittent phallus into two types based on the presence
(Type B) or absence (Type A) of a blind cavity. Type B phalluses are similar in
shape to Type A but have a blind tubular cavity, like the invaginated finger of
a glove, when flaccid, and tend to be twisted in a spiral from the base to the
tip when erect. This categorization, based on the presence/absence of a blind
cavity, appeared to make some evolutionary (phylogenetic) sense at one time,
but more recent work on the anatomy and histology of tinamou phalluses
(Oliveira and Mahecha 2000), as well as the best current phylogenies of birds
(Fig. 3.3), suggest that the blind cavity may be an adaptive trait, possibly with
some erectile function. Thus, the distinction between Type A and B phalluses
does not appear to be particularly useful from a phylogenetic perspective.
Like the phalluses of crocodilians and chelonians (Fig. 3.4), all intromittent
true phalluses in birds are composed of fibrous, erectile tissue that arises from
the ventral wall of the proctodeum and have a ventral sulcus (ejaculatory
groove) along which the semen travels during ejaculation (e.g., Figs. 3.5-3.10).
[In birds, the cloaca has three compartments separated from each other by
folds: (1) the proctodeum, nearest the vent, (2) the urodeum, a narrow zone
that the urogenital ducts empty into (except in tinamous and rheas; Oliveira et
al. 2003), and (3) the coprodeum, into which the rectum empties (King 1981b).]
When the phallus is erect, the lips of the ejaculatory groove also become
engorged, sealing the groove and thus preventing the spillage of semen.
Intromittent phalluses all have a fixed base of fibrous tissue and a free conical,
or tubular portion (composed of two fused fibrolymphatic bodies) that is
everted during erection and copulation. Such phalluses usually curve
towards the male’s left, and thus during copulation will most likely deposit
sperm into the female’s left oviduct, which in birds is the functional one (King
1981a). The fixed base of the phallus has lymphatic spaces that fill and
become dilated during erection (Oliveira and Mahecha 2000); vascular bodies
in the floor of the urodeum provide the lymph that engorges the phallus,
Fig. 3.5 Intromittent true phallus of the male Spotted tinamou in its erect state: A.
Caudal end. B. Left side view. Modified from Oliveira, C. A. and Mahecha, G. A. B.
2000. Annals of Anatomy 182: 161-169, Figs. 18 and 19.
Anatomy and Evolution of Copulatory Structures !
possibly through the action of the cloacal sphincter muscle. The base of the
phallus also has glandular tissue that secretes mucous that lubricates the
phallus and facilitates both eversion and, presumably, copulation (Komárek
and Marvan 1969). Below we review the taxonomic distribution of phalluses
among extant birds; classification and species numbers are taken from
Dickinson (2003).
3.3.2.1 Palaeognathae
Males of all species of Palaeognathae studied so far have an IO that is similar
to the crocodilian phallus (Fig. 3.4C), characterized a conical base of fibrous
tissue, and a bend to the bird’s left when erect due to the two fused
fibrolymphatic bodies being laterally asymmetrical (e.g., Figs. 3.5-3.8). In all
species studied so far, the female also has a phallus that is much smaller than
that of the male and does not extrude from the cloaca (e.g., Fig. 3.6B).
Tinamidae (tinamous, 47 species). Tinamous were classified by King (1981a)
as having a Type A phallus (i.e., lacking a blind cavity), based on work by
Müller (1836) and Gerhardt (1933) on Crypturellus cinnamomeus. However, a
recent anatomical and histological study of the Spotted tinamou (Nothura
maculosa) clearly documents the presence of a blind cavity (Fig. 3.5A; Oliveira
and Mahecha 2000) like that in the Anseriformes, rheas, emus, and
cassowaries. It is quite possible that other species of tinamou possess such a
cavity but there is too little information available (even in Gerhardt 1933) to be
certain.
The phallus of the Spotted tinamou is the best studied in this family, based
on a sample of 26 males captured at different times of the year (Oliveira and
Fig. 3.6 North Island kiwi (Apteryx australis mantelli): A. Intromittent true phallus
of the male. B. Vestigial phallus (clitoris) of the female. Modified after Caithness,
T. A. 1971. International Zoo Yearbook 11: 206-208, Figs. 2 and 3.
Mahecha 2000). To describe the morphology of the phallus, Oliveira and

Mahecha (2000) conducted modern histological and anatomical studies of
birds with the phallus both flaccid and erect (the latter induced by cloacal
massage). In this species, the phallus is composed of a fixed, fibrous, conical
base attached to the floor of the proctodeum and a tubular portion with a
blind-ended tube at the tip (Fig. 3.5A). When the phallus is not erect, it lies
entirely within a phallic pouch in the floor of the proctodeum. The erect
phallus is distinctly coiled in a spiral (Fig. 3.5B) and extends about 3 cm out
of the male’s vent during copulation, directed towards the male’s left.
The fixed base of the phallus of both Spotted and Red-winged tinamous
(Rhynchotus rufescens) also has intraepithelial plasma cells that increase at
least 8-fold in number during the breeding season (Oliveira et al. 2003). These
cells contain granular material indicative of immunoglobulin accumulation
that might be important for an immune response to protect the phallus tissue
from infection resulting from sexually transmitted diseases. Alternatively,
secretions from these cells may be added to the seminal fluid and thus protect
sperm within the female’s reproductive tract (Oliveira et al. 2003).
Apterygidae (kiwis, 3 species). Except that it is asymmetrical and curves to
the male’s left, the phallus of male kiwis (Fig. 3.6A) is very similar to that of
crocodilians and chelonians (Fig. 3.4). The phallus of the female (i.e., clitoris)
is very small and cannot be everted from the cloaca (Fig. 3.6B), making this a
relatively easy trait for sexing these externally monomorphic birds (Caithness
1971).
Casuariidae (cassowaries, 3 species). Phalluses have been observed in the
Southern (Casuarius casuarius) and Dwarf Cassowaries (C. bennetti), and in the
latter were described as being indistinguishable from the phalluses of rheas
and emus (Gerhardt 1933).
Dromaiidae (emus, 3 species). Emus are reported to have the same phallus
structure as rheas (Müller 1836, Gerhardt 1933).
Struthionidae (ostrich, 1 species). The large, bright red phallus of the male
Ostrich (Struthio camelus) is up to 20 cm long when flaccid and extrudes 40 cm
or more out of the male’s cloaca when erect (Fig. 3.7A; Gerhardt 1933). Even in
its flaccid state, it has to be partly protruded from the cloaca to allow
defecation as it takes up so much space in the cloacal lumen that it blocks the
opening of the ureter. At rest, it lies within a wide pocket in the ventral wall of
the proctodeum.
As in other paleognaths, the walls of the ejaculatory groove (Fig. 3.7B) in
the Ostrich are composed of erectile tissue that fills with lymph during
erection, and presumably seals the lip of the groove to form a tube down
which the semen passes. During erection a pair of retractor muscles (m. lecator
phalli) extrude the flaccid phallus from its pocket (Fig. 3.7A), followed by the
vascular bodies filling the phallus with lymph. King (1981a), however, notes
that the literature on this lymphatic mechanism (and the tissues involved) is
old, equivocal, and biased. It is unlikely that the mechanisms of erection in
Anatomy and Evolution of Copulatory Structures #
Fig. 3.7 Intromittent true phallus of the male Ostrich: A. Erect phallus extruded
from vent, with dissection showing musculature. B. Cross section of A (at about
the dotted line) showing asymmetrical sizes of the left and right fibrolymphatic
bodies. Not drawn to scale (especially since the erect phallus should be about
40 cm long, or about 10 times the width of the vent), nor were scale bars shown on
the original drawings. Modified after King, A. S. 1981a. Pp 107-147. In King, A. S.
and McLelland, J. (eds), Form and Function in Birds, vol. 2. Academic Press,
London, Figs. 3.3b and 3.4d.
ostriches is different from that in the other paleognaths but this deserves some
study using modern techniques
Rheidae (rheas, 2 species). Müller’s (1836) description of the phallus of the
Greater rhea (Rhea americana) is still the best and most detailed (Fig. 3.8). The
other species of rhea has not been studied but its phallus should have
essentially the same structure. In the Greater rhea, the resting phallus has an
orifice at the tip that leads to a blind tube or cavity, as in both the Spotted
tinamou and the Anseriformes. When the phallus is erect, about half of this
blind cavity is evaginated. The proximal end of this cavity is continuous with
the phallic sulcus, and when the phallus is at rest (i.e., flaccid) part of the
phallic sulcus is actually pulled into the cavity.
Unlike the other paleognaths studied so far, the fibrous bodies at the base
of the rhea’s phallus are spirally intertwined, and the free tubular portion of
the phallus continues in a slight left-turning spiral throughout its length
(Fig. 3.8). In addition a band of elastic fibers (lig. elasticum phalli) runs along
the entire length of the everted phallus and probably retracts the phallus
during detumescence (Müller 1836; Gerhardt 1933).
Fig. 3.8 Intromittent true phallus of the male Rhea. Not drawn to scale, nor was
there a scale bar shown on the original drawing. Modified after Briskie, J. V. and
Montgomerie, R. 1997. Journal of Avian Biology 28: 73-86, Fig. 1a, who redrew this
from King, A. S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form
and Function in Birds, vol. 2. Academic Press, London, Fig. 3.3d.
3.3.2.2 Neognathae
Galloanserae (452 species)
The Galliformes and Anseriformes comprise the Galloanserae (Sibley and
Ahlquist 1990), recognizing that these two avian orders are sister taxa (Fig.
3.3). Like the paleognaths, both of these orders possess a true phallus but only
in the Anseriformes, and probably in the Cracidae and Megapodidae, is it
intromittent. Thus the common ancestor of these two orders most likely had
an IO but the size of this phallus became reduced and no longer involved in
intromission in the galliform lineage leading to the Phasianide, Numididae,
and Odontophoridae, and possibly in the Megapodiidae (Fig. 3.3).
Anseriformes (162 species)
Anatidae (ducks, geese, swans; 158 species). The family Anatidae is distin-
guished by having (i) the species with the longest (relative to body size) IO of
any bird (McCracken 2000), (ii) the species with the best-studied IO, and (iii)
by far the largest number of species for which there are quantitative data on
IO size and morphology (Coker et al. 2002). Not only is the Anatidae the most
speciose family of birds with IOs, but it displays a wide range of mating sys-
tems and male reproductive tactics and thus provides a useful model for
studying the evolution of IOs in relation to sexual selection. Coker et al. (2002)
have made an excellent start at this but there is still much to be done. The
Anatidae are also relatively easy to keep and study in captivity, even while
breeding (Johnsgard 1978), and so would lend themselves well to both critical
Anatomy and Evolution of Copulatory Structures %
experiments about IO function in relation to structure and detailed anatomi-

cal/physiological study during the critical period when males copulate.
The phallus of the male Mallard is probably better studied than that of any
other bird, save possibly the non-intromittent phallus of the domestic chicken.
At rest, the Mallard’s phallus lies coiled within a thin peritoneal sac in the
ventral wall of the proctodeum; when erect, the base of the phallus fills the
male’s vent and the phallus extrudes 4 cm out of the cloaca in wild birds (but
8 cm in domesticated stock; Rautenfeld et al. 1974). The right fibrolymphatic
body is much larger that the left and the phallus twists 3-4 turns in a left
spiral from the base to the tip (Fig. 3.9). These two bodies are fused and thus
continuous with each other but are separated at the surface by a deep
ejaculatory groove (phallic sulcus) along which semen travels during
ejaculation. The surface of the phallus is relatively smooth at the base but
within a half turn of the spiral the surface of the fibrolymphatic bodies
becomes cornified with rough transverse ridges about 2 mm apart. The tip of
the IO has a small opening into a blind cavity, typical of Type B avian
phalluses (Fig. 3.9).
The Mallard phallus becomes erect by filling with lymph from two small
(1 ¥ 4 cm) vascular bodies in the cloacal wall near the base of the phallus.
Peristaltic contractions of the cloacal sphincter increase the flow of lymph to
the fibrolymphatic bodies (Guzsal 1974). The swelling of these bodies seals
the lips of the phallic sulcus, converting it into a closed tube such that semen
can leave the phallus only at the tip. The phallus detumesces when the cloacal
sphincter relaxes and lymph flows out of the phallus into general circulation.
Fig. 3.9 Intromittent true phallus of the male Mallard as viewed from the left side
when fully erect. No scale bar shown on the original drawing. Note that the right
fibrolymphatic body is much smaller than the left, and that the ejaculatory groove
(phallic sulcus) is between them. Modified after Briskie, J. V. and Montgomerie, R.
1997. Journal of Avian Biology 28: 73-86, Fig. 1a, who redrew this from King, A. S.
1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form and Function in
Birds, vol. 2. Academic Press, London, Fig. 3.6.
This process is assisted by contractions of two pairs of muscles as well as by

elastic fibers in the phallus that were stretched during erection. Detumescence
begins with the immediate invagination of the base of the phallus, then over
about 30 s the entire phallus is retracted into the cloaca by the action of the
retractor muscles and the elastic fibers. Finally, over a 2-4 min period, the
whole flaccid phallus is folded back inside the peritoneal sac, aided by the
antiperistaltic action of the cloacal sphincter as well as by mucous that is
secreted from the glandular base of the phallus.
Coker et al. (2002) studied the IOs of 54 species of Anatidae for which they
had detailed, scaled drawings of IOs (one per species) made from formalin-
preserved museum specimens of males taken during the breeding season. As
they did not work with fresh or live specimens, it is unknown whether the
species that they studied have phalluses that are similar in structure to that of
the Mallard. From each drawing, they measured the flaccid length of the IO,
estimated its circumference, and quantified the numbers and heights of knobs
and ridges (Fig. 3.10, Plate 3.1) on its surface. In the species studied, flaccid IO
length varied from 1.25 cm in the Red-breasted goose (Branta ruficollis) to an
incredible 28.5 cm in the Australian blue-billed duck (Oxyura australis). IO
length was not related to body length (r = –0.11, P = 0.36) in this sample of
species, suggesting that variation in size might be adaptive. Indeed, the largest
geese and swans had among the smallest IOs (e.g., 2.4 cm in the Canada
goose, Branta canadensis, and 2.3 cm in the Tundra swan, Cygnus columbianus),
whereas the relatively small stiff-tailed ducks (subfamily Oxyurinae) had
among the largest (e.g., 23.6 cm in the Ruddy duck, O. jamaicensis). Even
between closely-related species of similar size, the IO can be quite different in
Fig. 3.10 Intromittent true phallus of the North American ruddy duck, showing
knobs and ridges (not to scale). The base of the phallus is to the left and the view
is from the right side of the phallus. Modified after Coker, C. R. et al. 2002. Auk 119:
403-413, Fig. 1.
Anatomy and Evolution of Copulatory Structures '
size (e.g., 9.2 cm in the Common eider, Somateria mollissima, versus 15.0 cm in
the King eider, S. spectabilis; 9.2 cm in the White-faced whistling duck,
Dendrocygnus viduata, versus 18.8 cm in the Black-bellied whistling duck, D.
autumnalis). There was also considerable variation in both the number
(density) and size of knobs and ridges on the surface of the IOs, with the
density of ridges strongly negatively correlated with IO circumference (r = –
0.86, P <0.05). Thus larger IOs tended to have more knobs than ridges (Fig. 3.11).
The stiff-tailed ducks have long been known to have large IOs especially in
relation to their body size. Indeed, the IO of the Australian blue-billed duck
has been described as being so large that, after copulation, the male rolls on
his back and prods his long, detumescing IO back into his cloaca with his bill
(Marchant and Higgins 1990). Males of both the Australian blue-billed duck
and the North American ruddy duck apparently often preen their IO after
copulation (McCracken 2000). In this same subfamily, McCracken et al. (2001)
recently discovered a male Argentine lake duck (O. vittata) with an IO that was
42.5 cm long in its everted, flaccid state, fully as long as the duck itself (Plate
Fig. 3.11 Results of Principal Component Analysis on IO sizes and surface

structures (see Fig. 3.10) in the family Anatidae, grouped according to the
presumed frequency of forced extrapair copulations (FEPCs). Each data point
represents one species; outlines encompass each mating system type, with the
range of monogamous birds shaded to improve clarity. Modified after Coker, C. R.
et al. 2002. Auk 119: 403-413, Fig. 2.
CMYK

CMYK
CMYK
Plate 3.1 A,B Intromittent true phallus of the male Argentine lake duck: A.
Extending from male, pulled out to its full extent but not erect. B. Showing knobs
Plate 3.1 Contd. ...

CMYK
Anatomy and Evolution of Copulatory Structures !
3.1A)! McCracken (2000) had previously described the IO of this species as

averaging 22.3 cm (range 19.0-24.5 cm, n = 4 males), thus suggesting that this
one male had a particularly long IO. Despite its length, the width of the IO
averaged only 6.5 mm (range 4.5-8 mm) at its base. In this species, the inverted,
detumescent IO lies entirely within a blind pouch in the ventral wall of the
cloaca, as in other anatids. The base of the IO is covered with a dense array of
large, sharp, black-tipped white spines, most dense on the ventral side of the
IO, and pointing outward and backward toward the cloaca when everted
(Plate 3.1B). Thus copulation may proceed by the male everting the first 1-2 cm
of his IO and inserting this into the female where the spines would clasp the
female’s cloaca while the male everts the rest of his IO inside the female
(McCracken 2000). Spines also occur along a series of spiral grooves extending
along the full length of the everted IO (Plate 3.1B).
Anseranatidae (magpie goose; 1 species). To the best of our knowledge, the
phallus of the Magpie goose (Anseranas semipalmata) has never been studied,
but there is no reason to expect that it differs much in structure from the
typical anatid phallus.
Anhimidae (screamers; 3 species). The phallus of screamers has never been
studied, but looks superficially like that of the Mallard (R. M., unpublished
data).
Galliformes (290 species)
Megapodiidae (scrub fowl and brush turkeys; 22 species). The Australian
brush-turkey (Alectura lathami), the Malleefowl (Leipoa ocellata), and species in
the genus Aepypodius are all reported to have an intromittent phallus, whereas
at least some other megapodes in the genus Megapodius apparently do not
(Brom and Dekker 1992; Jones et al. 1995), though no details are available on
the source of these observations. It is unknown what sort of phallus occurs in
other genera of this family. If Fig. 3.3 correctly depicts the evolutionary
relationships in the Galloanserae, and if the Cracidae do have an anatid-like
IO (see below), then the absence of an IO in some megapodes would be an
additional instance of the independent evolutionary loss of the intromittent
function of the avian phallus. Understanding the patterns of phallus
evolution in the megapodes, particularly the evolutionary transformation
from an intromittent to a non-intromittent phallus, presents an interesting
challenge that might provide some useful insights into the reasons for IO loss
in birds.
Plate 3.1 Contd. ...

and spines in everted but flaccid state. C. Copulation in the Red-breasted
merganser (Mergus serrator): typical of waterfowl that copulate on the water, the
female’s cloaca is submerged, so the male phallus may help to prevent water from
entering the cloaca and damaging sperm during copulation. Original photographs
by Kevin McCracken (A, B) and Iztok Škornik (C).
Cracidae (guans and currasows; 50 species). Gerhardt (1933), as well as

Gadow and Selenka (1891), report that cracids have a phallus resembling that
of the Mallard but details are sketchy and some modern work is really needed
on this taxon. Gadow and Selenka (1891) report on three independent
accounts of a cracid phallus from the early literature. In one of these accounts,
the phallus is described as a spiral structure about 35 mm long with an
ejaculatory groove and an opening (presumably a blind cavity) at the tip.
Gerhardt (1933) studied, but did not dissect, a museum specimen of the Black
currasow (Crax alector), and describes a flaccid phallus that resembled that of
the Mallard and was 2.4 cm long with a blind cavity that could be everted to
increase the total length to 3.9 cm.
Given our current understanding of the relationship of the Cracidae to the
other Galloanserae (Fig. 3.3), a detailed study of phallus anatomy within this
taxon would be particularly useful. It would not be too surprising if the cracid
phallus resembled that of the anatids since they share a common ancestor but
further study might help us to understand how the intromittent phallus was
lost in the clade comprising Phasianidae, Odontophoridae and Numididae
because this clade also shares a common ancestor with the cracids (Fig. 3.3).
Numididae (Guineafowl; 6 species). The guineafowl are expected to have a
non-intromittent phallus like that of the chicken but its structure has never
been described.
Odontophoridae (New World quails; 32 species). The New World quails are
also expected to have a non-intromittent phallus like that of the chicken but
its structure has never been described.
Phasianidae (pheasants and grouse; 180 species). All of the Phasianidae are
expected to have a non-intromittent phallus like that of the chicken and turkey
but there have not, to the best of our knowledge, been any studies of any other
species in this family. The phalluses of the domestic chicken and turkey have,
however, been very well studied. Both species have a non-intromittent phallus
on the ventral lip of the vent that, even when not erect, can readily be seen by
manually depressing this lip. This phallus appears to be homologous in
structure to the intromittent true phalluses of the Neognathae and
Palaeognathae.
In the domestic chicken, the erect phallus is roughly heart-shaped with a
median groove (Fig. 3.12A) into which the paired deferent ducts discharge
semen directly. During erection of the phallus, both the vent and the floor of
the proctodeum evert for a few seconds exposing the phallus externally. The
chicken phallus has vascular bodies embedded in the cloacal wall in the same
position as those of the Mallard, attesting to their common evolutionary origin,
though their actual structures are substantially different (see King 1981a for
details). The lymphatic channels of the vascular bodies connect with the
lymphatic channels of both the phallic bodies and the lymphatic folds. Thus
erection of the chicken phallus is accomplished by engorgement with lymph,
as in the Mallard, and results in the eversion of the phallic area of the vent
Anatomy and Evolution of Copulatory Structures !!
Fig. 3.12 Non-intromittent true phalluses of male A. Domestic chicken. B.

Domestic turkey. Not drawn to scale, nor were scale bars shown on the original
drawings. A modified after King, A. S. 1981a. Pp 107-147. In King, A. S. and
McLelland, J. (eds), Form and Function in Birds, vol. 2. Academic Press, London,
Fig. 3.8a; B modified after Briskie, J. V. and Montgomerie, R. 1997. Journal of Avian
Biology 28: 73-86, Fig. 1e, who drew this from a photograph in King, A.S. 1981a.
Pp. 107-147. In King, A.S. and McLelland, J. (eds), Form and Function in Birds, vol.
2. Academic Press, London, Fig. 3.10b.
and the adjacent cloacal floor. Both the phallic bodies and the lymphatic folds
swell to meet along the midline of the phallus, thus creating a median groove
(Fig. 3.12A) down which the semen flows during ejaculation. Immediately
after ejaculation, lymph drains from the phallic bodies and lymphatic folds
via the lymphatic vessels of the pudendal artery (Nishiyama 1955).
The phallus of the domestic turkey is structurally and functionally similar
to that of the chicken but differs in having two prominent prismoid humps
separated by a deep furrow (Fig. 3.12B). The lymphatic folds extend obliquely
across the floor of the proctodeum and have 3-4 prominent oblique ridges on
their surface.
Neoaves (about 9500 species)
There is no strong evidence for a true phallus in any of the Neoaves, and
direct evidence for a truly intromittent copulatory structure is only convincing
for the Coracopsis parrots. During the breeding season, male Greater (C. vasa)
and Lesser vasa parrots (C. nigra) of Madagascar and the Comoro Islands
have a very large, heavily vascularized cloacal protrusion (Fig. 3.13A). This
large fleshy bag is usually everted during copulation when it becomes dark
red in color and extends 50 mm or more out of the cloaca. During one
copulation the male mounted the female’s back and his cloacal protrusion
appeared to completely enter the female’s cloaca, which stretched to
accommodate it (Wilkinson and Birkhead 1995). After intromission, the male
slipped off the female’s back but their cloacas remained interlocked for
104 min. When the male and female eventually disengaged, both sexes ejected
a small volume of liquid and a small (5-15 mm) phallus-like organ was
Fig. 3.13 Pseudo-phalluses of male A. Vasa parrot. B. Buffalo weaver. C. Superb

fairy wren. Arrows indicate the opening to the cloaca. Modified after Briskie, J. V.
and Montgomerie, R. 1997. Journal of Avian Biology 28: 73-86., Fig. 1c, d, f.
observed at the tip of the cloacal protrusion. Some detailed anatomical work is
needed to assess the structure of this apparent phallus.
The remaining extant bird species in the Neoaves do not appear to have a
phallus that is homologous to that of the Palaeognathae and the Galloanserae.
Thus the true phallus appears to have been lost during the early evolution of
this entire clade (Fig. 3.3). Although the literature from the 19th century does
suggest some vestigial phallic structures might be present in some species of
Neoaves (see King 1981a for details), it is far from certain whether these are
homologous to the true phallus.
For example, in the Emberizidae, Wolfson (1954a) reported that a pair of
papillae formed by the proctodeal wall could be protruded from the vent when
the cloacal protuberance was squeezed. These papillae meet and formed a
median groove that carried semen during ejaculation. Wolfson (1954a)
suggested the structure was analogous to the phallus of the rooster and
functioned in the same manner. However, it is not clear whether these
papillae are intromittent or whether they simply assist in passing the ejaculate
to the female. An intromittent function has been proposed by a variety of
authors but at present the exact positioning of the papillae during copulation
is unknown.
Papillae have now been observed in a variety of other passerine species
(Briskie 1993; Birkhead et al. 1991; Birkhead and Hoi 1994; Nakamura and
Matsuzaki 1995; Lombardo 2001; Chiba and Nakamura 2003). Although
papilla size varied slightly across a small number of species measured by
Briskie (1993), this variation did not appear to be related to differences in
social mating system. The length of the papillae in the Alpine accentor
(Prunella collaris) increases in the breeding season (Chiba and Nakamura
2003), suggesting that the increased size may be important for its functioning
during copulation. However, no seasonal changes in the size of the papillae
were found in the Tree swallow (Tachycineta bicolor; Lombardo 2001). As the
size of the papillae in all species examined so far has been relatively small
(about 1-3 mm long), it is unlikely that they penetrate very far into the female’s
reproductive tract even if they are intromittent. More research is needed to
Anatomy and Evolution of Copulatory Structures !#
confirm the presence and anatomical/histological details of papilla-like

structures in other Neoaves (especially non-passerines), and to determine the
exact role they play in sperm transfer.
3.3.3 Bird Cloacal Protuberances and other Structures

During the breeding season, the cloacal region of male passerine birds
enlarges to such a degree that a protuberance forms as an outpocketing of the
ventral abdominal wall (Fatio 1864; Salt 1954; Wolfson 1952, 1954a;
Middleton 1972). In most species, this cloacal protuberance is formed
primarily by the vastly enlarged seminal glomera (the distal ends of the ductus
deferens). The seminal glomera function in sperm storage, and Wolfson (1954b)
found that sperm are kept cooler in the cloacal protuberance as a result of its
position outside the body cavity. However, it has also been suggested that the
cloacal protuberance may function as a copulatory organ by elevating the
cloaca from the body wall, thereby facilitating contact with the female genital
area (Wolfson 1954a).
The cloacal protuberance varies in size across species, and reaches its
largest dimensions in those species with highly promiscuous mating systems
in which males are subject to intense sperm competition (Nakamura 1990;
Birkhead et al. 1991; Briskie 1993; Mulder and Cockburn 1993; Schulze-Hagen
et al. 1995; Tuttle et al. 1996; Castro et al. 1996; Dixon and Birkhead 1997).
Although such large cloacal protuberances are clearly the result of large
seminal glomera containing large numbers of sperm, the enlarged size of the
cloacal protuberance might also function to increase the efficiency of
copulation in these species (Birkhead et al. 1993). Those species that copulated
more frequently sported larger cloacal protuberances (Birkhead et al. 1993), but
there was no relation between the duration of copulation and cloacal
protuberance size.
It is interesting to note that, in the Bearded tit (Panurus biarmicus), the cloacal
protuberance is formed primarily from gelatinous connective tissue overlain
by muscle layers, and not by the seminal glomera (which remain within the
body cavity; Birkhead and Hoi 1994). As a result, the extension of the cloacal
protuberance outside the body cavity cannot play a role in the temperature
regulation of stored sperm in this species. Thus this cloacal protuberance may
function primarily to improve ejaculate transfer by longer and better-
positioned cloacal contact (Sax and Hoi 1998).
The enlarged cloacal protuberance of the New Zealand stitchbird
(Notiomystis cincta) likewise contains only the ejaculatory ducts and not the
seminal glomera (Castro et al. 1996). Low et al. (2005) found that the cloacal
protuberance in this species not only increased in size over the breeding
season but also changed orientation such that the opening to the vent rotated
anteriorly by about 60 degrees. This “cloacal erection” was suggested to
facilitate ejaculate transfer as it would allow males to more directly align the
opening of their cloaca with that of the female during their face-to-face
copulation (Low et al. 2005).
!$ Reproductive Biology and Phylogeny of Birds
In a few species, the cloacal region has become modified further in ways
that suggests some copulatory function. The skin beside the cloaca of both
Red-billed (Bubalornis niger) and White-billed buffalo weavers (B. albirostris) of
Africa have become highly modified to form an external phalloid organ (Fig.
3.13B) that is employed during copulation, although it is now known not to
be intromittent (Winterbottom et al. 1999, 2001). This organ lies immediately
anterior to the cloaca, is composed of connective tissue, has no sperm ducts,
and is non-erectile. In the Red-billed buffalo weaver, the length of the male’s
phalloid organ averages 15.7 mm (±0.29 mm SE, n = 109) whereas the female’s
is much smaller (6.1±0.19 mm, n = 68). Interestingly, resident males had a
significantly longer phalloid organ than non-residents, and residents with a
harem of females had a significantly longer organ than those without a harem
(Winterbottom et al. 2001). During copulation the male’s phalloid organ is
rubbed vigorously against the female and appears to stimulate the male to
orgasm and ejaculation (Winterbottom et al. 1999).
In male Superb fairy wrens (Malurus cyaneus), the cloacal protuberance has
a cartilaginous projection from its anterior surface (Fig. 3.13C; Mulder and
Cockburn 1993). The location and phallus-like shape of this projection
suggest that it may be a copulatory structure but its function is unknown.
Other species of fairy wrens appear to have a similar projection (Tuttle and
Pruett-Jones 2004) but details are lacking.
Detailed studies of the dynamics of sperm transfer are needed in species
with both large cloacal protuberances and pseudophalluses before their true
nature can be determined. With only a handful of species studied in any
detail, it is likely that a variety of other modifications to the external genitalia
in the Neoaves may be discovered by careful observers.
3.4 ADAPTIVE SIGNIFICANCE OF IOs

To date, seven different hypotheses have been proposed to explain the pattern
of presence/absence in the intromittent phalluses of birds. The true avian
phallus was probably lost only once during the evolution of birds (Fig. 3.3)
and is thus absent in all of the Neoaves, comprising >95% of extant avian
species. Reasons for this loss remain obscure, in part because of the paucity of
data on both the anatomy and physiology of phalluses in extant species, but
also because of the technical (and ethical) difficulties in conducting critical
experiments and the lack of power in a comparative analysis with only two
states. The evolutionary history of avian IOs, on the other hand, is a little more
complex in that these copulatory structures have originated at least once in
birds (in the vasa parrots) and may have been lost three times if both the
phylogeny and the data on the presence/absence of IOs shown in Fig. 3.3 is
correct: once in the common ancestor of the Phasianidae-Numididae-
Odontophoridae clade, once (at least) in the Megapodiidae, and once in the
lineage leading to the Neoaves, the large sister clade of the Galloanserae
(Fig. 3.3). This pattern of loss/gain is correct only if all of the Cracidae have
Anatomy and Evolution of Copulatory Structures !%
an IO and the IO has been lost once in the Megapodiidae, but more work is
needed to confirm this. In addition to this pattern of IO loss/gain, copulatory
structures have arisen independently in the buffalo weavers, and possibly the
fairy wrens but neither of these pseudophalluses appears to be intromittent.
In this section, we summarize the various hypotheses proposed so far to
explain the evolutionary loss of the IO in most birds, and evaluate the
evidence for and against each of them. Because the IO appears to have been
lost only three times in the evolutionary history of birds, we cannot employ
modern comparative methods to evaluate these hypotheses. Instead, we draw
on a variety of anatomical, physiological, and behavioral data at the species
and family level in birds, and we use some recent data on variation in IO size
within species and families to address these hypotheses. We have classified
the hypotheses as being the result of natural and sexual selection and we
present them individually but there is clearly no reason that more than one of
them could not explain the loss of IOs. Thus, for example, different hypotheses
may explain the maintenance of IOs in different taxa. In addition, the
disappearance of the IO in avian evolutionary history logically requires that
IOs are costly, and that cost must be due to either development or
maintenance. All of these hypotheses have previously been evaluated in detail
(Briskie and Montgomerie 1997, 2001). We therefore provide only a brief
summary here plus new information that has come to light since those
previous reviews.
3.4.1 Natural Selection Hypotheses

H1 Water Damage
Lake (1981) suggested that an IO might prevent water from entering the
female’s cloaca during copulation where it would either damage sperm (via
osmotic shock), dilute the ejaculate, or wash it out of the cloaca. Thus IOs
would be favored in species that copulate on the water (e.g., Plate 3.1C), but
lost, due to some cost (e.g., see H4 and H5), in lineages that copulate out of the
water. This hypothesis gains some support from the fact that the majority of
the Anatidae, which have IOs, copulate on the water, whereas almost all
species in the many other families of aquatic birds that copulate on land do
not have IOs (Briskie and Montgomerie 1997). On the other hand, many birds
that copulate on the water (e.g., some puffins, auklets, murrelets, and
phalaropes) also lack an IO and do not seem to be at a particular
disadvantage with respect to water damage, though at least a few species lift
their cloacas clear of the water when copulating (Volume 6B, Chapter 6). On
balance, the water damage hypothesis might provide an explanation for the
maintenance of an IO in lineages that typically copulate on the water, but it
cannot explain variation in IO size in those taxa nor can it explain why the IO
has been lost in the majority of bird species.
H2 Genital Contact
King (1981a) suggested that an IO might provide an advantage for sperm
transfer in species where cloacal contact is made difficult by the bird’s
anatomy or its environment. Thus large-bodied, long-legged, and flightless

birds (who may be less able to use their wings for balancing) might have
difficulty maintaining cloacal apposition during sperm transfer. Briskie and
Montgomerie (1997) suggested that this explanation was weak because even
birds with an IO employ several methods to maintain genital contact and
there was no clear pattern with respect to body size, long-leggedness, and the
presence of an IO.
Nevertheless, it is clear that the IO of vasa parrots is essential for
maintaining their long period of genital contact as the male dismounts the
female. It also seems to us that an IO would provide some selective advantage
to males when cloacal contact is difficult to maintain, especially during forced
copulations and interference from rival males. Coker et al. (2002), for example,
found that ducks and geese with high levels of forced extrapair copulations
were more likely to have longer IOs, and IOs with more and larger knobs,
than monogamous species (Fig. 3.14A,B,C) where forced extrapair copulations
are thought to be rare. In the subfamily Oxyurinae, in particular, males often
display in groups to females and copulation can be quite tumultuous
(McCracken 2000), so a relatively large, spiny IO might help a male to
maintain genital contact with a female.
H3 Copulation Duration
Wesolowski (1999) proposed that the loss of an IO in most birds would result
from selection for shorter copulations, either to reduce the period when
copulating pairs would be vulnerable to predation or to minimize the period
when the male would have to balance unsteadily on the female’s back (as in
H2 above). Contrary to expectations from this hypothesis, males in species
with an IO mounted females for shorter durations during copulation than
those without an IO, controlling for body size (Briskie and Montgomerie
2001). Even within the non-passerines, species without an IO do not mount
for shorter periods than those with an IO (Fig. 3.15A). Thus there is, so far, no
empirical support for this interesting idea. Wesolowski (2001) correctly
pointed out that the amount of time that a male spends mounting a female
may not be an accurate index of the duration of copulation, but accurate data
on the actual duration of cloacal contact in birds are rare (Volume 6B, Chapter
6) so this idea is difficult to test quantitatively without more data.
H4 Flight Costs
The loss of an IO might be expected in flying animals to reduce flight costs
and increase maneuverability. Indeed, many birds with an IO are flightless or
relatively poor fliers. With few exceptions, though, the mass of a bird’s IO is
unlikely to be more than a tiny proportion of its body mass and thus its
influence on flight would be negligible. Höhn (1960), for example, showed
that the mass of the Mallard’s IO at the height of the breeding season was
only 0.3% of the male’s body mass. Thus this hypothesis may only explain
the extremely large IOs in ostriches, which are flightless.
Anatomy and Evolution of Copulatory Structures !'
Fig. 3.14 Size and structure of the male intromittent organ (IO) in species from the
family Anatidae in relation to the expected frequency of forced extrapair copulations
FEPCs: A. IO length, controlling for variation in body size. B. Percent of the IO
surface that is covered by knobs or ridges (see Fig. 3.10). C. Size of knobs or ridge.
D. Showing residual IO size in relation to residual testes mass (both controlling for
body size), where the latter is an index of the intensity of sperm competition.
Redrawn from data in Coker, C. R. et al. Auk 119: 403-413, Figs. 3 and 4.
H5 Sexually Transmitted Diseases

Briskie and Montgomerie (1997) suggested that sexually transmitted diseases
(STDs) might pose a significant cost to the maintenance of an IO in birds. They
argued that STDs might be especially problematic in birds because they are
the only homeotherms that have a common cloaca for both defecation and
reproductive functions. Thus, in birds, the male inserts his IO into a warm site
through which fecal material passes, potentially an excellent environment for
the proliferation of pathogens (e.g., Stewart and Rambo 2000). Wesolowski
(1999) argued that STDs were unlikely to be prevalent in most birds, based on
Lombardo’s (1998) suggestion that STDs should be rare in birds because, in
seasonal breeders, the dispersal rates of highly virulent STDs should be low.
Unfortunately, there are few hard data to address this idea but we do know
Fig. 3.15 Tests of adaptive hypotheses to explain the pattern of presence (black
bars and symbols) and absence (open bars and symbols) of intromittent organs
(IOs) in birds: A. In non-passerines there is no difference in mean copulation
duration (controlling for body mass) between species with (n = 16) and without (n
= 167) an IO (ANCOVA on log-transformed variables, F1,180 = 0.03, P = 0.86; bar
graphs show least squares means±95%CL). B. Male and female contributions to
incubation in relation to the presence/absence of male IO (data from 10 families
with and 86 without an IO). C. Relation between the volume of one egg and female
body mass in species with (Anseriformes) and without (Galliformes) an IO in the
Galloanserae. A from data in Briskie, J. V. and Montgomerie, R. 2001. Journal of
Avian Biology 32: 184-187, Fig. 1; B and C from data in Briskie, J. V. and
Montgomerie, R. 1997. Journal of Avian Biology 28: 73-86, Figs. 3 and 4.
Anatomy and Evolution of Copulatory Structures "
that virulent STDs have been isolated in the domestic chicken (Sheldon 1993),
and that bacteria are sexually-transmitted in the Red-winged blackbird
(Agelaius phoeniceus; Westneat and Rambo 2000). Moreover, both Spotted and
Red-winged tinamous have plasma cells in the epithelium of the fixed base of
the IO, suggesting an immunoprotective function (Oliveira et al. 2003). The
dramatic increase in the number of these cells during the breeding season
indicates enhanced immune function during this period, potentially to protect
against STDs. While there are other potential explanations for the function of
these cells, this finding does raise the intriguing possibility that birds with
IOs are particularly susceptible to STDs.
Given that IOs are clearly not needed for internal fertilization in birds,
Briskie and Montgomerie (1997) argued that selective pressures due to the
costs of development and maintenance, especially in the face of STDs, would
favor the disappearance of the IO unless there was a particular benefit to
retaining it. Two such benefits could accrue as a result of sexual selection via
male-male competition or female choice, as follows.
3.4.2 Sexual Selection Hypotheses

H6 Sperm Competition (Male-Male Competition)
Briskie and Montgomerie (1997) proposed that IOs may be favored
particularly when there is competition among males to fertilize a female’s ova.
They argued that IOs, and longer IOs, would allow a male to deposit his
sperm higher up the female’s reproductive tract, closer to the site of sperm
storage at the utero-vaginal junction. In addition, the spikes, knobs and ridges
on some IOs might facilitate the displacement or removal of sperm of rival
males from the female’s urogenital tract.
Briskie and Montgomerie (1997) tested this hypothesis indirectly by
comparing the incidence of IOs in species with male, female, and biparental
incubation, arguing that species with male-only care would be more-strongly
selected to ensure their paternity and thus maintain an IO. This prediction
was supported by an analysis at the family level (Fig. 3.15B).
Interestingly, the IO of domesticated Mallards (mean length 8 cm) is about
twice as large as in the wild bird (mean length 4 cm). Rautenfeld et al. (1974)
suggested that this difference was due to the increased sexual activity of the
domesticated form but we see no reason that IO size should be influenced by
sexual activity alone. Rather it seems to us that this difference in IO size may
be due to increased sperm competition in domestic flocks, where groups of
males have constant access to females in the absence of male territoriality, and
possibly because of reduced costs of both development and maintenance of an
IO in captivity. This apparently evolutionary change of IO size in domestic
mallards deserves some in-depth research.
Coker et al. (2002) also support this sperm competition hypothesis as an
explanation for the interspecific variation in size of anatid IOs. They analyzed
data on the IO size of 54 species of ducks and geese in relation to both mating
systems and relative testes size (controlling for body size), an index of the
intensity of sperm competition (Briskie and Montgomerie, Chapter 9). Mating

systems of 28 of these species in relation to the expected rate of forced
extrapair copulations (FEPC) were classified as 1 = monogamous (presumably
no FEPCs), 2 = rare FEPCs, 3 = frequent FEPCs, and 4 = polygynous or
promiscuous (presumably FEPCs common), based on extensive natural
history observations of the species studied. Both the presumed incidence of
FEPCs (Fig. 3.14A) and the relative testes mass of males were significantly
positively correlated with IO size (Fig. 3.14D). As noted above, males in
species that were more likely to engage in male-male competition also had
more and larger knobs and spikes on their IOs (Fig. 3.14B, C). Inceased
number and sizes of knobs, spikes, and ridges on an IO would (i) enhance a
male’s ability to stay inside the female’s cloaca while being harassed by rival
males, (ii) allow a male to fully erect his IO inside the female, and (iii)
potentially enhance a male’s ability to displace or extract the sperm of rival
males who had previously recently copulated with the female, much in the
manner of the odonate IO (Waage 1979). While males of all the species studied
by Coker et al. (2002) have an IO, the patterns they uncovered show that both
the size and structure of the avian IO are quite variable interspecifically and
thus appear to respond to selection resulting from sperm competition. In the
absence of sperm competition, it is therefore at least plausible that the phallus
would be reduced in size over evolutionary time, due to costs of IO
development and maintenance, until it is no longer intromittent. Given the
potential advantage for even a small IO in the Anatidae to prevent water
damage to sperm, we might not expect to see the disappearance of the IO in
species that copulate on the water. Thus, it would be interesting to compare
the IO sizes of species that copulate on and off the water, while controlling for
the intensity of sperm competition. Similarly, IO size and structure in relation
to mating patterns need to be examined in both the cracids and tinamous, two
groups in which the large number of species would enable some rigorous tests
of this hypothesis.
H7 Female Choice
Briskie and Montgomerie (1997) also suggested that the evolutionary loss of
an IO in birds may have been driven by female choice. They reasoned that, if
females could somehow prefer males that did not force copulations, the IO
might serve no selective advantage. This hypothesis rests on four
assumptions: (i) that IOs are not needed for internal fertilization in birds, (ii)
that the main function of male IOs in birds is to force inseminations and/or to
ensure that semen is deposited well into the female’s reproductive tract, (iii)
that females have a mechanism to reject or reduce the fitness of males that they
prefer not to mate with, and (iv) that IOs are too costly to develop and
maintain if they do not provide any advantage to males that possess them.
Assumption (i) is clearly true, and assumptions (ii) and (iv) are supported by
some limited evidence as presented above. In accordance with assumption
(iii), female birds potentially have the ability to abort or discard at relatively
little cost any ova that are fertilized by non-preferred males. Since the ova of
Anatomy and Evolution of Copulatory Structures "!
all birds are fertilized individually at intervals of usually 24-48 h (Howarth

1974), and there appears to be last male sperm precedence (Birkhead 1998),
females have the potential to identify the paternity of each egg laid and lay it
outside their nest, thus selecting against disfavored males.
Briskie and Montgomerie (1997) reasoned that the costs of selective
abortion or abandonment of ova/eggs would be related to their size relative to
the female’s body size, and thus such a tactic would be most feasible for
females that laid relatively small eggs. Consistent with this prediction, they
found that females in species where the male has an IO lay relatively large
eggs compared to species without male IOs (Fig. 3.15C). Indeed this pattern is
particularly striking in the Galloanserae where male Anatidae have an IO,
frequently engage in forced copulations, and females generally lay very large
eggs, whereas in the Phasianidae, Odontophoridae and Numididae females
lay relatively small eggs, males have a non-intromittent phallus, and forced
copulations are less frequently observed. In other words, if eggs are large and
costly to produce because they are large relative to body size, then females
may be reluctant to abort or abandon such eggs, even if they are sired forcibly
by males that the female does not prefer as a sire. In these species with
relatively large eggs, males retain an advantage by forcing copulations and
maintaining an IO, while in the smaller-egged species, the IO is proposed to
have disappeared because females are able to control fertilizations through
refusing to raise an unwanted male’s offspring.
Like the sperm competition hypothesis, this female choice hypothesis pre-
dicts that IO size will be positively correlated with the frequency of forced
extrapair copulations, but makes the unique prediction that IO size will also
be positively related to the costs of egg production. Certainly among the
Anatidae, the Oxyurinae have relatively large eggs compared to other species
(Johnsgard 1978), and they also have the largest IOs, but a more comprehen-
sive analysis of this prediction is clearly needed.
3.5 FUTURE DIRECTIONS

Despite research on avian copulatory organs that spans almost two centuries,
we still have only the most rudimentary knowledge of IO structure and
function in all but a few species. Even the taxonomic distribution of IOs and
phalluses among taxa has not been comprehensively documented, including
even some uncertainty about whether or not males in the Cracidae and
Megapodidae possess an IO. There is clearly a need for more detailed
anatomical work. Though such studies have not been popular for some time,
modern anatomical techniques (e.g., Oliveira et al. 2000, 2004; Oliveira and
Mahecha 2003), and the various hypotheses about the adaptive significance
of IOs in birds presented above, make this a potentially attractive field of
study.
Some of the adaptive hypotheses summarized here could also be tested
with experimental studies of captive birds. Many species in the Anseriformes
and Galliformes can readily be bred in captivity so that hypotheses about
water damage, sperm competition, female choice, and STDs might be tested
with some clever experiments. There is certainly much to be learned about the
incidence of STDs in birds and their effect on the anatomy, physiology, and
costs of maintaining phalluses. With controlled experiments, it should also be
possible to quantify the influence of IO size and structure on the outcome of
sperm competition, especially within species in which there is natural
variation in IO size (e.g., domestic versus wild Mallards). Selection
experiments with domesticated Mallards would add some useful insights into
the heritability and evolvability of IO size in birds.
Because the taxonomic distribution of phalluses and IOs in birds is
relatively simple, with both being largely ancestral traits (Fig. 3.3), there is
virtually no scope for the sorts of comparative studies that have given us some
insights into the evolution of other reproductive traits in birds (e.g., Briskie et
al. 1997; see Chapter 9). Nonetheless, considerable progress can still be made
in our understanding of both IO evolution in birds and the reasons for the
loss of IOs in most of this Class by simply conducting more comprehensive
anatomical research. Half a century ago , Fisher (1955) lamented the fact that
‘The “modern” trend in biological sciences seems all too often to imply that
“anatomy as such” may be overlooked in the evolution of the “better and more
accepted” avenues of approach to biological problems.’ Plus ça change... The
time is ripe for some detailed anatomical work on avian copulatory structures,
informed by modern phylogenies and some of the hypotheses about their
adaptive significance that we have outlined above. We hope this chapter will
inspire an enterprising graduate student to take up this challenge.
3.6 ACKNOWLEDGMENTS
We are grateful to Meghan Goodchild, Kristen Scott, and Christina Cliffe for
help in compiling data and searching out references; and to Tim Birkhead and
Barrie Jamieson for numerous excellent comments on the manuscript. Our
work on avian reproductive tactics is supported by grants from the Natural
Sciences and Engineering Research Council of Canada (to R. M.) and the
University of Canterbury (to J. V. B.).

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CHAPTER
4
Developmental Anatomy of
the Female Reproductive Tract
Monika Jacob1 and Murray R. Bakst2
4.1 INTRODUCTION
In vertebrates, the reproductive system arises as bilateral anlagen, and
consequently, paired genital organs are commonly found in the adult. In
birds, the female reproductive system is unique. Although paired anlagen
appear, only the left genital primordia further develop to functional organs
except in birds of prey (order Ciconiiformes (=Falconiformes); families:
Cathartidae (American vultures), Accipitridae (kites, eagles, hawks, and
allies), Falconidae (caracaras and falcons). In cases of persisting right genital
organs, double oviducts are less frequently observed than double ovaries.
However, according to Kinsky (1971) they have also been found in other
orders than Falconiformes.
The reason for the unilateral development of female genital organs might be
to reduce weight for flying (see discussion by Gilbert, 1979). The question than
arises why the falconiforms allow themselves the luxury of two genital tracts.
To reduce weight their hard-shelled eggs are perhaps relatively small and are
laid down at an early stage of development. The weight of the egg in relation
to the maternal body weight in falconiforms varies from 2.75% in the White-
tailed eagle, Haliaeetus albicilla (Accipitridae) to 8.5 % in Falco (Falconidae) and
thus is at a lower level compared with non-falconiforms (Starck 1965). A
relatively long incubation time and a small number of eggs laid are also found
in falconiforms in comparison with other birds. Yet, these data do not yield an
entirely satisfactory explanation for either a single or a double female genital
tract. When ovulation occurs, the mature follicular oocyte is released from the
ovary and is received by the oviduct. This tract surrounds the ovum with the
albumen, the shell membranes, and the shell to form the characteristic avian
1
Abteilung für Anatomie und Embryologie, Ruhr-Universität Bochum, Bochum, Germany
2
Biotechnology and Germplasm Laboratory, ARS/USDA, Beltsville, Maryland 20705 USA
egg. In addition to forming and transporting the egg, the oviduct is the site of
sperm storage, sperm transport, fertilization and early embryonic
development. (See Chapter 11 for more details about the role of the oviduct in
reproduction.).
In this chapter we focus on the development and differentiation of the left
oviduct from the Müllerian (paramesonephric) duct and the concurrent
regression of the right Müllerian duct. We further present the macroanatomy,
histology and ultrastructure of the oviduct in hens. Data are mainly based on
studies of the domestic fowl, Gallus domesticus, since these birds have been
systematically studied. Only fundamental peculiarities of wild birds (as far as
known) are mentioned in this chapter. A link is made to the Wolffian duct
and the mesonephros, which form the reproductive tract in the male, but does
not further develop in females. Only remnants of these anlagen may be found
in hens.
4.2 EARLY MÜLLERIAN DUCT DEVELOPMENT

The Müllerian ducts, the anlagen of the female genital tracts in vertebrates,
arise in both sexes by inductive activity of the Wolffian (mesonephric) ducts.
The first sign of Müllerian duct development is a thickening of the coelomic
epithelium adjacent to each Wolffian duct in stage 19 HH chick embryos [HH
designates Hamburger and Hamiliton (1954) normal table of staging chicken
embryos]. This thickened epithelium forms the bilateral Müllerian ridge
(Fig. 4.1A-C) extending from cranial to caudal on the lateral side of the meso-
nephros.
Interestingly, nephrostome-like depressions are found within the cranial
part of both ridges (Fig. 4.1C, D). They are localized caudal and lateral to a
group of external (coelomic) glomeruli (Fig. 4.1A, C), which are well developed
even in the pronephros of higher vertebrates, including humans, and are
comparable to the glomeruli of the other kidney generations (Jacob et al. 1977,
1979, 1986). Peritoneal funnels of cranial mesonephric tubules are also found
adjacent to the external glomeruli (Jacob 1986; Hiruma and Nakamura 2003).
The nephrostome-like depressions at the lateral side of each cranial
Fig. 4.1 Gallus gallus, Fowl. A. Transverse section of the rostral part of the
Müllerian ridge (arrow) from a stage 23 HH (4 days) embryo. Scale bar = 50 µm.
WD, Wolffian duct; EG, external glomerulus B. Schematic drawings of serial
sections from the rostral part of the Müllerian ridge (arrows) adjacent to the Wolffian
duct (WD). Note the foveolae within the epithelium of the Müllerian ridge. Scale bar
= 100 µm. C. Scanning electron micrographs of the mesonephros (Mn) from a
stage 24 HH (4, 5 days) embryo. Irregularly arranged external glomeruli
(arrowheads) are found on the medial side of the mesonephros near the
mesenterium (Mt). Note the nephrostomal-like foveolae within the rostral part of the
Müllerian ridge. Scale bar = 100 µm. Go, Gonads; *, Müllerian ridge D. Detail from
the Müllerian ridge with nephrostomal-like openings. Scale bar = 10 µm. Original.
Developmental Anatomy of the Female Reproductive Tract #
Fig. 4.1
Fig. 4.2 Gallus gallus, Fowl. A. Scanning electron micrograph from a section
through the rostral part of the mesonephros (stage 26 HH, 5 days). The arrow
Fig. 4.2 Contd. ...

Developmental Anatomy of the Female Reproductive Tract #!
mesonephros represent only shallow epithelial invaginations from which

cells detach to form the anlage of the paired Müllerian duct (Fig. 4.1B).
According to Abdel-Malek (1950) they may considered as pronephrostomes
that have already shifted laterad while the pronephric tubules have already
degenerated. Similarly, in primitive vertebrates, like Ichthyophis
(Gymnophiona: Amphibia), pro-and mesonephric nephrostomial tubules are
found with an outer ciliated funnel at the lateral side (Semon 1890, 1892). The
fate of nephrostomal tubules was intensely studied by Wrobel and Süß (2000)
in larvae of the Gymnophiona (Ichthyophis kohtaoensis) as well as in bovine
embryos. Their comparative studies furnished evidence that the
nephrostomial tubules are involved in the formation of the Müllerian duct
infundibulum. It can be speculated that the lateral depression of the coelomic
epithelium in chick embryos might be a relicts of such an outer funnel, but the
complete evidence for this is lacking. Abdel-Malek (1950) has suggested that a
highly pronounced nephrostome at the posterior part of somite 17 eventually
forms the abdominal ostium of the oviduct in the chicken. However, the
caudal nephrostome-like depressions fuse and form the abdominal ostium of
the oviduct (Jacob et al. 1999, 2000). Such a “funnel field” is shown in Fig.
4.2A in a six-day chick embryo (stage 28 HH). One day later (stage 30 HH), a
deep oval ostium has formed (Fig. 4.2B), which is subdivided by a crest of
rounded cells presumably growing inwards to form the ostium. Therefore, the
old opinion which up to now is found in textbooks of embryology (e.g.
Hamilton 1965), that the cranial part of the Müllerian duct develops by fusion
of the lips of a longitudinal groove arising within the cephalic end of the tubal
ridge does not hold true for higher vertebrates, including birds. Our results
argue for an outgrowth of the Müllerian duct anlage from the funnel region
since at early developing stages, the abdominal aperture as well as the
Müllerian duct proper represent highly proliferating tissues as revealed by the
BrdU-anti BrdU reaction (Fig. 4.2C, D).
Fig. 4.2 Contd. ...

indicates the funnel region with several nephrostomal-like foveolae. On the cut
plane, the Wolffian duct (WD) and the anlage of the Müllerian duct (MD) are visible.
B. The same region in a stage 30 HH (7 days) embryo. One oval ostium with a
crest of cells has formed. C. and D. BrdU-anti-BrdU reaction through the funnel
region and a more caudal part of the MD, note the high proliferation within the MD
anlage (asterisks) as compared with the WD. E. Electron micrograph from the not
yet fully canalized part of the Müllerian duct (MA) in a stage 26 HH embryo. The
arrow indicates a nephrostome-like foveola. F. The tip of the Müllerian duct (MD)
with a group of densely packed mesenchymal cells from a stage 25 HH (4.5-5
days) embryo. G. At stage 28 HH (5,5-6 days) the caudal part of the MD is found in
close vicinity to the WD, but separated from the coelomic epithelium (CE) by a wide
acellular space. The wall of the WD, which is in contact with the Müllerian duct, is
double-layered and much thicker on the opposite side, both duct share a common
basal lamina. Scale bars in A, B. E and G = 10 µm, in C and D = 50 µm, and F =
2,5 µm. Original.
Fig. 4.3 Gallus gallus, Fowl. A-C stage 28 HH A. Scanning electron micrograph
from a section through the rostral part of the Müllerian duct (MD). A secondary duct
Fig. 4.3 Contd. ...

Developmental Anatomy of the Female Reproductive Tract ##
Thus, the Müllerian duct develops from a placodal-like funnel region,

which invaginates to form a pit. Here cells detach from the coelomic epithe-
lium and grow caudad (Fig. 4.2E). At first a solid cord of cells is established
(Fig. 4.2F). The caudal migrating end still preserves a mesenchymal, rod-like
configuration without a basal lamina. The cranial part transforms to a cana-
lized, epithelial duct (Fig. 4.2G), which gradually opens into the deepened
ostium.
A graphic reconstruction based on serial sections from a stage 28-chick
embryo has illustrated that caudal to the abdominal ostium additional
apertures appear at intervals of about 100 µm (Jacob et al. 1999). They are
connected with the main Müllerian duct by accessory ducts (Fig. 4.3A).
Balfour and Sedgwick (1878) already described multiple openings of the
cranial end of the Müllerian duct, and considered them as parts of a
rudimentary head kidney (pronephros) in the chicken. According to Hamilton
(1965), two or more coelomic apertures of the oviduct usually occur near the
ostium on the fifth day since the groove-like anterior part of the Müllerian duct
does not close uniformly. However, such a mechanism could not be observed
in our studies.
The Müllerian duct elongates very rapidly. At stage 28 HH it has a length
of about 2 mm. The caudal part is found in close vicinity to the Wolffian duct
and both ducts are enclosed by a common basal lamina (Fig. 4.3B, 4.2G). At
the same time, a broad space filled with a dense fibrillar matrix has been
established between the Müllerian duct and coelomic epithelium (Fig. 4.3C).
Because of their close relation, it has been speculated that cells from the
Wolffian duct contribute to the Müllerian anlage (see for discussion Didier,
1973). However, our own experimental and morphological data have clearly
shown that Wolffian duct cells do not invade the Müllerian duct epithelium.
Isotopic grafts of the tip of the Wolffian duct between chick and quail embryos
provided almost normally developed embryos. In such cases a normal chick-
derived Müllerian duct develops adjacent to the quail Wolffian duct, but no
quail (Wolffian duct) cells, possessing their characteristic distribution of
heterochromatin, are found within the Müllerian duct (Fig. 4.3D).
Fig. 4.3 Contd. ...
(2) is found beside the primary duct (1). B. The immunostaining of laminin exhibits
the common basal lamina between Wolffian duct (WD) and MD. C. The electron
micrograph reveals the dense fibrillar matrix between MD and coelomic epithelium.
D. Transverse section through a chimera with grafted quail WD at the place of the
chick duct. Feulgen staining demonstrates the quail nuclei within the WD
epithelium. The MD only contains only chick cells. Courtesy of Dr. H. J. Jacob. E
and F. Scanning electron micrograph from a transverse section (for technique see
Jacob et al. 1999) through the rostral part of the MD separated from the WD by
circular layer of mesenchyme. The detail shows a dense fibrillar matrix between
the mesenchymal cells. Scale bars in A, B, E = 10 µm, in C = 1 µm and D = 50 µm,
and F = 5 µm. Original.
Fig. 4.4 Gallus gallus, Fowl. A. Sagittal section through the mesonephos and
Müllerian duct (MD) of a stage 32 HH (7,5 days) embryo. In contrast to the Wolffian
Fig. 4.4 Contd. ...
Developmental Anatomy of the Female Reproductive Tract #%
Nevertheless, the developmental relationship between Wolffian and Müllerian

duct has teratological importance in all vertebrates. Gruenwald (1941), first,
delivered experimental evidence that destruction of the Wolffian duct results
in kidney and Müllerian duct aplasia. Likewise in Emx-2 and GATA-3
deficient mice with absent Wolffian ducts, Müllerian ducts fail to develop (see
Saino 2003).
During further development, a mesenchymal sheath surrounds the
epithelial duct (Fig. 4.3E). According to Didier (1973), and our own
observations, the mesenchyme of the Müllerian duct is formed by epithelio-
mesenchymal transformation of the pseudostratified epithelium of the
Müllerian ridge. The basal lamina ruptures, cells form pseudopodia and
detach from the epithelium to surround the epithelium of Müllerian duct.
Between the concentric layers of mesenchymal cells a dense extracellular
matrix is established (Fig. 4.3F).
Up to the eighth day (stage 33/34 HH), the development of the right and
left, female and male duct is identical. The Müllerian duct still runs parallel to
the Wolffian duct (Fig. 4.4A). But a dense mesenchymal tunic separates it from
the dilated Wolffian duct. The latter has a wide lumen, and is surrounded by
loose mesenchyme. The epithelium of the Müllerian duct has thickened and is
now pseudostratified (Fig. 4.4B). It is highly proliferative as indicated by many
mitotic figures. On the eighth day, the involution of the rostral part of the
mesonephros has started. Thus, the anlage of the ostium abdominale is now
found behind the lung anlage (Fig. 4.4C) and is attached to the mesonephros
by a double-layered fold (ligament) of serosa epithelium. The middle part of
the Müllerian duct projects over the lateral surface of the mesonephros,
surrounded by the serosa epithelium of the former Müllerian ridge, which has
now a flat appearance like the other parts of the splanchnopleure (Fig. 4.4D).
The data here presented have mainly been obtained from the order
Galliformes (species Gallus gallus and Coturnix coturnix japonica) but similar
developmental processes were also found in the order Anseriformes in the
Bean goose, Anser anser, and the domestic duck. A homology of avian
Müllerian duct development with that of amphibians was postulated by
Didier (1973). We believe that the early development of the paramesonephric
ducts is similar in all vertebrates including humans.
Fig. 4.4 Contd. ...
duct (WD), the MD is surrounded by dense mesenchyme. Scale bar = 100 µm.
B. Detail of the MD shows a pseudostratified epithelium with many mitotic figures.
Scale bar = 10 µm. C Oblique transverse section of a stage 34 HH (8 days) embryo.
Arrows, Müllerian duct; G, gonad; L, liver; Lu, lung; Ms, mesonephros; St, stomach.
The funnel region of the MD lies dorsal to the lung anlage. Scale bar = 0.5 mm.
D. Detail of the left MD. Scale bar = 10 µm. Original.
4.3 REGRESSION OF THE RIGHT MÜLLERIAN DUCT AND

DIFFERENTIATION OF THE LEFT ONE
The early development of the two Müllerian (paramesonephric) ducts is
identical in both sexes. At day seven they reach the cloaca with the compact
distal end, but there is no opening into the cloaca prior to hatching. An
occluding plate between oviduct and cloaca even persists up to the first
breeding season (from Gilbert, 1979). In the male embryo, both Müllerian
ducts, and in the female embryo, the right Müllerian duct undergos regression
starting at the eighth day of development. According to Lutz-Ostertag (1954)
the regression of the male duct occurs in two phases: first, the epithelium
undergoes cell death; and second, the ducts are transformed into dense
mesenchyme.
The process of regression is obviously highly conserved in vertebrates as
has been described in morphological studies in reptilians (Austin 1989, 1995),
birds (Forsberg and Olivecrona 1963) and mammals (Dyche 1979; Trelstad et
al. 1982; Wartenberg 1985). In male embryos apoptosis appears to be the main
cellular mechanism in Müllerian duct regression.
The central molecule in the apoptotic machinery is the anti-Müllerian
hormone (AMH) also named Müllerian-inhibiting substance (MIS) that is a
member of the transforming growth factor-beta (TGF-beta) superfamily (see
Behringer 1995, Visser 2003, Rey et al. 2003) (see also Chapter 12). Like other
TGF-beta family members, AMH appears to signal through two
transmembrane receptors, both serine/threonine kinases. The AMH type II
receptor, which has been clearly defined, binds to the ligand, and, it is
proposed, triggers the formation of a complex with an assumed type 1 receptor
ALK2 (activin-like kinase). The signaling pathway to activate the
proapoptotic gene probably uses cytoplasmatic Smads (mothers against
decapentaplegic related gene product) proteins (Clarke et al. 2001).
The AMH type II receptor is exclusively expressed in the mesenchymal
sheath surrounding the epithelial lining of the Müllerian duct, whereas
apoptotic cells are localized within the epithelium (Baarends et al. 1994;
Roberts et al. 1999). Thus, AMH functions indirectly via a paracrine
mechanism.
One of the first morphological features in Müllerian duct regression is the
loss of the basal lamina (Trelstad et al. 1982) and the condensation of the
mesenchymal sheath (Wartenberg 1985). These processes start in the male
chick embryo around the eighth day of development. The formation of an
“epithelial cuff” by the condensed mesenchyme is shown in Fig. 4.5C in a 9-
day-old embryo. As compared with the female ducts (Fig. 4.5A, B) the lumen
has narrowed, and the diameter of the duct is decreased. Apoptotic bodies
appear within the epithelium. Figure 4.5D from an 11.5-day-old male embryo
reveals that the epithelium has disappeared. Only dense mesenchyme mark
the former location of the Müllerian duct.
The molecular and cellular events during Müllerian duct regression were
demonstrated by Allard et al. (2000) including apoptosis and epithelial-
Developmental Anatomy of the Female Reproductive Tract #'
Fig. 4.5 Gallus gallus, Fowl. A. Transverse section through the right Müllerian duct
(MD) of a stage 36 HH (10 days) female embryo at the level of the gonad without
sign of regeression. B. Section of the left MD at stage 33/34 HH (8 days) at the
same level revealing many mitotic figure in the epithelium. C Transverse section of
the right MD of a stage 35 HH (9 days) male embryo. The diameter is smaller than
that of a comparable duct in the female. Note also the narrow lumen and the
apoptotic bodies. D. At stage 37/38 HH (11, 5 days) the MD (here the left side) is
replaced by dense mesenchyme. Scale bars = 25 µm. Original.
mesenchymal transformation. b-catenin, in association with lymphoid

enhancer factor 1, is suggested to be involved in this process. Furthermore,
Roberts et al. (2002) propose potential roles of the matrix metalloprotease
MMP2 in mediating duct regression. Not all cells of the regressing Müllerian
duct undergo apoptosis. Viable cells transform into mesenchyme and are able
to migrate into other locations such as the mesonephric tubules (Hutson et al.
1984; Austin 1995).
The mechanisms leading to the regression of the right Müllerian duct in
female chick embryos are probably quite similar to those in male duct
regression with AMH as a trigger. While in mammals, AMH is synthesized
only in Sertoli cells immediately after testicular differentiation and in ovarian
granulosa cells after birth, the situation is quite different in birds. Prior to
14 days of development, AMH is produced in the ovary. The left ovary
synthesizes AMH at a level similar to that seen in the testis (Hutson et al.
1981). Its maximum level is at 14 days of development, latter than the testes,
Fig. 4.6 Gallus gallus, Fowl. Schematic drawing to show the differentiation of the
left and the regression of the right MD. A. Stage 34 HH (8 days). Both MD are
equally formed. B. Stage 37 HH (11 days). The regression of the right MD has
Fig. 4.6 Contd. ...

Developmental Anatomy of the Female Reproductive Tract $
but at a time when the right ovary (ovotestis) is drastically reduced in size
(Teng 1987). It is this peak in AMH synthesis by the left ovary that may
account for regression of the right ovary. Similarly, chick AMH mRNA
expression in the ovary was found to peak at the seventeenth day but in a
lower amount than in the testis (Carré-Eusèbe et al. 1996).
In mammals, the onset of AMH expression depends on SOX9 -a member of
the SRY gene family. However, in the chick embryo AMH is expressed in
males one day before SOX9 transcripts appear and SOX9 is never expressed
in females (Oreal et al. 1998). Furthermore, the chicken differs from mammals
in that AMH is expressed in dispersed medullary cells of the indifferent gonad
(Oreal et al. 2002). With the early differentiation of the left ovary AMH
concentrates in the outer medullary zone whereas a dispersed expression is
preserved in the right ovary. Since AMH expression in indifferent and female
gonads is not correlated with factors as in testis or follicles, Oreal et al. (2002)
and Lasala et al. (2004) postulated a bird-specific control mechanism of AMH
transcription.
The apoptotic pathway in the female right Müllerian duct is described as
caspase-3 mediated (Teng 2000) and shows the typical DNA laddering due to
internucleosomal fragmentation. Recently, Ha et al. (2004) have shown that
MMP2 (matrixmetalloprotease) mRNA of the right female Müllerian duct was
significantly higher than on the left side at days 15 to 18 of incubation
coinciding with the time of regression, and that diethylstilbestrol could
decrease MMP2 expression.
The female left Müllerian duct and even its right duct (Teng 1987) are much
more resistant to AMH than the male ducts, and it is therefore obvious that a
special protection exist. Many studies have shown that estrogenic hormones
prevent the induction of left duct regression (Wolff, Et 1939; Hutson et al. 1982,
1985). While estrogen is synthesized in both male and female gonads in chick
embryos (Woods and Erton 1978), it is much higher in the ovary than in the
testis especially on the twelfth to the fifteenth day of incubation (Guichard et
al. 1977). Furthermore, nuclear estrogen receptors were found to be higher in
the female left Müllerian duct (MacLaughlin et al. 1983).
The development of the Müllerian duct has been intensively studied by
Lutz-Ostertag (1954). Figure 4.6 is based upon her data. In the 8-day-old
Fig. 4.6 Contd. ...
started cranially. The right ovary is already smaller than the left. The mesonephros
is well developed, and both Wolffian ducts (WD) are functional. C. Stage 40 HH
(14 days) with regression of the cranial part of the right MD. D. Stage 45 HH
(20 days). On the left side, dilatation of the caudal MD. On the right side,
rudimentary duct without ostium abdominale. Reduced size of mesonephros, but
the WDs are still present. E. Adult: The different parts of the oviduct have developed
on the left side. On the right side, small appendix of rudimentary duct. The
metanephros with upper, middle, and lower lobes is formed. The mesonepros is
involuted. Only rudiments of the WD persist. Drawing after Lutz-Ostertag 1954
made by K. Barteczko.
embryo (Fig. 4.6A), both ducts have equal length. The mesonephroi are well
developed with both gonads symmetrically positioned at their medial sides.
The first difference in length is seen in the 10-day-old embryo and becomes
clearly visible in the 11-day-old embryo (Fig. 4.6B). While the left gonad has
grown, the right gonad began to regress.
After 14 days of incubation (Fig. 4.6C) the size of the right ovary is
significantly reduced and the right Müllerian duct has regressed nearly
completely with only its caudal part evident. But during the period of
regression the abdominal ostium is suggested to remain open (Lutz-Ostertag
1954). The mesonephros is still functioning although some nephrons are
involuted (Volle and Beaumont 1964), and both Wolffian ducts are well
developed.
Prior to hatching (Fig. 4.6D), the right oviduct is still rudimentary with a
length of about 4.5 mm while the left one has grown steadily and has
differentiated into several regions. The future shell gland (uterus) can be
distinguished as a dilatation of the tract near its distal end. The mesonephroi
are reduced in size with a big left and a small right ovary attached on the
ventral and medial side. The Wolffian ducts are likewise preserved.
Histological examination of the oviductal mucosa shortly after hatching
(Fig. 4.7A-C) shows well-developed primary folds with secondary folding
starting. Cell strands of the dense lamina propria contact the basal lamina.
They appear to help in glandular formation. The nonciliated epithelium is
irregular and consists of columnar or pseudostratified cells. Berg et al. (2001)
demonstrated that exposure of female quail embryos to ethynyloestradiol
induces precocious differentiation of the epithelium and tubular glands in
immature birds
In the adult chicken (Fig. 4.6E), the mesonephros is replaced by the
metanephros. Rudiments of the Wolffian ducts might persist. The left ovary
lies adjacent to the upper part of the kidney (metanephros) and ventral to the
aorta. It has an irregular shape resulting from different growth of follicles. The
left oviduct, which is straight and small before sexual maturity, increases
enormously in length and in diameter in the breeding season. The oviduct
differentiates into five morphologically distinct regions: infundibulum,
magnum, isthmus, the prominent shell gland, and vagina. The caudal part,
the vagina is separated from the cloaca proper by the occluding plate, which
is ruptured after mating or at the onset of egg production. On the right side a
rudimentary Müllerian duct persist as an appendix of the cloaca.
The normal growth of the right and left female Müllerian duct is
summarized in Table 4.1. The data are adapted from Teng (1987) and Lutz-
Ostertag (1954). The latter are measured in White Leghorn embryos. The
differences in absolute values may be due to different methods or different
strains. However the ratios are similar.
An overview of the development of the oviduct in duck embryos was
likewise presented by Lutz-Ostertag (1954). Up to day 9, the female Müllerian
ducts are of equal length. The regression of the right duct starts in day-10
Developmental Anatomy of the Female Reproductive Tract $!
Fig. 4.7 A. Longitudinal section of the left oviduct from a one-day-old chicken. The
future magnum is dilated. The mucosa reveals primary folds with a dense lamina
propria. B. Detail from a well-developed primary fold with secondary ones that
appear leaf-like. C. The luminal epithelium is columnar and non-ciliated. Cells of
the dense stroma attach the basal lamina. D. Transverse section through the distal
region of the right oviduct and the rudimentary Wolffian duct (*) (WD) between ureter
(U) and oviduct (O). The undifferentiated oviduct reveals a columnar epithelium with
a thick basal lamina surrounded by a dense mesenchymal layer. E. Detail of the
WD to show the well developed columnar epithelium and the small mesenchymal
layer. Scale bars: A, B, D = 100 mm, C and E = 25 mm. Original.
Table 4.1 The normal growth of Müllerian duct (MD) in female chick embryos
Days of Length of left MD Length of the right MD

incubation (mm) (mm)
6 3.8 3.8
8 5.3 5.3
(7.5) (7.5)
10 7.5 7.5
(10.6) (9.6)
11 9.0 7.8
(11.5) (8.7)
14 104 6.4
(17.9) (4.8)
16 19.2 4.7
(21.5) (4.6)
21 22.2 4.4
(28.0) (7.5)
The data are taken from Teng (1987) and, in parenthesis, Lutz Ostertag (1954)
embryos in the same manner as in the chick embryo. Apoptotic bodies are
visible reaching their maximum during days 14 and 15. The regression is
terminated at the nineteenth day of incubation with a rudiment similar in
appearance to that observed in the chick.
4.4 THE RIGHT MÜLLERIAN DUCT AFTER HATCHING

While the left oviduct is a length of 22 or 28 mm respectively (see Table 4.1) at
the time of hatching, the right oviduct is rudimentary with a length of 4 to
9.3 mm according to authors such as Benoit 1950 (cited by Hlozankova and
Zalenka 1978), Lutz-Ostertag 1954; Romanoff 1960; Teng 1987. Figure 4.7D
shows a persistent right oviduct shortly after hatching. It is poorly
differentiated with a columnar epithelium, a thick basal lamina, and a dense
mesenchymal sheath.
Observing 63 female chicks (hybrid combination Ross 1), Hlozankova and
Zelenka (1978) found a rudimentary right oviduct in all animals. Their length
and width increase in relation to the growing hen. At 15 months, their average
length was of 93.1 mm and the diameter had enlarged three-fold (see Table
4.2). However, there was also significant variation in shape and size. Two
narrowings separate an ampulla, which could be compared with the shell
gland, from the caudal retroperitoneal part and from a thin cranial cap-like
Table 4.2 Size of the right Müllerian duct (MD) after Hlozankova and Zelenka (1978)
Age in days 22 36 57 450

Average length (mm) 17.3 23.7 24.8 93.1
Average width (mm) 4.6 7.1 6.9 13.8
Developmental Anatomy of the Female Reproductive Tract $#
part that extends into the meso (ligament). In some hens this upper part was
separated from the ampulla by connective tissue. The rudiments are always
filled with a clear liquid and in some cases they have dilated to a cystic
structure. Interestingly, when the occluding plate is ruptured there is a clear
fluid released from the vagina (unpublished observations, MB).
Sell (1959) found well-developed right oviducts in 80% of the White
Plymouth Rock hens examined. They possessed all regions of a normal
oviduct, but in all cases the infundibulum was shorter than normal, and eggs
were only found in the left oviduct. The incidence of a fully functional
persistent right oviduct is described in White Leghorn by Bickford (1965), and
in the ring necked pheasant by Purohit et al. (1977).
4.5 RUDIMENTS OF MESONEPHROS AND WOLFFIAN DUCT IN

FEMALES AFTER HATCHING
The mesonephros is functional until 3 days before hatching (Wendler 1965).
The regression of mesonephric tubules continues up to the 3rd or 4th week
after hatching (Budras 1972), but rudiments persist even in old adults. This
has been described in detail by Kummerlöwe (1931) for many wild birds such
as Turdus and Accipiter. Budras (1972) intensely investigated the remnants of
mesonephros in Gallus. He always found cranial tubules (epoophoron) located
between adrenal gland and ovary and some of them are supposed to grow
into these adjacent organs. Inside the capsule of the adrenal gland and at the
border between ovarian medulla and cortex they transform into interrenal
(adrenal cortical-like) and interstitial nodules, respectively. The epoophoron
seems to be an additional source of sex-steroid producing cells similar to the
corpus luteum of mammals (Budras 1972). The pathological significance of
epoophoron-derived tumors with virilisation of hens has been described by
Boring and Pearl (1918).
Because of the close relationship between the mesonephros and gonads,
several authors (Witschi 1935, 1956, cited by Carlon et al. 1983; Carlon et al.
1983) claimed that, as in other vertebrates, the part of the chick embryo’s
mesonephros which does not form nephrons contributes to the medullar cells
of the gonads. Rodemer et al. (1986), using chick-quail chimeras, showed that
from the third to the sixth segment, the ventromedial part of the differentiating
mesonephros participates in the formation of stromal cells to the gonads.
The mesonephric (Wolffian) ducts persist in female mammals as Gartner’s
ducts and can be the source of hydatids of Morgagni (Motta 1966; Jacob and
Barteczko 2005) or cysts. Glandular acini opening in Gartner’s duct were also
found in the camel (Shehata 1978), and are discussed as accessory glands of
the female genital tract. In female birds rudimentary Wolffian ducts persist as
permanent structures running parallel with the ureter within the dorsal
ligament of the oviduct. King (1975) stated that the adult hen possesses on the
right side a potentially viable and complete system of the male duct including
the rete, the epoophoron and the mesonephric duct, while the fate of the left
duct remains uncertain. In contrast to Domm (1927), Brode (1928) and Kar
(1947) (all cited by King 1975) concluded that the left mesonephros and duct
involuted nearly completely before hatching. We found in the one day-old
female chicken (Fig. 4.7D, E) on both sides a patent Wolffian duct with a
narrow lumen surrounded by a cuboidal or columnar epithelium and some
layers of mesenchymal cells. At the beginning of the breeding season the
Wolffian ducts respond to androgenic hormones produced by the female and
might be prominent in many avian species (for references see Gilbert 1979).
Yet, no information is available about the functional significance of such
gland-like structures.
4.6 STRUCTURE OF THE HENS OVIDUCT DURING EGG PRODUCTION

4.6.1 Overview
The oviduct in mature birds in egg production is a relatively large tubular
organ that has three primary functions all within the realm of reproduction.
These include the following: egg formation; sperm selection, storage and
transport; and, fertilization and early embryonic development. Briefly, at
ovulation the fimbriated region of the infundibulum guides the ovum to the
ostium abdominale, the opening of the oviduct. Then within minutes, sperm,
if present, make contact with the inner perivitelline layer (IPVL) initiating the
process of fertilization. Whether fertilized or not, the ovum accrues secretory
material released from the infundibular mucosa forming the thin continuous
layer and the more extensive outer perivitelline layer (OPVL). Within 15-
20 min, the ovum, now referred to as an egg-mass, enters the magnum, which
is the largest of the five oviductal regions. The egg albumen proteins are
synthesized in the magnal tubular glands and secreted (see Burley and
Vadehra, 1989 for a comprehensive review on albumen composition and
chemistry). After about 3 hr, the albumenous egg mass enters the isthmus for
75-90 min where the inner and outer shell membranes are formed. The egg
mass then reaches the uterus (shell gland) and during the next 18-20 hr the
calcareous shell is deposited and there is a movement of “plumping” fluid
(uterine transudate) into the egg mass. At oviposition the hard-shelled egg
mass is expelled through the vagina, which serves as a conduit between the
uterus and cloaca. The vagina also plays a vital role in sperm selection,
transport and storage. If fertilized, and depending on the species, the egg at
oviposition contains a blastoderm with 20,000 to 60,000 cells. The time from
ovulation to oviposition is referred to as the “ovulatory cycle” and in domestic
birds it is about 23-25 hr. These events are summarized in Fig. 4.8 and
presented in greater detail in Chapter 11 of this volume.
To more fully understand and appreciate these functions, a comprehensive
understanding of the tissue and cell structures and their interrelationships is
necessary. The descriptions provided will focus on the anatomy, histology
and to a lesser extent the ultrastructure of each oviductal segment. Unless
otherwise stated, the description will be that of the commericial turkey. For
CMYK
Developmental Anatomy of the Female Reproductive Tract $%

CMYK
CMYK
Fig. 4.8 Drawings illustrating formation of the hard-shelled egg in Gallus gallus
and the segments of the left oviduct corresponding to the different stages of the
ovulatory cycle. A. Length of the segment in mm and schedule of the ovulatory cycle
in minutes. B. Alteration of the egg mass during passage along the oviduct.
C. Segments of the oviduct with transverse sections (D) and details of the mucosa
(E). a, left ovary; b, infundibulum; c, funnel-like fimbriated region of the infundibulum
showing an ovum (t) entering the ostium abdominale; d, tubular neck region of
infundibulum; e, magnum; f, isthmus with pars translucens (g) and the tubular
shell gland (h); I, uterus (shell gland); k, vagina; u, continuous and outer
perivitelline layer; v, albumen; x, shell membrane; z, shell. (From: Komarek V,
Malinovsky L, Lemez L: 1982. Anatomia avium domesticarum et embryologia galli.
Part 2. Priroda, Bratislava,Tab. LVI, with permission of the publishers).
CMYK
more detail discussion on the overall anatomy of the oviduct refer to Aitken
(1971), Hodges, (1974), and King (1975). Anatomical nomenclature will follow
that suggested in Handbook of Avian Anatomy: Nomina Anatomica Avium
(Baumel, 1993).
4.6.2 Infundibulum
The infundibulum is the most anterior segment of the oviduct. It is divided
into a funnel like region, the fimbria (also referred to as the ampulla), which
grasps the ovulated ovum and guides it into the ostium abdominale, and the
more elongated, tubular neck region (also referred to as the chalaziferous
region). The neck gradually merges with the proximal segment of the magnum.
If fertilization is to take place, sperm must be present in the funnel or upper
neck region.
The surface mucosa is thrown into a series of primary and smaller second-
ary folds clearly more voluminous and longitudinally orientated at the neck
region than at the fimbria, which are shorter and more randomly orientated
(Bakst and Howarth 1975). The mucosal folds in the neck region begin to form
widely scattered subepithelial tubular glands just caudal to the fimbria (Fig.
4.9A, B). Moving caudally into the neck region the tubular glands proliferate
into dense clusters and eventually mix with clusters of the larger tubular
glands characteristic of the proximal magnum (Fig. 4.10A, B). Secretions de-
rived from the infundibular tubular glands contribute to the formation of the
continuous layer and OPVL. Together, these tertiary investments (formed by
oviducal secretions) envelop and add strength to the IPVL as well as serving
as a block to pathological polyspermy (see Chapter 11).
The pseudostratified columnar epithelium lining the mucosa surface at the
fimbriated region is composed of a dense array of ciliated cells that more
caudally give rise to alternating ciliated and secretory cells in the neck (Fig.
4.10B). At the base of some neck folds, nonciliated cuboidal predominate and
in cross section appear as ‘glandular grooves’. When these folds are apposed
to an egg mass the nonciliated cells forming the glandular grooves are
exteriorized and clearly visible (Fig. 4.9A).
True tubular glands are observed throughout the neck region and resemble
the tubular glands of the the magnum and isthmus (Fig. 4.10A, B). Structurally
the tubular glands of the neck region are compound multicellular glands. The
spherical secretory granules densely packed in the apical half of the secretory
Fig. 4.9 A. When apposed to an ovum, the mucosa of the midregion of the
infundibulum becomes distended exposing its nonciliated basal surface. An
opening to a tubular gland is observed (arrow) in this scanning electron
micrograph. After Bakst, M.R. 1978. Poult. Sci. 57: 1065-1069. Fig. 2. B. A squash
preparation of unfixed infundibular mucosa from the same region as Fig. 4.9A is
viewed by differential interference contrast (DIC) microscopy. With the plane of the
optical section just subjacent to the surface epithelium portions of several tubular
glands (arrows) are observed among the capillaries in the loose connective tissue.
After Bakst, M.R.1994. Biology of Reproduction 50: 987-992, Fig. 5.
Developmental Anatomy of the Female Reproductive Tract $'
Fig. 4.9
Fig. 4.10 A. A squash preparation of unfixed infundibular mucosa from caudal

neck region is viewed by DIC microscopy. Secretory granules are abundant only in
the supra-nuclear cytoplasm of the secretory cells permitting an unobstructed view
Developmental Anatomy of the Female Reproductive Tract %
cells when secreted presumably are the source of the tertiary investment
around the ovum.
It has long been speculated that the infundibulum is a secondary sperm
storage site (see Bakst et al., 1994 for review) but this role has been recently
questioned (see Chapter 11). Regardless of the role of the infundibulum in
sperm storage, it is the site of fertilization. To be successful, sperm must find
their way to the 3 mm diameter germinal disc at the surface of the ovum,
interact with the appropriate sperm receptors on the IPVL surface before the
OPLV is formed.
4.6.3 Magnum
In egg production the magnum is responsible for the synthesis and secretion
of the albumen proteins (see Burley and Vadehra 1989 for a discussion on
albumen chemistry). Visually, the longitudinally orientated folds are high,
voluminous and ivory in color prior to the passage of an egg mass.
Immediately after passage, the folds appear somewhat diminished in size and
have a pale reddish-brownish coloration. This volume change is due to the
depletion of secretory material from the tubular glands. However, full volume
and the ivory coloration of the folds are replenished prior to the next
ovulation.
The pseudostratified columnar epithelium lining the mucosa surface
contains a near equal distribution of non-ciliated secretory and ciliated cells.
Using transmission electron microscopy (TEM), its clear that the non-ciliated
secretory cells are mucin secreting cells, reminescent of goblet cells (Blom
1973; Sandoz et al. 1976). Tubular glands are prominent in sections of the
magnum and clearly present at all levels of the folds. Like the neck of the
infundibulum, the tubular glands are compound multicellular glands lacking
a transition between the tubular gland and surface epithelium (see Blom
1973). Toward the distal 2-3 cm of the magnum, the mucosal folds appear less
voluminous and the goblet cells in the surface epithelium appear to be the
dominant cell type. This is sometimes referred to as the mucous part of the
magnum. The transition between the magum and isthmus is abrupt and easily
recognized by eye as a 1-3 mm wide, nearly translucent band, the pars
translucens.
4.6.4 Isthmus
Slightly shorter and smaller in diameter than the magnum, the isthmus
synthesizes and secretes an array of proteins and glycoproteins that form the

of the nuclei (arrow). B. From the same region as Fig. 4.10A, a section containing
the ciliated surface epithelium (C) and several tubular glands are observed.
Secretory granules are abundant only in the supra-nuclear cytoplasm of the
secretory cells permitting an unobstructed view of the nuclei (arrows). Original.
shell membranes. Given this mix, Burley and Vadehra (1989) point out that it
was incorrect to refer to the shell membranes are being composed of keratins.
The longitudinally orientated mucosal folds are somewhat smaller than the
folds of the magnum and contain tubular glands endowed with a dense array
of secretory granules. Like the magnum, the surface epithelium is composed of
both non-ciliated secretory cells and ciliated cells arranged as a
pseudostratified columnar epithelium. Interestingly, the openings of the
tubular glands are slightly larger than those observed in the magnum and by
SEM appear as dimples (Bakst and Howarth 1975) or shallow depressions
(Blom 1973) distributed on the mucosal surface.
There was disgreement in the literature regarding the caudal aspect of the
isthmus and whether it should be considered part of the isthmus or uterus
(Fig. 4.11A). The mucosal folds of the tubular shell gland resembles that of the
isthmus. However, the tubular glands may (Richardson 1935) or may not
(Aitkin 1971) differ from the uterine pouch. Solomon (2002) reviewed the
arguments and suggested that the term “tubular shell gland” as it was
commonly used. This term concurs with that found in Nomina Anatomica
Avium (Baumel 1979) “Pars cranialis uteri” suggesting that the tubular segment
caudal to the isthmus is the cranial aspect of the uterus.
4.6.5 Uterus
Also referred to as the shell gland, the uterus has two distinct regions, the
more cranial tubular shell gland (see previous paragraph), and the pouch-like
portion of the uterus. Less distinct is the recessus uterinus, described as a funnel
shaped, greyish white color at the caudal end of the uterus (Baumel 1979). Its
mucosa appears to be more a transitional zone between the uterovaginal
junction (UVJ), the latter being the most cranial aspect of the vagina defined
by the presence of sperm storage tubules (SST). These structures are best
appreciated when the recessus uterinus, UVJ, and cranial half of the vagina are
collectively dissected free of connective tissue and laid straight (Fig. 4.11A).
Each region becomes clearly visible after they are liberated from the
enveloping connective tissue.
The mucosa folds are not clearly longitudinal but form leaf-like structures
that seem to be arranged more circumferentially. This circumferential
orientation is lost as the connective tissue band surrounding the pouch is
dissected away. The pseudostratified columnar epithelium contains nearly an
equal distribution of non-ciliated secretory cells and ciliated cells. The tubular
glands of the uterine pouch are less voluminous than in the isthmus or
magnum.
4.6.6 Vagina
The most caudal segment of the oviduct, the vagina, is coiled and folded and
held in that complex configuration by a dense covering of connective tissue
(Fig. 4.11B). This same connective tissue also binds the coprodeum, the most
cranial compartment of the cloaca, in juxtaposition with the vagina. It should
Developmental Anatomy of the Female Reproductive Tract %!
Fig. 4.11 A. After isolating and stripping off the connective tissue around the uterus
and vagina, the recessus uterinus (oval) and extended vagina are observed. Also
observed is the Pars cranialis uteri, the tubular shell gland (box). The uterovaginal
junction is just caudal to the recessus uterinus, and is defined by the presence of
sperm-storage tubules (SST). After Bakst, M.R. 1998. J. Exp. Zool. 282: 618-626,
Fig. 4. B. The vagina (encircled) has been cut free of the connective tissue that
envelops it with the coprodeum (upper arrow). However, the dense connective
tissue still keeps the vagina as a tightly coiled structure. The lower arrow highlights
the ostium cloacale oviductus sinistri (Baumel 1993), the opening of the left oviduct.
After Bakst, M.R. 1993. Chapter 2, Manipulation of the Avian Genome, Fig. 4.
Fig. 4.12 A. A squash preparation of unfixed UVJ mucosa viewed by DIC

microscopy showing the midregion of a sperm storage tubule containing a few
sperm. Basal nuclei (arrows) and more apical lipid droplets characterize this
simple columnar epithelium. After Bakst, M.R. 1993. Chapter 2, Manipulation of the
Avian Genome, Fig. 10. B. The complexity of the mucosal folds in the distal vaginal
is observed in this section. Note where the folds are tightly apposed the lumen is
not discernible (arrow). Original.
Developmental Anatomy of the Female Reproductive Tract %#
be noted that with the removal of the connective tissue binding the distal
uterus, vagina and coprodeum and subsequent straightening of the vagina, at
no time was there observed a thickening of the Tunica muscularis (MB, personal
observations). Thus, the existence of a vaginal sphincter around the cranial
aspect of the vagina analogous to the mammalian cervix (Baumel 1979)
should be questioned.
About 24 nearly parallel primary folds are longitudinally orientated and
are further subdivided into secondary and tertiary folds. The complexity of the
mucosal folds is best appreciated in histological sections (Fig. 4.12B). Tightly
apposed folds with seemingly interdigitating ciliated surfaces were often
observed with one or more sperm between the cilia. By SEM, the mucosa
surface appears to be densely ciliated although nonciliated secretory cells are
also observed in its pseudostratified columnar epithelium. At the juncture of
the distal vagina with the urodeum, the central zone of the cloaca in which
the urogenital ducts empty, the ciliated epithelium of the vagina ends rather
abruptly and is replaced with the secretory (mucin) cells of the urodeum.
Unlike the other oviductal segments, the vaginal segment does not possess
tubular glands. However, the UVJ at the cranial end of the vagina is defined
by the presence of SST (Fig. 4.12A) These differ from the tubular glands seen
elsewhere in the oviduct as the SST are composed of simple columnar non-
secretory cells characterized by a basal nuclei and an accumulation of
intracellular lipid. A more detailed account of SST structure and function is
found in Chapter 11. The development of the SST was recently examined by
Holm and Ridderstrale (2002) in quail and found to coincide with the
development of the oviduct at maturation. Sperm-storage tubule numbers,
spatial distribution, and morphology (Birkhead and Moller 1998) as well as
the spatial and temporal patterns of sperm filling and emptying (Briskie 1996;
Bakst and Vinyard 2002) appear to vary between and within species.

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n n
CHAPTER
5
Endocrinology of Reproduction
G.E. Bentley1, K. Tsutsui2 and J.C. Wingfield3
5.1 INTRODUCTION
Birds reproduce in virtually all habitats on earth from the poles to the equator
and alpine meadows to the driest deserts. Because all avian species are
oviparous, they are restricted to land for breeding purposes. Thus, the only
major ecosystem in which birds are unable to nest is the ocean. Nonetheless,
marine habitats provide rich trophic resources for birds nesting on islands
and coastal areas. Restriction of the reproductive process to the oviparous
mode might suggest that birds have an equally restricted hormone control
system. Compared with other vertebrate taxa this may be true but the
complexity of avian breeding cycles and the broad spectrum of habitats in
which they nest indicates great flexibility in these endocrine control systems.
We approach the hormonal control systems of the class Aves in four major
parts: 1) types of environmental signals that influence reproduction and how
they are perceived; 2) the hypothalamus as an integrator of environmental
information from the external and internal environments, biological clock, etc.,
that through neuroendocrine and neural secretions affects all aspects of repro-
duction; 3) the hypothalamo-pituitary unit that transduces environmental
information processed by higher centers into endocrine secretions; 4) the func-
tional gonads (ovary and testis) themselves. We do not provide much detail
on the gonadal histology as this topic is covered in chapters 2, 4 and 7 in this
book. This is an exciting era in neuroendocrinology as new peptides discov-
ered in recent years provide even more flexibility in control mechanisms. This
is timely in an age when global climate change, human disturbance and
pollution require organisms to be even more flexible in how they cope with
1
Department of Integrative Biology, University of California, Berkeley, California 94720,
USA.
2
Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-
8521, Japan.
3
Department of Biology, University of Washington, Box 351800, Seattle, Washington,
98195, USA.
such events. We hope that this review will focus new attempts to explore the
breeding strategies and cycles of vertebrates in general.
5.2 ENVIRONMENTAL SIGNALS AFFECTING REPRODUCTION

Environmental signals that are indicative of transitions of life history stages
(such as breeding) required in the near or immediate future can be used to
trigger appropriate responses of the individual (Wingfield et al. 1999;
Wingfield 2004). In other words, environmental signals can be used to:
(a) Orchestrate development (ontogeny) of organisms and respond to
environmental changes that require adjustment of developmental
trajectories. Maternal effects are also important at this time.
(b) Regulate development, maintenance and regression of morphological,
physiological and behavioral traits characteristic of life history stages
(e.g. migration, breeding, molt) throughout the adult life cycle.
(c) Regulate when adjustments of homeostasis are to be made in advance of
predictable changes in the environment.
(d) Signal when alternate behavioral and physiological patterns should be
triggered in response to unpredictable and disruptive events.
(e) Adjust social interactions to maximize an individual’s ability to gain
access to shelter, food resources, territories, mates etc.
There is a complex cascade of events involved in the perception, integration
and transduction of environmental signals into chemical signals (e.g.
hormones, neurotransmitters) that then initiate appropriate morphological,
physiological and behavioral adjustments (Ball 1993). There are five major
processes involved:
1. Specialized receptors for specific external environmental signals that
then transduce them into neural events.
2. Neural pathways from the specialized receptors project to target areas in
the brain that integrate the environmental information in relation to
internal physiological state (biological clock, social status; nutritional
state, disease etc.).
3. These target areas of the brain then signal specific neurons in the
hypothalamus to release neurosecretions that initiate a hormonal
cascade that in turn triggers appropriate adjustments of morphology,
physiology and behavior.
4. Neurosecretions and enzymes within the CNS can have separate
regulatory effects on neurosecretions from the hypothalamus into the
blood circulation. Note also that internal signals (from the biological
clock, social status; nutritional state, disease etc.) may also act at the
level of neurosecretions within the CNS.
5. Peripheral, blood borne, hormones (especially steroids) may feedback to
act on the brain and alter responsiveness to environmental signals at
either the specialized receptor, target brain area, or neurosecretory
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neuron levels. Again, these effects may be further regulated by the

biological clock, social status; nutritional state, disease etc.
It should also be borne in mind that possible neural pathways for
environmental signals may be very different among the types of signal (e.g.
food, temperature, social etc., Fig. 5.1). Furthermore, these neural pathways
may not always be stimulatory. Some may inhibit neurosecretion, others may
release an inhibition rather than stimulate directly. Clearly an almost limitless
Fig. 5.1 Proposed pathways of transduction for proximate information that may
regulate reproductive function. In homeothermic vertebrates such as birds, direct
effects of environmental cues on peripheral endocrine cells are not common (an
example is the effect of gut contents on the hormone secreting cells of the gastric
mucosa, Norris 1997). Most environmental information from the physical
environment, social interactions and internal cues are transduced through the
hypothalamo-pituitary unit. Note that some environmental cues may be signalized
directly through central (CNS) or autonomic (ANS) nervous systems (e.g.
behavioral responses to social interactions, ejaculation when mating, oviposition
etc.). Others may be signalized through neuroendocrine secretions directly (i.e.
from the pars nervosa). Most appear to be transduced through the hypothalamo-
pituitary unit and have cascading effects on physiology, morphology and behavior
associated with reproductive development, actual nesting and finally termination of
breeding. An example of this would be the effects of changing photoperiod on
GnRH and gonadotropin release that directly regulate gonadal function. Original.
spectrum of specialized receptors, neural pathways and neuromodulator

mechanisms exist, but we still know very little about these critical first steps
in response to environmental signals.
Next, the steps in the cascade of events that takes place after an individual
is exposed to environmental signals, and the hormonal cascades that follow
will be defined further. The types of environmental signals regulating
transitions of life history states may vary markedly. For example, signals
emanating from other individuals may be perceived by sensory receptors and
transduced by areas of the CNS that are very different from physical
environmental signals. Exactly how an organism perceives relevant
environmental signals, and the influences those signals have, can be
classified in two major ways. Firstly the effects may be directly on cells that
respond with a physiological or morphological response (e.g. gut cells
responding to changes in food composition, e.g. Norris 1997) or are signalized
through the CNS (Fig. 5.1). Environmental cues perceived by the CNS allow
the individual to respond neurally (e.g. via the autonomic nervous system) or
by the neuroendocrine/endocrine system (i.e. secrete hormones). In the latter
case neural signals are transduced into neuroendocrine secretions that then
influence the rest of the endocrine system by blood borne hormones (Wingfield
et al. 1999).
External cues that are signalized may be perceived by various sensory
modalities—visual, tactile, auditory, chemical (taste/smell) electrical, deep
brain photoreceptors and baroreceptor. Many other sensory receptors remain
to be identified such as those for detection of temperature responses, humidity
etc. In addition to external cues, internal signals also have important
influences on the neuroendocrine and endocrine systems. Examples are blood
and stored levels of amino acids, free fatty acids, sugars, vitamins etc. Other
examples include osmoregulatory functions (salt/water balance), the immune
system and disease, endogenous rhythms (“internal clock”) and aging. One
can imagine many potential internal signals. Even here, however, the
mechanisms by which such signals are perceived may not always be intuitive.
5.2.1 The Reproductive Life History Stage and Its Organization

There are three major phases to any life history stage (Jacobs and Wingfield
2000; Wingfield 2004; Fig. 5.2). The development phase consists of maturation
of the reproductive system itself, as well as the activation of reproductive
behaviors including establishment of a breeding territory, attraction of a mate
etc. The mature capability phase (Fig. 5.2) is when actual nesting can begin.
At this time the gonads are mature and females are ready to begin the final
maturation of ovarian follicles leading to yolk deposition and ovulation. There
are two major sub-phases, sexual and parental, with complex patterns of sub-
stages within them that can be repeated when re-nesting after failure of one
breeding attempt, or when raising multiple broods within a season.
Interactions between these sub-phases occur when the sexual phase
terminates (the clutch is complete) and parental behavior begins. Because the
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Fig. 5.2 Phases and sub-stages of the reproduction life history stage in birds. The
development phase consists not only of development of the reproductive system
itself, but also establishment of a breeding territory, attraction of a mate etc. The
mature capability phase is when actual nesting can begin. There are two major
sub-phases, sexual and parental, with complex patterns of sub-stages within them
that can be repeated when re-nesting after failure of one breeding attempt, or when
raising multiple broods within a season. Finally the reproduction life history stage
is terminated and the reproductive system regresses (often to a completely infantile
state), reproductive behaviors wane and the next life history stage (e.g. molt)
develops. Modified from Wingfield, J. C., Jacobs, J. D., Tramontin, A. D., Perfito, N.,
Meddle, S., Maney, D. L. and Soma, K. 1999. Pp. 85-128. In K. Wallen and J.
Schneider (eds), Reproduction in Context. M.I.T. Press, Cambridge, Massachusetts,
Fig. 4.2.
reproductive system remains mature throughout this process, switches

between sub-phases and the sub-stages involved (e.g. courtship and
copulation, nest building, incubation, feeding young etc.) can be rapid. Finally
the reproduction life history stage is terminated (Fig. 5.2) and the reproductive
system regresses (often to a completely infantile state), reproductive behaviors
wane and the next life history stage (e.g. molt) develops.
5.2.2 Classification of Environmental Signals Affecting

Reproduction
The spectrum of environmental signals known to influence reproduction in
vertebrates is truly daunting. However, it is possible to classify these factors
into three major types (Fig. 5.3, Wingfield 1980, 1983; Wingfield et al. 1999,
note that the signals from unpredictable events are not included here). Initial
predictive information (e.g. the annual change in day length) triggers the
development phase of the reproductive life history stage, maintains mature
capability and then regulates termination at the end of the breeding season
(Fig. 5.3). Local predictive information (e.g. temperature, food supply, rainfall)
may inhibit or accelerate effects of initial predictive information. These cues
are also important to time onset of actual nesting at mature capability, and
adjust timing of the termination phase (Fig. 5.3). All social interactions are
clustered under synchronizing and integrating information that can influence
the development phase and support functions such as territorial behavior,
synchronize mates as they begin nesting and integrate transitions between
sub-phases such as sexual to parental behavior (Wingfield 2006, Fig. 5.3).
Fig. 5.3 Different types of environmental signals from the physical environment
and from social interactions can influence all three phases of the reproduction life
history stage. Initial predictive information initiates gonadal development, maintains
reproductive capacity and then terminates the breeding season. Local predictive
and synchronizing and integrating information can influence all three phases too.
Thus knowing phase of the reproduction life history stage will be an important
determinant of how, for example, a given behavioral interactions will affect
neuroendocrine and endocrine mechanisms. Modified from Wingfield, J. C.,
Jacobs, J. D., Tramontin, A. D., Perfito, N., Meddle, S., Maney, D. L. and Soma, K.
1999. Pp. 85-128. In K. Wallen and J. Schneider (eds). Reproduction in Context.
M.I.T. Press, Cambridge, Massachusetts, Fig. 4.3.
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There is also strong evidence that behavioral interactions regulate the

termination phase. Note how types of environmental signals act at different
phases of the reproductive life history stage. This may indicate very different
mechanisms by which a specific type of environmental signal may affect
different phases (Wingfield et al. 1999; Wingfield 2004).
5.3 THE HYPOTHALAMUS AS A TRANSDUCER OF

ENVIRONMENTAL CUES INTO HORMONAL SIGNALS
The avian hypothalamus and the hypothalamo-pituitary unit are functionally
composed of both the nervous and endocrine systems. They have been
described in depth frequently (e.g. Wingstrand 1954; Oksche and Farner 1974;
Mikami 1986; Scanes et al. 1984) and show clear homologies with other
vertebrate taxa. A photomicrograph of this region in the White-crowned
Sparrow (Zonotrichia leucophrys gambelii) is shown in Fig. 5.4.
5.3.1 Hypothalamus
The hypothalamus extends from the lamina terminalis, rostral to the optic
chiasm, to the supramamillary decussation. The base and lateral walls of the
third ventricle provide useful markers (Fig. 5.4). This region has been divided
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into preopticohypothalamic, tuberal, and mamillary regions (e.g. Kuenzel and
van Tienhoven 1982), and most of the hypothalamic nuclei, neuroendocrine
Fig. 5.4 Sagittal section of the hypothalamo-pituitary unit in the white-crowned

sparrow, Zonotrichia leucophrys gambelii. Modified from Farner, D. S., Wilson, F. E.
and Oksche, A. 1967. Pp. 529–545. In L. Martini and W. F. Ganong (eds),
Neuroendocrinology, vol. 2. Academic Press, New York, Fig. 2.
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fibers, tracts and associated glial and ependymal cells are contained within it.
Neuroendocrine cells within the hypothalamus project to the pars nervosa
and the median eminence where they form terminal fields next to blood capil-
laries (Fig. 5.5, from Farner et al. 1967; Wingfield and Farner 1993). Avian
hypothalamic nuclei are rather diffuse, perhaps numbering 15-20, although
only a few have been well studied (Fig. 5.5, from Farner et al. 1967). Note also
that regions of the avian telencephalon and diencephalon have undergone a
major revision of nomenclature (Reiner et al. 2004).
It is through these brain regions that many environmental signals perceived
by sensory receptors are received as neural transmissions. These signals are
converted into neuroendocrine secretions that then influence morphology,
physiology and behavior (Scanes et al. 1984; Ball 1993; Ball and Balthazart
2002). Most environmental information from the physical environment, social
interactions and internal cues are transduced through the hypothalamo-
pituitary unit. Note that some environmental cues may be signalized directly
through central (CNS) or autonomic (ANS) nervous systems (e.g. behavioral
responses to social interactions, ejaculation when mating, oviposition etc.).
Others may be signalized through neuroendocrine secretions directly (i.e. from
the pars nervosa). Most appear to be transduced through the hypothalamo-
pituitary unit and have cascading effects on physiology, morphology and
behavior associated with reproductive development, actual nesting and
finally termination of breeding. An example of this would be the effects of
Fig. 5.5 Schematic diagram of the major components of the hypothalamic-pituitary

unit in birds (c.f. Fig. 5.1) and diagram of some avian hypothalamic nuclei. Modified
after Farner, D. S., Wilson, F. E. and Oksche, A. (1967). Pp. 529-582. In L. Martini and
W. F. Ganong (eds.), Neuroendocrinology, vol. 2. Academic Press, New York, Fig. 1.
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changing photoperiod on gonadotropin releasing hormone (GnRH) and other

peptides (Fig. 5.6, see below) and gonadotropin release that directly regulate
gonadal function (Fig. 5.6).
5.3.2 Median Eminence

The median eminence is where neurohormones from hypothalamic
neuroendocrine cells are released into the hypophysial blood portal system to
stimulate or inhibit release of anterior pituitary hormones. This is a major
switch point as environmental information relayed through hypothalamic
neurons triggers a hormonal cascade that affects many aspects of an
organism’s life cycle. In birds the median eminence is formed by the ventral
floor of the hypothalamus, emerging posteriorly from the optic chiasm and
forming the infundibular stalk (Figs. 5.4 and 5.5). It receives axons from
hypothalamic nuclei and also contains glial cells. There are specialized
ependymal cells that terminate on portal capillaries in juxtaposition with
terminals of axons (Figs. 5.4, 5.5 and 5.7). Additional neurons of hypothalamic
origin may also be found but they are not neuroendocrine (Kobayashi et al.
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Fig. 5.6 A schematic representation of how signalized environmental information

transduced by the brain results in secretion of hypothalamic peptides such as
avian gonadotropin-releasing hormone-I (GnRH-I) and Lamprey GnRH-III (lGnRH-
III) that stimulate gonadotropin release. Note that avian gonadotropin-inhibitory
hormone (GnIH) inhibits at least luteinizing hormone secretion. These cascading
effects regulate gonadal development and function such as secretion of sex
steroids, avian inhibins etc. Original.
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1970). Typically, hypothalamic neuron terminals filled with neurosecretory

granules are separated from primary capillaries by two basement membranes
(Fig. 5.7). These capillaries, that drain to the hypophysial portal system, have
many fenestrations of the endothelial cells as found in capillaries of other
endocrine tissues. Note that the perivascular spaces between the basement
membrane and the capillary endothelium also contain cells and sometimes
erythrocyctes (Fig. 5.7, Mikami et al. 1970). There are also pericytes that orient
spirally around endothelial cells (Fig. 5.7). They may have a function as a
mechanical support of the portal vessels as well as a contractile function
regulating blood flow (Mikami et al. 1970). Additionally, the endothelial cells
may have protrusions into the capillary lumen that have been suggested to
have a role in regulation of blood flow. In some species, the median eminence
and the primary portal capillaries are encased by the pars tuberalis of the
adenohypophysis (Kobayshi et al. 1970; Vitums et al. 1964; Mikami et al. 1970).
5.3.3 Hypothalamic Nuclei and Tracts

Secretory neurons in the hypothalamus synthesize a number of hormones that
are released into the peripheral blood circulation (e.g. those of the pars
nervosa) and have effects largely on contraction of smooth muscle and water
balance in the kidney. Neurosecretions from the median eminence enter a
specialized blood portal system that vascularizes the pars distalis (anterior
pituitary or adenohypophysis). These secretions either stimulate or inhibit the
synthesis and secretion of hormones from endocrine cells within the pars
distalis. They are commonly referred to as “releasing” or “inhibiting”
hormones or factors (Scanes et al. 1984). The hypothalamic neurons, often
clustered in groups (nuclei), project their axons to the pars nervosa and/or
the median eminence (Fig. 5.5) forming tracts and terminal fields where axons
may contact basement membranes of capillaries (Fig. 5.7). The median
eminence narrows caudally into the tubular infundibular stalk terminating in
the pars nervosa (Figs. 5.4 and 5.5). Neurosecretory material is transported
through this stalk via the supraoptico-paraventriculo-hypophysial tract to the
pars nervosa (see Wingstrand 1951, 1954; Oksche and Farner 1974 for detailed
review).
Many tracts have been indentified in the avian hypothalamus (Wingstrand
1951). Important examples are (1) the tractus supraoptico-hypophyseus,
which originates in the supraoptic region and terminates in the pars nervosa;
(2) the tractus tuberohypophyseus projecting from the infundibular nucleus to
the median eminence; (3) the tractus hypophyseus posterior, mostly receiving
fibers from the posterior hypothalamus; and (4) the tractus hypophyseus
anterior, consisting of fibers from nuclei in the lateral hypothalamus, and
from the paraventricular and preoptic areas that terminate in the median
eminence. It is likely that there are many differences among species. The
systematic use of immunocytochemical methods and in situ hybridizations for
mRNA coding for peptides may resolve these issues.
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Fig. 5.7 A. Electron micrograph of anterior median eminence (AME) and primary
capillary (PCp) of a photostimulated white-crowned sparrow, Zonotrichia leucophrys
gambelii. B. Enlargement of primary capillary (PCp) that is widely extended on the
surface of the median eminence and separated from it by two layers of basement
membrane (Bm). Fenestrations (arrows) of the endothelial cells (En) are evident. A
Pericyte (Pc) with a large nucleus is in the lower left corner. Perivascular spaces
(PVS) are found between the basement membrane and the capillary endothelium.
From Mikami et al. (1970). Zeitschrift für Zellforschung 106: 155-174. Springer-
verlag, Berlin, Fig. 2.
5.3.4 Hypothalamo-hypophysial Portal System

There are two networks of primary portal capillaries arising separately from
the ventral surfaces of the anterior and posterior divisions of the median
eminence (Fig. 5.5). Anterior and posterior portal veins may be enclosed as a
bundle by the pars tuberalis of the adenohypophysis, flowing to secondary
capillary plexuses and sinuses in the rostral and caudal lobes respectively
(Fig. 5.5, e.g. Vitums et al. 1964, 1966; Duvernoy et al. 1969, 1970; Singh and
Dominic 1975). It is assumed that this portal system delivers neurohormones
from neurons in specific areas of the hypothalamus to discrete areas in the
pars distalis of the adenohypophysis (Vitums et al. 1966), although as yet
there is no actual physiological evidence that supports this hypothesis
(Wingfield and Farner 1993).
The portal vessels may be the sole vascular supply to the pars distalis, and
there is no unequivocal evidence of direct hypothalamic or other innervation,
although recently such projections have been claimed to exist. The late Donald
S. Farner described this hypothalamo-hypophysial system very well as
follows: “defined hypothalamic nuclei contain cells of different functions
despite their near-uniform appearance when examined by classical
cytological and cytochemical techniques. The same may hold for fiber systems
within anatomically defined tracts. A crude analogy can be drawn with
industrial cities, railways and highways, and destinations. Factories
(perikarya) in several cities (hypothalamic nuclei) produce a variety of
products (neurohormones) some of which are produced in more than one city.
It then follows that railways and highways (axons and tracts) are involved in
delivery of a variety of products (neurohormones etc.) to different
destinations—neurohemal organs, synapses, cerebrospinal fluid etc. The
analogy is of course incomplete because nothing is included about
communication and transport of materials to the factories” (from Wingfield
and Farner 1993).
5.3.5 Pars Nervosa

The caudal portion of the infundibulum of the hypothalamus, just posterior to
the median eminence, forms the pars nervosa (Figs. 5.4 and 5.5, Wingstrand
1951; Oksche and Farner 1974). The lumen may be open or closed and the
neuroendocrine pars nervosa contains branched terminals and fibers of the
supra-optico-paraventriculo-hypophysial tract, glial cells, pituicytes, and
ependymal cells. The pars nervosa is a neurohemal organ, like the median
eminence, from which hypothalamic neurohormones can be transferred into
circulating systemic blood (Wingfield and Farner 1993). In birds there are two
pars nervosa hormones—arginine vasotocin (AVT) and mesotocin (MT).
Magnocellular bodies in the pre-optic, supra-optic and paraventricular
regions of the hypothalamus synthesize these peptides and immunocy-
tochemical techniques show each cell body expresses one hormone—either
mesotocin-like or vasotocin-like. Note that all these cell types are found in the
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hypothalamic nuclei containing magnocellular neurons (Oksche and Farner

1964; Wingfield and Farner 1993).
5.3.6 Hormones of the Pars Nervosa

Mesotocin (Ile-8-oxytocin, MT) and arginine vasotocin (AVT) were first
demonstrated in extracts of posterior pituitaries in Domestic fowl (Gallus
gallus), Turkey (Meleagris galloparvo) and Goose (Anser anser). Both MT and
AVT are amidated nonapeptides with a disulphide bridge and amino acid
substitutions at positions 3 and 8 (Acher et al. 1993 for review). They are
found in all flying birds studied to date as well as ratites, e.g. Ostrich (Struthio
camelus) (Lazure et al. 1987; Michel et al. 1990a). The genes also express
neurophysins that have specific properties of binding neurohypophysial
hormones, carry them down the axon to terminal fields, and play a role in
their release by exocytosis. Neurophysins are of two major types and appear
to occur on a one-on-one molar ratio with the hormones they carry.
Neurophysin I binds mesotocin. Neurophysin II binds AVT (George 1980;
Michel et al. 1990ab; Hamann et al. 1992; Acher et al. 1993). Neurophysins are
synthesized in perikarya, transported axonally, stored and released with the
hormones themselves. They have a high degree of homology of amino acid
sequence with mammalian neurophysins. The precursor molecules
themselves are biologically inactive. Enzymatic cleavage produces the active
peptides in the Golgi apparatus and in secretory granules prior to release.
Neurophysin bound to hormone requires about 2-3 hours to migrate from the
magnocellular neurons to terminal fields in the pars nervosa.
In general, AVT and MT have the following functions (see George 1980 for
review): 1. Water balance, through flow in semipermeable membranes. 2.
Regulation of blood pressure. 3. Contraction of smooth muscle in gonadal
ducts. 4. Effects on sexual behavior and aggression (Goodson and Adkins-
Regan 1999). Note that actions 2 and 3 are on contraction or relaxation of
smooth muscle. AVT has well known actions on contraction of smooth
muscle, especially in the oviduct at oviposition. In support of this view,
plasma levels of AVT increase at oviposition in chickens perhaps mediated by
prostaglandins in oviductal tissue (see George 1980; Acher et al. 1993 for
reviews). In addition to the action of AVT, galaninergic innervation of the
uterine oviduct may be involved in oviposition in quail (Li et al. 1996; Tsutsui
et al. 1997, 1998; Sakamoto et al. 2000). There is also evidence that injections of
AVT in pigeons increase plasma levels of free fatty acids, glucose and growth
hormone (George 1980). The biological effects of MT in birds are less well
known. Both MT and AVT can act as neurotransmitters since such neurons
project to many sites in the CNS including the median eminence (Acher et al.
1993). AVT has also been shown to increase male sexual behavior in the
pigeon (Kihlström and Danninge 1972) and male zebra finch (Taenopygia
guttata) (Goodson and Adkins-Regan 1999) and on aggression (Goodson
1998a,b; Goodson and Adkins-Regan 1999), as has been well demonstrated in
other vertebrates such as amphibia (e.g. Moore 1987). More recently, there is
some evidence that AVT can act to attenuate behavior- and crowding-induced
aggression via a cardiovascular mechanism, rather than a receptor-mediated
central mechanism, in European starlings (Sturnus vulgaris) (Nephew et al.
2005a). The mechanism of action of MT in birds is not well known.
5.4 ADENOHYPOPHYSIS (= ANTERIOR LOBE OF

THE PITUITARY GLAND)
Unlike the pars nervosa (neurohypophysis), the adenohypophysis is not part
of the brain but derived from pharyngeal tissue and becomes associated with
the median eminence during embryonic development (Wingstrand 1951;
Gorbman et al. 1983; Norris 1997). The avian adenohypophysis consists of the
pars tuberalis and pars distalis. There is no pars intermedia (Wingstrand
1951; Gorbman et al. 1983; Norris 1997).
5.4.1 Pars Distalis and Pars Tuberalis

Cells of the pars distalis are typically arranged in cords (Fig. 5.8). Using histo-
logical and cytochemical techniques a system of putative cytophysiological
functions for cells of the avian pars distalis has been developed (for extensive
reviews see Tixier-Vidal and Follett 1973; Mikami 1986; Mikami et al. 1975).
The early investigations on the functions of these cells involved surgical
removal of gonads, thyroid glands, and adrenals. Later, the morphology of
secretory granules and other ultrastructural features of secretory cells were
studied using electron-microscope techniques. Now immunocytochemical
and in situ hybridization techniques provide more definitive evidence. Here
we will focus on the gonadotropin and prolactin secreting cells as the major
types directly involved in control of reproduction.
Histological investigations with light and electron microscopy combined
with reproductive state and following castration, lead to the conclusion that
there are two types of gonadotropes in the rostral and caudal lobes of the pars
distalis. We now know that these cell types secrete the gonadotropins follicle-
stimulating hormone (FSH) and luteinizing hormone (LH) that regulate
gonadal function (e.g. Goldsmith and Follett 1980; Norris 1997; Kirby et al.
2005). These conclusions seem to be at least partially sustained because in
chickens, in situ hybridization studies show that FSH and LH appear to be
produced by different cells. In situ hybridization of mRNAs of the b subunit of
LH match immunocytochemical localization in pituitaries at different stages
of reproduction in white-crowned sparrows (Fig. 5.9, e.g. Kubokawa et al.
1994).
Recent immunocytochemical evidence indicates that prolactin secreting
cells (lactotropes) are found primarily in the rostral lobe of the pars distalis
(Mikami 1986). Note that the term lactotrope is misleading in birds because
most species do not lactate (except for crop milk in columbiiformes), and
prolactin has many other actions (Wingfield and Farner 1993; Norris 1997).
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Fig. 5.8 Area of the caudal lobe (A and C) and cephalic lobe (B and D) of an adult
white-crowned sparrow, Zonotrichia leucophrys gambelii. Note cells arranged in
cords. From Matsuo, S.-I., Vitums, A., King, J. R. and Farner, D. S. (1969). Zeitschrift
für Zellforschung 95: 143-176. Springer-verlag, Berlin, Fig. 3.
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Fig. 5.9 In situ hybridization for LH b-subunit mRNA (right) and immunocytochem-
istry of LH (left) in anterior pituitaries of White-crowned Sparrows, Zonotrichia
leucophrys gambelii. SD = short day (16L 8D) LD = long day (20L 4D). From
Kubokawa, K., Ishii, S. and Wingfield, J. C. (1994). General and Comparative Endo-
crinology 95: 42-51. Academic Press, New York, Fig. 6.
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5.4.2 Hormones of the Pars Distalis and their Targets

There are three major types of hormones in the pars distalis: the glycoproteins,
the pro-opiomelanocortin derived peptides, and the polypeptides. Some have
direct actions on morphology, physiology and behavior, whereas others have
tropic effects on peripheral endocrine glands. A detailed account of all three
types is beyond the scope of this review and we will focus on gonadotropins
and prolactin.
There are three glycoprotein hormones secreted by the pars distalis and all
have both early and late (trophic) cell responses. Luteinizing hormone (LH)
regulates sex steroid production as well as ovulation. Follicle-stimulating
hormone (FSH) promotes spermatogenesis and ovarian follicle maturation. It
also regulates secretion of a gonadal protein inhibin, that feeds back
negatively to regulate FSH secretion. Thyroid-stimulating hormone (TSH)
regulates thyroid hormone synthesis and secretion (see Goldsmith and Follett
1980; Norris 1997). All three hormones share a characteristic structure of two
subunits, a and b. The a subunit is shared by all three hormones whereas the
b subunit is specific. For example, if subunits are disassembled, it is possible
to recombine an FSH a subunit with a TSH b subunit and still get good TSH
biological activity. The b subunit provides specificity, but association of a and
b subunits is essential for binding to receptor and biological action (e.g.
Gorbman et al. 1983; Baeulieu and Kelly 1990; Norris 1997).
There are two related polypeptides secreted by the pars distalis, one is
growth hormone that regulates growth (through release of insulin-like growth
factors—IGFs), as well as lipid metabolism. The other is prolactin that has a
vast spectrum of actions including reproductive function, lipid metabolism
and osmoregulation (Goldsmith and Follett 1980; Norris 1997).
5.4.3 The Hypothalamo-Adenohypophysial-Gonad Axis

A summary of the interrelationships among the brain, anterior pituitary and
gonad is presented in Fig. 5.6. This follows the general vertebrate system (e.g.
Gorbman et al. 1983; Wingfield and Farner 1993). The scheme can be referred
to as a summary guide and quick reference for the complex relationships with
morphology, physiology and behavior discussed below.
Synthesis and release of the gonadotropin LH is regulated by a
hypothalamic releasing factor—gonadotropin-releasing hormone, GnRH (also
called luteinizing hormone releasing hormone, LHRH, see Dunn and Millam
1998; Sharp and Ciccone 2005 for reviews). Hypothalamic control of FSH
secretion is less clear because injections of GnRH have little effect, at least in
chickens (Kirby et al. 2005). Evidence for GnRH in birds comes from
measurements of biological activity and content of the avian hypothalamus
by determining the ability of hypothalamic extracts to release LH in vitro. In
some cases the released LH was measured by a second bioassay (e.g. 32P
uptake in chick testis (e.g. Erickson 1975) in White-crowned sparrows
(Zonotrichia leucophrys gambelii) or by radioimmunoassay either in the
incubation medium (e.g. Bicknell and Follett 1975) in Japanese quail (Coturnix
japonica), or in blood after injection of hypothalamic extracts in vivo in Z. l.
gambelii (Wingfield and Farner 1993). In the 1980s, it was found that the
hypothalamus of the domestic fowl had two distinct GnRH molecules,
designated chicken GnRH-I and GnRH-II (cGnRH-I and -II) that differed from
mammalian GnRH by one and three amino acid substitutions (King and
Millar 1982a,b; Miyamoto et al. 1984). It has since been shown that the two
chicken GnRHs are also found in the hypothalamus of wild species, European
starling, and Song sparrow (Melospiza melodia) with cGnRH-I predominating
(Sherwood et al. 1988). The cDNA encoding for the pre-pro-GnRH molecule
has been sequenced for the domestic fowl (Dunn et al. 1993). Both have potent
effects on release of LH in male and female Song sparrows in a more or less
dose dependent fashion. However, in the domestic fowl, cGnRH-I and -II had
different potencies in releasing LH (Sharp et al. 1987).
In general, cGnRH-I is located in the pre-optic region of the brain with
axonal projections (fibers) to the median eminence, where the terminal fields
are located. The oculomotor complex of the midbrain is the location of the
cGnRH-II neurons, which are typically less polar in shape than the cGnRH-I
neurons and as a result have few processes extending from the cell bodies (for
distribution see Millam et al. 1993, Millam et al. 1998; Teruyama and Beck
2000). Despite a long-held acceptance of the notion that cGnRH-II neurons are
not hypophysiotropic, there have been some reports in quail that cGnRH-II ir-
fibers are present in the POA, the lateral septum (Millam et al. 1993), and the
median eminence (D’Hondt et al. 2001; Millam et al. 1998; van Gils et al. 1993;
Clerens et al. 2003). However, there has only been one published report of
cGnRH-II in the ME of songbirds (Stevenson and MacDougall-Shackleton
2005), while others find no evidence of GnRH-II in the same area (Meddle et al.
2006), and experiments in chickens suggest that the median eminence is a site
of release of cGnRH-I, but not cGnRH-II, into the hypophysial portal
vasculature (Sharp et al. 1990). Thus, the distribution and function(s) of
cGnRH-II have remained somewhat enigmatic, although it should be noted
that central administration of cGnRH-II, but not cGnRH-I enhances
copulation solicitation in female white-crowned sparrows (Maney et al. 1997).
Thus, cGnRH-II might act in a neurotransmitter role to influence reproductive
behaviors, as has also been postulated for some mammalian species (Temple
et al. 2003; Kaufmann and Rissman 2004). Hypothalamic hormones released
from the median eminence circulate in the portal system and are quickly
degraded before they reach the peripheral circulation.
Avian LH and FSH were first purified from adenohypophyses of the
domestic fowl by Stockell-Hartree and Cunningham (1969), and later by
Furuya and Ishii (1974), and Sakai and Ishii (1980). Bioassay studies show
that as in mammals, LH acts primarily on the endocrine gonad and FSH on
the gametogenic gonad (e.g. Brown et al. 1975; Furuya and Ishii 1976; Brown
and Follett 1977; Maung and Follett 1977; Follett et al. 1978). FSH acts in
synergy with testosterone on spermatogenesis. Note that in Zonotrichia l.
Endocrinology of Reproduction ''
gambelii and many other photoperiodic songbirds testis size shows large
variation in size with season (e.g. Wingfield and Farner 1993). In the
regressed, non-breeding testis, semiferous tubules are inactive and regressed
and cells of Leydig are undifferentiated. However, in the reproductively active
males, seminiferous tubules are greatly enlarged and have bunches of
spermatozoa. Leydig cells are active but not conspicuous between the greatly
enlarged tubules. The epididymis, deferent duct (vas deferens) and seminal
glomus show marked development in the breeding season. The cloacal
protuberance (vent) can also become greatly enlarged, but this varies with
species (see Birkhead and Møller 1992 for review).
Using purified avian gonadotropins, specific radioimmunoassays (RIAs)
have been developed for chicken LH (Follett et al. 1972), Turkey LH (Burke et
al. 1979); and for chicken FSH (Scanes et al. 1977; Sakai and Ishii 1985). With
the exception of the FSH assay developed by Scanes et al. (1977) these
gonadotropin RIAs have proved to be applicable to plasma samples from a
wide range of avian species (see Follett et al. 1978; Goldsmith and Follett 1980;
Sakai and Ishii 1985, 1986). Isolation of LH and FSH from truly wild species
appears to be restricted to the ostrich in which LH and FSH fractions have
potent activity in both avian and mammalian bioassy systems (Bona-Gallo et
al. 1983). Furthermore, LH from the ostrich is far more potent than FSH in
stimulating secretion of testosterone from immature Mallard (Anas
platyrhynchos) testes suggesting that as in domesticated species, increased
androgen secretion is highly specific for LH (Chase 1982).
Putative cDNA sequences for chicken LH b-subunit and Japanese quail LH
b- and a-subunits have been cloned (Noce et al. 1989; Ando and Ishii 1994).
The predicted amino acid sequences reveal some homology with mammalian
forms of LH including the positions of proline residues which Ishii (1988,
1990) suggests may be related to species specificity of LH and its receptor. The
chicken cDNA clone has been used to measure changes in mRNA for b-LH
subunit during photostimulated gonadal growth in Zonotrichia l. gambelii
(Kubokawa et al. 1994). The cDNA sequence encoding the extracellular
component of LH receptor in testes of Japanese quail has also been
characterized. This domain of the receptor is thought to be important for
binding to the LH molecule. The predicted sequence of amino acids was about
70% homologous with mammalian LH receptor sequences, although the
positions of cysteine residues and potential N-linked glycosylation sites were
virtually identical to those of mammals (Akazome et al. 1994). Such
conservation suggests extensive homology of molecular conformation of the
LH receptors across phylogeny.
A number of investigations (summarized in Lofts and Murton 1968, 1973;
Wingfield and Farner 1993) as well as others, indicate that cells of Leydig are
present in all phases of the annual cycle of periodically breeding species, and
that their numbers and activity correlate reasonably well with sexual
behavior. This conclusion is consistent, with few exceptions, with measured
plasma levels of LH and testosterone, e.g. in Zonotrichia l. gambelii (Wingfield
and Farner 1978a,b), European starling (Ball and Wingfield 1984), and others
(see Wingfield and Farner 1993; Wingfield et al. 1999 for reviews).
Steroid biosynthesis in avian gonads is essentially similar to that of
mammals. In all steroid secreting cells the precursor molecule is cholesterol
(Furr and Pope 1970). The actions of LH after binding to a receptor on a
steroid synthesing cell is to activate adenylate cyclase resulting in an increase
in cAMP levels in the cell (e.g. Kubokawa et al. 1994). cAMP then activates
protein kinases resulting in phosphorylations of proteins associated with
mobilization of cholesterol droplets with the cell. The critical first step is
enzymatic cleavage of the cholesterol side chain to give pregnenolone (e.g.
Ozon 1972a). Next is the conversion of the hydroxyl group at carbon 3 to a
ketone group to give progesterone under the action of an enzyme D5, 3b-
hydroxysteroid-dehydrogenase (HSD). Histochemical methods to visualize
activity of this enzyme were used widely in the past as an indication of
steroid synthesis because this enzyme catalyzes an important step from
biologically inactive to active steroid hormones.
Progesterone may, in some circumstances and in specific cells, be secreted
itself. In other cases it it hydroxylated at carbon 17 to give 17a-
hydroxyprogesterone (e.g. Furr and Pope 1970; Ozon 1972a) that is then con-
verted to androstenedione by cleavage of the side chain and dehydrogenase to
give a ketone group at carbon 17. Simple hydroxylation at carbon 17 then
gives testosterone that may be secreted (but note that it can be converted to
various metabolites in the periphery, see below). Both androstenedione (since
it is so easily converted to testosterone) and testosterone may also be substrate
for an enzyme aromatase that results in aromatization of ring A (with loss of
carbon 19, and hydroxylation at carbon 3) to give estradiol-17b (e.g. Ozon
1972b). Aromatase is widespread in avian tissues including gonads, steroid
sensitive secondary sex characters, and brain (e.g. Schlinger and Arnold
1991,1992). The most common metabolite of estradiol-17b is estrone. The latter
is generally regarded as being much less potent than estradiol, although
plasma levels of estrone are sometimes measured.
The avian ovary is unusual in that only one develops in the vast majority
of species. This is usually the left ovary, although in Apterygidae both ovaries
are apparently functional (see Lofts and Murton 1973; Welty and Baptista
1988). A cohort of ovarian follicles is recruited each breeding season and they
develop to a stage in which they contain white yolk, but are still far from
reaching an ovulatory state (King et al. 1966; Kern 1972; Lofts and Murton
1973). For each laying sequence (or clutch) a sub-cohort of these partially
developed follicles begin sequestering yellow yolk from blood. This process is
called vitellogenesis. Vitellogenin is synthesized in the liver and is stimulated
by estradiol-17b. It is a dimer molecule of about 480 kDa containing complexes
of protein-bound phosphorus and lipoproteins. The liver also produces other
lipid rich proteins that are precursors to yolk (Griffin et al. 1984). Note that
once these yolk precursors reach the ovary they must pass through the
capillary wall as well as the basal lamina of the granulosa and granulosa
cells themselves that surround the oocyte. Yolk precursors appear to be

transported by receptor mediated endocytosis (Griffin et al. 1984). Ovarian
follicles grow rapidly as yolk is deposited in layers (Kern 1972; Lofts and
Murton 1973; Griffin et al. 1984) in a sequence according to size and they are
ovulated one at a time as they mature (Wells and Gilbert 1984). Usually one
follicle is ovulated each day (although there is great variation in ovulation
times—see Welty and Baptista 1988). The ovulated egg is “captured” by the
ostium (also known as infundibulum—not to be confused with the
infundibulum of the hypothalamus) of the oviduct. Fertilization and
deposition of the first layer of albumen occur here. The ovum passes down the
oviduct through highly differentiated regions that have specific functions.
Further albumen is laid down in the magnum, and membranes surround the
developing egg in the isthmus. On reaching the shell gland (also known as
uterus) a shell and pigment is deposited (e.g. Solomon 1983). Finally
oviposition occurs through the vagina and cloaca. Note that since there is
only one ovary and one oviduct, further ovulations cannot occur unless
oviposition has occurred (e.g. Sharp 1980).
It is possible that medullary and cortical interstitial cells of the theca are
structurally identical, separate designations for them may be useful only
because they may be functional at different times. Studies of the ovary show
follicles as well as the theca and granulosa layers of follicles having steroid
secreting capacity. Interstitial cells in the ovarian stroma also may secrete
steroids. Atretic follicles also occur because not all follicles of a cohort selected
for development are actually ovulated. Once the clutch is complete, other
follicles with white yolk that have never entered the final maturation
pathway, degenerate. Typically they collect lipid droplets and regress.
Histochemical techniques that visualize 3bHSD (the enzyme that regulates
conversion of pregnenolone to progesterone—and thus the conversion of a
biologically inactive steroid to an active one) show potential steroid
synthesizing activity of theca, granulosa and stromal cells (for reviews see
Gilbert 1971; van Tienhoven 1983; Wells and Gilbert 1984; Johnson and Tilly
1990).
Evidence suggests that interstitial cells of the theca interna and/or theca
granulosa of the follicle secrete estrogens, progesterone, and testosterone.
Indeed, Huang et al. (1979) have proposed a model that attributes an
interaction between these two types of cells in the secretion of these hormones
involving an interaction of LH and FSH on theca and granulosa cells
respectively. The sex steroids testosterone, and also estradiol provide negative
feedback signals to the gonadotropes in the pars distalis as well as GnRH
neurons in the hypothalamus to inhibit GnRH and gonadotropin secretion.
There is a marked decrease in sensitivity to steroid feedback at least in male
Zonotrichia l. gambelii during gonadal maturation that allows a general
increase in gonadotropin and sex steroid secretions (Matt 1983).
5.4.4 Actions of FSH on Follicular Development

Ovarian Growth Factors
Inhibin, a peptide first demonstrated in the Sertoli cells of seminiferous
tubules, is under the control of FSH and can inhibit the secretion of FSH from
the pars distalis. There is also evidence that granulosa cells may secrete a
substance similar to inhibin. This action seems to be specific for inhibition of
FSH release while LH secretions remains unchanged. Since then there is now
extensive evidence for an inhibin-activin system in the regulation of ovarian
folliculogenesis in birds. They are members of a conserved transforming
growth factor b super family that play intragonadal roles via autocrine and
paracrine actions. As yet, there is no evidence that activins act on the pituitary
(Knight et al. 2005). There is also evidence for paracrine effect of a growth
factor (GDF9) from the oocyte itself that also plays an important role in oocyte
development—possibly during the yolk uptake phase (Johnson et al. 2005).
5.5 PINEAL BODY (EPIPHYSIS CEREBRI)

The pineal gland (epiphysis) is located in the dorsal brain and made up of a
stalk and rudimentary photoreceptors complete with visual pigments (rhodop-
sins). In birds there is a reduction in sensory neurons, but some sympathetic
aminergic innervation may remain. This innervation is involved in the regula-
tion of melatonin secretion—the main product of the pineal gland. Melatonin
is an indoleamine synthesized from serotonin by the action of N-
acetyltransferase to N-acetylserotonin and by hydroxyindole-O-
methyltransferase (HIOMT) to melatonin (e.g. Gorbman et al. 1983; Norris
1997).
At least in diurnal species melatonin is secreted at night (i.e. light inhibits
release) and injections of melatonin can reverse activity rhythms. Transplants
of pineal glands from individuals entrained to one rhythm can induce a
similar rhythm in the recipient suggesting some autonomy. Indeed, pineals
cultured in vitro can retain rhythmicity and even entrain to changing
photoperiod (Cassone and Menaker 1984). Note, however, that the retina can
also produce melatonin and thus the eyes may also be important.
The avian pineal body is a circumventricular organ that shows great
morphological diversity (e.g. Quay and Renzoni 1963; Quay 1965; Renzoni
1965), and in general consists of a stalk that arises from the roof of the
diencephalon in the region between the habenular and posterior commissures,
expanding dorsally to become the pineal organ. It thus lies close to the interior
surface of the dorsal wall of the brain case in a triangular area delimited by
the posterior margins of the cerebral hemispheres and the anterior margin of
the cerebellum. In some species such as owls (Strigidae), the pineal gland is
lacking or is a degenerate remnant (Breucker 1967; Renzoni 1968). The avian
pineal has been studied in no less than 100 species of 13 orders (for
cytological details on some selected wild species (see Quay and Renzoni 1963;
Oksche and Kirschstein 1969; Ueck 1979).
Endocrinology of Reproduction !
Few investigations addressed annual changes in pineal function, but in the

House sparrow (Passer domesticus) Ralph and Lane (1969) found no evidence
for seasonal changes. However, Barfus and Ellis (1971) found that the activity
of pineal hydroxyindole-O-methyl transferase of P. domesticus was lowest
during the breeding season. Similar results were obtained in the Baya weaver
(Ploceus phillipinus) (Saxena et al. 1979) and the Indian tree pie (Dendrocitta
vagabunda) (Chandhuri and Maiti 1989).
Although direct illumination apparently does not alter electrical activity of
the avian pineal body (Ralph and Dawson 1968; Morita 1975; Homma et al.
1980), secretion of melatonin is an endogenous circadian function that is
entrained by photoperiod (e.g. Ralph et al. 1967; Ralph 1976; Binkley et al.
1977; Menaker et al. 1978; Cassone and Menaker 1984). Chicken pineal cells in
culture show an endogenous oscillator that controls the circadian rhythm of
serotonin N-acetyltransferase activity and contain a rhodopsin-like
photoreceptor (Deguchi 1982). Although pineal activity may be affected by
light in some species, whether this can be viewed as evidence that this organ
is directly photosensitive in all has been questioned (Oliver and Baylé 1982)
and there is now evidence suggesting extensive interspecific differences (cf.
Cassone and Menaker 1984). There are several investigations claiming both
anti- and progonadal effects of the avian pineal (for critical review see Binkley
1988). Unlike in mammals, there is little evidence of a significant direct role of
the pineal in the regulation of gonadal function in birds. However, recently
published findings point to the possibility that melatonin is, to some degree,
involved in regulation of reproduction [see later section on gonadotropin-
inhibitory hormone (GnIH)]. Pinealectomy of male Baya weavers resulted in
increased rate of photoperiodically induced testicular growth (Balasubramanian
and Saxena 1973), and when mature males were transferred to short days, the
pinealectomized group showed partial testicular regression in comparison
with complete regression in the sham operated group. The authors interpreted
these results as effects of removal of an inhibitory effect of melatonin on the
hypothalamo-hypophysial-gonadal axis. However, because the pineal body is
a component of the avian circadian system (Cassone and Menaker 1984), it is
possible that a shift in phase angle between a daily cycle in photosensitivity
and the daily photoperiod could explain the results (e.g. Gwinner et al. 1981).
Other components of the avian circadian system (Gaston and Menaker
1968) include the suprachiasmatic nucleus, retina, and deep encephalic
(hypothalamic) photoreceptors (e.g. Cassone and Menaker 1984; Underwood
and Siopes 1985), all have a common developmental origin in the
diencephalon, and they are photoreceptive or receive photic information
(Menaker 1982; Cassone and Menaker 1984; Underwood and Siopes 1985).
More recently there has been a renaissance of interest in the possibility that
melatonin is involved in seasonal processes in birds, including regulation of
the reproductive axis. In a study involving the use of melatonin antiserum,
Ohta et al. (1989) concluded that melatonin is involved in at least the initial
stages of photoperiodism in quail (i.e., early in the dark phase), and that the
timing of suppression of plasma melatonin is critical to gonadal development.

In a contradictory study, injecting melatonin into quail exposed to long day
lengths (and thereby simulating a short-day melatonin signal) did not inhibit
the long-day-induced gonadal growth (Juss et al. 1993). The fact that one study
(Ohta et al. 1989) took the approach of shortening the melatonin signal during
short days and another (Juss et al. 1993) lengthened the melatonin signal
during long days might prove fundamental to the differences in results,
especially if it is true that the timing of the melatonin signal is critical to the
ensuing effect. It is possible that studies on melatonin and the reproductive
axis in birds have been hampered by the modes and timing administration of
melatonin, making interpretation of the findings difficult at best. At any rate,
it seems obvious that the role of melatonin in reproduction in birds is
somewhat more complex than its role in mammalian reproduction.
Whatever its actions are, it is likely that melatonin affects a suite of
physiological systems in addition to the reproductive system. For example
Guyomarc’h et al. (2001) suggest that melatonin supplementation might affect
food intake and fat deposition and thereby be responsible for the slight
inhibition of sexual development that they observed in European quail
(Coturnix coturnix). Perhaps the most convincing, and least confusing, effects
of melatonin upon the avian reproductive system stem from two recent
studies. The first study indicates that in chickens the effects of melatonin on
plasma LH can be described very simply (Rozenboim et al. 2002). These
authors demonstrated very large reductions in plasma LH (up to 70%) after
injections of relatively high doses of melatonin into castrated white leghorn
roosters. Additional experiments in that study indicated that melatonin
administration inhibited plasma LH in a time- and dose-dependent manner,
and that the effects persisted for as long as melatonin was administered
(Rozenboim et al. 2002) . The second study is described in section 5.7.2 (Ubuka
et al. 2005), and is concerned with the dose-dependent regulation of
gonadotropin-inhibitory hormone by melatonin.
5.6 SEASONAL BREEDING, THYROID HORMONES AND PROLACTIN

5.6.1 Photoperiodism and Photorefractoriness
In northern temperate zones, most bird species have evolved mechanisms to
coincide their breeding with periods when environmental conditions are
optimal for the raising of young. Often this depends on seasonal food supply
and/or climatic conditions. In general, the further north an individual lives,
the more imperative it becomes to coincide sexual maturity and raising of
young with favorable conditions and to avoid breeding during less suitable
periods. Any environmental factor that predictably signals the onset of a
change in ecological conditions can act as a “proximate factor”—as long as it
can be perceived and measured by the organism. The most reliable proximate
factor is the changing length of photoperiod throughout the annual cycle.
Other proximate factors as rainfall, food availability and temperature may
Endocrinology of Reproduction #
advance or delay the breeding period to varying degrees (Gibb 1950; Kluijver
1951; Immelman 1971; Wingfield 1980, 1983; Wingfield et al. 1983; Wingfield
and Farner 1993). Thus, most non-tropical birds have a very well-defined
annual breeding season which is regulated precisely by changing
photoperiod. This was first demonstrated in the junco (Junco hyemalis) by
Rowan in 1925. These types of environmental signals have been called initial
predictive information (such as photoperiod that initiates the reproductive
process and sustains it) and local predictive information that acts as an
inhibitor or accelerator (Fig. 5.3, see Wingfield et al. 1999).
Photoperiodism in birds involves a physiological system that regulates not
a change in responsiveness to day length, as it is often perceived, but a change
in the quality of the response to changing day length (Nicholls et al. 1988;
Wilson and Donham 1988). At one time of the year the neuroendocrine system
responds to the appropriate long day length with physiological cascade that
includes a dramatic increase in gonadotropin secretion, gonadal growth, and
a range of hormone-dependent processes, including behavior. This state of
responsiveness to long day lengths in birds is known as photosensitivity.
However, temperate-zone birds develop a change in response to these
stimulatory day lengths over time, so that at other times of the year the same
day length maintains a state of reproductive inactivity (for reviews see
Nicholls et al. 1988; Wilson and Donham 1988; Wingfield and Farner 1993;
Dawson et al. 2001). In birds, this state is known as photorefractoriness
following which GnRH cell bodies in the brain shrink and fibers emanating
from these towards the median eminence show a marked decrease in
immunocytochemical staining (Foster et al. 1987; Goldsmith et al. 1989;
Boulakoud and Goldsmith 1991). This has also been demonstrated in the
House sparrow (Hahn and Ball 1995). Thus, pituitary release of the
gonadotropins luteinising hormone (LH) and follicle-stimulating hormone
(FSH) is reduced to a minimum (Dawson and Goldsmith 1982, 1983) and the
gonads undergo marked regression (see Fig. 5.10). Parry and Goldsmith (1993)
have shown that increased synaptic input to the GnRH neurons is coincident
with the long-term photorefractory state. Other physiological effects
associated with photorefractoriness are a peak in plasma prolactin levels and
a post-nuptial moult (Goldsmith and Nicholls 1984a; b). Despite the lack of
gonadal activity and reproductive behavior during photorefractoriness, long
day lengths maintain this inactivity. Without a long day stimulus,
photorefractory birds develop what is termed photosensitivity, so that they are
once again able to grow their gonads in response to a long day stimulus. Seen
this way, it seems that the terms “photosensitive”, “photostimulated” and
“photorefractory” are somewhat misleading, given that the reproductive
system of photoperiodic birds is sensitive to, and exhibits a response to, the
ambient photoperiod no matter what it is; it is just the nature of the response
that changes from one time of year to another.
It might be envisaged that changing photoperiod determines when birds
can feed and how much they can take it. A decrease in food intake could be
Fig. 5.10 A. Immunocytochemically-localized gonadotropin-releasing hormone

(GnRH) neurons in the hypothalamus of Rufous-collared sparrows, Zonotrichia
capensis, from non-breeding (left panel) and breeding (right panel) individuals.
Note the increased number of neuronal processes and larger, more intensely
immunoreactive neurons in the breeding individual as compared to the non-
breeding individual. This reflects increased GnRH synthesis, transport and
release. B. Testes from non-breeding and breeding European starlings, Sturnus
vulgaris. Note the large change in testis volume (this mirrors the GnRH changes in
the brain) and the change in color (reason unknown but possibly a result of
circulating lymphocytes invading the regressing testis and depositing melanin).
Original.
responsible for such a dramatic change in reproductive state from one time of
year to another, but this is not the case. For example, in the European starling
(Sturnus vulgaris), photorefractoriness ensues as day length is still increasing
(Dawson and Goldsmith 1982) and therefore the opportunity to further
increase food intake still exists as photorefractoriness occurs. Also,
experimental manipulation with ad libitum food supply contradicts such this
theory. An example of this is a study carried out by Dawson et al. (1985). They
demonstrated that if non-photorefractory (i.e. photosensitive) starlings are
exposed to only seven “long” days, then this is often sufficient to provide the
photoperiodic drive for photorefractoriness to ensue some weeks later. It has
also been shown that the rate of onset of photorefractoriness is proportional to
the length of the photoperiod (Hamner 1971; Dawson and Goldsmith 1983). A
long day is that in which the duration of the light period is over and above a
“critical day length”, that being the length of day below which
photorefractoriness cannot be induced. The experiment by Dawson et al.
(1985) also indicates that even though photorefractoriness does not become
apparent for several weeks after exposure to long days, it is initiated rapidly
and the reproductive system continues to proceed towards a photorefractory
state regardless of subsequent photoperiod once initiation is complete. More
recently, Dawson (2001) provided evidence that a single long day can initiate
Endocrinology of Reproduction %
progression towards photorefractoriness. In the same way, a single long day

causes activation of the reproductive system in quail (Nicholls et al. 1983;
Meddle and Follett 1995), and song sparrows (Wingfield 1993).
Photorefractoriness is gradually dissipated by short days (i.e. day length
below the critical photoperiod), and “photosensitivity” is acquired. That is,
the bird’s gonads grow in response to long days again. In the wild,
photosensitivity is acquired in the autumn, when day length falls below
approximately 11.5 hr—resulting in some species what has been termed
“autumnal sexuality” (Murton and Westwood 1977; Lincoln et al. 1980;
Dawson 1983; Wingfield et al. 1987).
Birds that breed within the tropics (where 60% of birds live) have
traditionally been considered to be non-photoperiodic, since the annual
change in photoperiod is slight (Dittami and Gwinner 1985). Nevertheless,
tropical birds have been shown to be photoperiodic, e.g. African stonechats
(Saxicola torquata) (Gwinner and Scheuerlein 1999) and Zebra finches (Bentley
et al. 2000), although the experimental amplitude in photoperiod used in these
studies exceeded that of the tropics. However, two other studies suggest that
Spotted antbirds (Hylophylax naevioides) can respond to tropical changes in
photoperiod (Hau et al. 1998; Beebe et al. 2005). The strategy adopted by
tropical birds appears to be to remain in a physiological state of “readiness to
breed” for a large proportion of the year, and then to use non-photoperiodic
cues, such as rainfall or food abundance, to determine the exact time of
breeding. In zebra finches, for example, breeding can occur at any time of the
year, but breeding bouts are more intense during periods of rainfall (Zann et
al. 1995). Stonechats may respond to low light intensity as a predictive cue for
rainfall (Gwinner and Scheuerlein 1998). There is evidence that the HPG axis
of such birds remains for much of the year in a state somewhere between that
characteristic of photosensitivity and photostimulation in non-tropical
seasonally breeding birds, and that full functionality is triggered by the
relevant proximate cues (Perfito et al. 2006). Photorefractoriness does not seem
to occur in spotted antbirds (Beebe et al. 2005). Gonadal regression and molt
frequently follow breeding, but the extent to which the GnRH system is
“switched off” during this period is mostly unknown. One study on
Ecuadorean rufous-collared sparrows (Zonotrichia capensis) which experience
only 3 min photoperiod fluctuation over the year suggests that there are
changes in the GnRH system of a magnitude similar to that seen in obligately
photoperiodic birds (Moore et al., 2006).
Non-tropical northern hemisphere opportunistic breeders, such as
crossbills can breed at most times of the year (mainly between January and
August). Crossbills (Loxia curvirostra) feed on conifer seeds, the availability of
which appears to synchronize reproductive effort. However, these species do
have a fixed non-breeding period during fall, when the HPG axis appears to
be photorefractory and when molt occurs (Hahn et al. 1995b; Hahn 1998).
Even if opportunistic breeders are photoperiodic to a degree (Bentley et al.
2000), the relative importance of non-photoperiodic cues in “fine-tuning” the
timing of breeding varies and appears to be greater than that in obligately

photoperiodic species (Wingfield 1980).
5.6.2 Absolute and Relative Photorefractoriness

In birds that exhibit absolute photorefractoriness, e.g. European starling,
(Nicholls et al. 1988), and European rook (Corvus frugilegus) (Lincoln et al.
1980), breeding attempts are halted at a specific time of year and at a
consistent time after initial photoperiodic stimulation. At this point, the
gonads spontaneously regress, moult is initiated and hormonal changes occur
concurrently. There is a rise in plasma prolactin levels coincident with the
onset of moult (Dawson and Goldsmith 1982, 1983); plasma LH and FSH
levels decrease to a minimum as photorefractoriness occurs (Goldsmith and
Nicholls 1984a). Such changes occur even though day lengths are longer than
those which were originally stimulatory and often still increasing. In other
species where absolute photorefractoriness occurs—e.g. American crowned
sparrows (Zonotrichia sp.), Mallard (Anas platyrhynchos), Canary (Serinus
canarius)—changes associated with photorefractoriness happen when day
length is decreasing but is still longer than those at the time of reproductive
stimulation. Such a condition is termed absolute photorefractoriness because
no increase in day length, even up to 24 hr light per day, will cause a change
in the reproductive state once a photorefractory condition has been induced.
A condition similar to absolute photorefractoriness is relative photo-
refractoriness. The main difference is that once relative photorefractoriness
has been induced and the gonads have regressed, a subsequent substantial
increase in day length will once more initiate reproductive maturation—
without the need for a “sensitization”, or photosensitive, stage (Robinson and
Follett 1982). For example, if Japanese quail experience day lengths of over
11.5 hr, rapid gonadal development occurs. After about 3 months, and when
(in the wild) day length decreases below 14.5 hr, complete gonadal regression
occurs—in a similar manner to absolute photorefractoriness (Nicholls et al.
1988). However, if the day length is subsequently artificially increased further,
a full return to reproductive maturity occurs. Indeed, if quail are maintained
on any constant long day length, no form of photorefractoriness will be
elicited unless they experience a decrease in day length, for example, from 23
hr to 16 hr (Nicholls et al. 1988). This suggests a shift in the critical day length
in birds which are relatively photorefractory—a shift which appears to
depend on the photoperiodic history of such birds (Robinson and Follett
1982). There does not seem to be any change in critical day length in birds
that exhibit absolute photorefractoriness, regardless of their photoperiodic
history (Dawson 1987; Follett and Nicholls 1984, 1985)
The fact that there is such an apparent difference between bird species that
exhibit absolute photorefractoriness and those that exhibit relative
photorefractoriness, and that breeding seasons vary so widely among bird
species to suit their ecological needs has led to two schools of thought as to
how mechanisms controlling the termination of the breeding season have

evolved. The first school is that which argues for the separate evolution of
similar mechanisms in different bird groups (Farner 1964; Farner et al. 1977).
The idea behind such views is that although circadian periodicities became
established very early in evolution, the photoperiodic systems that have been
imposed upon them evolved much later and from multiple origins. The
alternative view is that proffered by Follett and Nicholls (1984, 1985), who
suggest that photorefractoriness has a single origin, but simple modifications
have made the phenomenon appear to be very different in separate bird
species. It is now generally accepted that relative photorefractoriness in quail
is closely related to absolute photorefractoriness in starlings, and that both
types of photorefractoriness involve long day-induced alterations to the
photoperiodic response; which will not occur without a functional thyroid
gland (Follett and Nicholls 1984, 1985). This explanation obviously dismisses
the idea of a change, or “shift”, in the critical day length. If critical day length
is shifted to a higher threshold during the onset of relative photorefractoriness
(i.e. more hours of light are required per day for gonadal maturation), then
gonadal regression is not long day-induced—as, by definition, critical day
length is the boundary between “long” and “short” days, with long and short
days having completely different photoperiodic effects. Similarities also exist
between photorefractoriness in juvenile and adult Red-legged partridge
(Alectoris graeca chukar), just as there are between juvenile and adult starlings
(Dawson et al. 1987; Williams et al. 1987; Creighton 1988). Homology has also
been suggested for the neural mechanisms controlling seasonal breeding in
Welsh mountain ewes and starlings (Nicholls et al.. 1988b). In addition, if
birds that exhibit absolute photorefractoriness are placed under short days
whilst in a photorefractory state, photosensitivity is regained over a period of
weeks. Before photosensitivity is fully regained, however, they are found to be
in a state of relative photorefractoriness (Hamner 1968; Nicholls et al. 1988a).
This demonstrates that the regaining of photosensitivity is a gradual process,
and it can be shown that the rate of recovery is proportional to the “shortness”
of short days, and to the length of time held on short days (Vaugien 1955;
Farner and Follett 1966; Steel et al. 1975; Turek 1975; Nicholls and Storey 1977;
Gwinner et al. 1988; Dawson 1991, Boulakoud and Goldsmith 1994, 1995).
5.6.3 Mechanisms Involved in the Detection and

Transduction of Day Length
Unlike other vertebrates, seasonally breeding mammals generally use a
combination of their eyes (retina) and pineal gland for photoreception and
transduction of the light signal (see Foster et al. 1989 for review). For example,
if sexually mature hamsters are blinded whilst held under long days, then
their gonads regress—even though they are still receiving the long-day light
stimulus (Reiter 1978). This indicates that ocular photoreceptors are
responsible for light detection and transduction. Although it has been unclear
as to the nature of the photoreceptors that fulfill this function (Foster et al.
1991), it appears that melanopsin is most likely involved (Hattar et al. 2003).
Non-mammalian vertebrates, on the other hand, transduce seasonal
changes in photoperiod via photoreceptors that are extraretinal and
extrapineal (see Groos 1982 for review). The first evidence for avian
extraretinal photoreceptors was shown by Benoit (1935a,b), who demon-
strated that simultaneous photostimulation of blinded and sighted ducks re-
sulted in equal testicular growth rates. Furthermore, the testicular response
could be abolished by covering the duck’s head with a black cap. Since then,
further experiments involving ducks (Benoit and Ott 1944), house sparrows
(Menaker and Keatts 1968) and American tree sparrows, Spizella arborea (Wil-
son 1991) have demonstrated convincingly that an extraretinal photoreceptor
participates in the reproductive responses of birds, and that there is little or
no retinal involvement in reproductive responses (Underwood and Menaker
1970; see also Foster and Follett 1985). Thus in birds, the detection of light for
reproductive purposes occurs via an extraretinal, hypothalamic pathway
(Menaker 1971). In an elegant experiment, Yokoyama et al. (1977) used fiber
optics to illuminate discrete areas of the hypothalamus and found that the
infundibular nucleus appeared to contain most photoreceptors in Zonotrichia
l. gambelii.
Despite numerous studies, it is still unclear precisely as to where the
photoreceptors lie in the avian brain. There is some evidence that extraretinal
photoreceptors may reside in the infundibular region of the hypothalamus
(Oliver and Baylé 1982) and in the parolfactory lobe of quail (Sicard et al.
1983). Additionally, Foster et al. (1985) demonstrated the involvement of a
rhodopsin-like photopigment, which has a maximum spectral photosensitiv-
ity at 492 nm (Foster and Follett 1985). More recent research involving
immunostaining of opsin (a protein involved in signal transduction by light
activation) has added weight to the idea that the deep brain photoreceptors
are located in the hypothalamus (in this case, the tuberal hypothalamus of the
Ring dove (Streptopelia risoria), duck and quail—Silver et al. 1988; Saldanha et
al. 2001). Thus, it appears that in birds, light must pass through the skull and
into the hypothalamus for transduction of its signal to occur.
For a “long day” signal to result in photostimulation, day length must be
measured with a high degree of accuracy. Birds, unlike mammals, do not seem
to require the melatonin as a time signal (from the pineal gland) to control
reproductive changes in response to day length (but see later section on
gonadotropin-inhibitory hormone, or GnIH). The physiological mechanism(s)
underlying measurement of day length are still unknown, but there are two
main models for which there are some supporting experimental data. The first
is the “hour-glass” model, in which it is hypothesized that an “hour-glass”-
like timer is set in motion by the onset of dusk or dawn, and a photochemical
accumulates during either the light or the dark phase. If a sufficient amount of
the photochemical accumulates, then a photoperiodic response is initiated.
The fundamental property of the hour-glass model is that it requires constant
resetting by the light/dark cycle. Thus, it would not operate under conditions
of constant light or constant dark. There is evidence that such a system
operates in some insects (for examples see Beck 1968; Lees 1973; Vaz Nunes
and Veerman 1984, 1986; Veerman et al. 1988). The second theory, which has
been shown to hold true for birds, was first proposed by Bünning in 1936 (see
also Pittendrigh and Minis 1964, 1971; Follett 1973; Elliott 1976). It assumes
that there is a circadian rhythm of responsiveness to light, i.e. for the first part
of the cycle (subjective day) the organism is insensitive to light; whereas
during the second part (subjective night) it becomes “photoinducible”. Should
light fall during the subjective night, then a “long-day” photoperiodic
response is initiated. Evidence that this “external coincidence model” applies
to avian photoperiodicity was first supplied by night-interruption
experiments on the House finch (Carpodacus mexicanus) (Hamner 1963, 1964),
which has been reinforced by experiments on other bird species (for examples
see Follett and Sharp 1969; Turek 1974; Follett et al. 1992). Another model of
how photoperiodic responses are initiated by day length is termed the
“internal coincidence model” (Pittendrigh and Minis 1964). This model
assumes that photoperiod entrains two circadian oscillators, one entrained by
dusk and the other by dawn, with photoperiodic time measurement a function
of the phase relationship between the two clocks. Thus, when the phase of the
two oscillators coincides, photoinduction occurs. Although there is little
experimental evidence to help distinguish between the latter two hypotheses,
Follett et al. (1974) in an elegant experiment looking at a surge in plasma LH
induced by the coincidence of light with the photoinducible phase in
Zonotrichia l. gambelii, showed that the responsiveness to light had a strong
circadian component with peak LH responses between 12 and 18 hours after
the subjunctive dawn. This is consistent with the external coincidence model.
Other experiments on Zonotrichia sp suggested that circadian rhythms in
corticosterone and prolactin represented internal coincidence because
injections of these hormones at different times of day had different effects on
breeding, fattening and migration (Meier et al. 1965; Meier 1972; Meier and
McGregor 1972).
Evidence against the external coincidence model comes from Bentley et al.
(1998), in an experiment where light intensity, rather than length of
photoperiod, was manipulated. Few investigations have incorporated
manipulation of light intensity rather than day length to affect reproductive
responses since Bissonnette’s study in 1931. It is difficult to draw definitive
conclusions from most early experiments on this subject, as mixtures of
natural and artificial light were used in the same days of treatment
(Bissonnette 1931); others omitted to control for different wavelengths of light
from sources of differing intensities (e.g. Bissonette and Wadlund 1933) [the
importance of wavelength in the avian photoperiodic response has been
established for some time (Ringoen 1942; Benoit and Ott 1944)]. There are even
reports of both low and high light intensities causing convulsions and death
in the African weaver finch (Rollo and Domm 1943)! So far, the most
comprehensive experiment of this type to have been carried out was by

Bartholomew (1949), in which house sparrows were subjected to different
light intensities under the same photoperiod. His study concluded that light
intensity can indeed modify the reproductive response of sparrows to day
length, but that there is a minimum intensity below which no photoperiodic
response can be evoked. In addition, there is an upper intensity threshold
above which there is no increase in rate of response, and intensity cannot be
substituted for day length per se. Work carried out on mammals indicates that
there are also light intensity effects on the mammalian endocrine system, such
as in ferrets (Marshall and Bowden 1934) and pigs (Griffith and Minton
1992). In the experiment by Bentley et al. (1998), photosensitive starlings
transferred from short days to long days of different light intensities
underwent graded reproductive responses according to the light intensity
they experienced. Testes size in the group in the lowest intensity (3 lux)
increased faster than that in controls on short days of normal intensity, but
they did not become photorefractory. Testes size increased in the groups on
13, 45, and 108 lux and subsequently became photorefractory. However, the
13- and 45-lux groups required more time to become photorefractory than did
the 108-lux group. The responses observed were similar to those seen in
starlings exposed to different photoperiods (e.g. 11 hr light:13 hr dark
[11L:13D], 13L:11D, 16L:8D, 18L:6D), even though all were on the same
18L:6D photoperiod. Initially, the results appear to challenge the external
coincidence model for photoperiodic time measurement, but consideration of
the phase response curve of the circadian rhythm of photoinducibility in
starlings and the way in which it might be affected by low light intensities
refute this challenge and reinforce the external coincidence model (see Bentley
et al. 1998 for discussion). For further details on recent advances in our
understanding of the molecular and cellular intricacies of the avian circadian
system, see Dawson et al. 2001; Brandstatter 2003).
5.6.4 Physiological Mechanisms that have been Suggested to

be Involved in the Control of Photorefractoriness
A seasonal change in hypothalamic sensitivity to gonadal steroid feedback
(Cusick and Wilson 1972; Sharp and Moss 1977; Stokkan and Sharp 1980a,b)
considers the idea that as gonadal steroid feedback increases under long days,
the hypothalamus becomes increasingly sensitive to this feedback and “shuts
down” completely, resulting in photorefractoriness. This was shown not to be
the case because gonadectomised Zonotrichia l. gambelii and European
starlings became photorefractory in the same way, and within the same time-
course as intact birds (Wingfield et al. 1980; Dawson and Goldsmith 1984).
The conclusion was drawn that gonadal steroid feedback is unimportant in
the induction and maintenance of photorefractoriness. Also, long days
override the inhibitory effect of gonadal steroids on hypothalamic GnRH.
There is no decrease in hypothalamic sensitivity to the inhibitory effects of
steroids (inhibition must increase as steroid output from the gonads increases
with long days) but the “photoperiodic drive” is increased to a greater level
than the inhibition by the steroids, producing a net positive effect (Matt 1983;
Nicholls et al. 1984). Furthermore, Wilson (1985) has demonstrated that in
American tree sparrows, hypothalamic sensitivity to inhibition by gonadal
steroids decreases, rather than increases, following exposure to long days.
It is possible that an inhibitory hormone terminates reproduction and
prolactin was thought to be a strong candidate for this (Dawson and
Goldsmith 1982; Ebling et al. 1982; Goldsmith 1983; Dawson and Goldsmith
1983), as there is always a peak in the level of plasma prolactin coinciding
with the physical manifestations of photorefractoriness. This theory is
unlikely, however, as the administration of exogenous prolactin does not on
its own cause the onset of photorefractoriness (Goldsmith 1985), although this
experiment was carried out using heterologous prolactin. Further evidence
against the idea that prolactin is involved in the control of photorefractoriness
is that if photosensitive starlings are thyroidectomized and transferred from
short to long days, photorefractoriness does not occur, and there is no rise in
plasma prolactin levels (Dawson and Goldsmith 1984). This indicates that
prolactin probably does not have a causal role in the instigation of
photorefractoriness, as the pituitary is not damaged by thyroidectomy and
hence the prolactin-secreting cells are intact (Goldsmith and Nicholls 1984b;
Dawson et al. 1985). However, this approach does not take into account the
fact that thyroid hormones may be required as “permissive” agents for the
release of prolactin as a result of photostimulation, thus resulting in
photorefractoriness.
It can be shown that prolactin is not responsible in itself for gonadal
regression. If photosensitive starlings are kept under a photoperiod of 11
hours of light and 13 hours of darkness per day (11L:13D), slow but complete
reproductive maturity ensues (Hamner 1971; Goldsmith and Nicholls 1984c).
With 11L, photorefractoriness does not occur because this is below the critical
day length for this species. When the birds are subsequently transferred back
to short days (6-8L), the gonads regress in size, but there is no increase in
plasma prolactin levels (Goldsmith and Nicholls 1984c). If such 11L birds are
put in to long days (18L) instead of being returned to short days,
photorefractoriness occurs, along with the associated rise in plasma prolactin.
Thus, prolactin is not responsible for gonadal regression in short days, but is
associated somehow (perhaps only temporally) with regression during
photorefractoriness. The timing of high levels of circulating prolactin is also
closely linked to plumage moult, and, where it occurs, premigratory fattening
and migration (Meier and MacGregor 1972; Meier 1972; Dawson and
Goldsmith 1983). A clear-cut experiment demonstrating that prolactin is not a
causative agent of photorefractoriness was performed by Dawson and Sharp
in 1998. European starlings were actively immunized against vasoactive
intestinal polypeptide (VIP), the prolactin releasing hormone in birds, or
against prolactin, during a photo-induced breeding cycle. VIP-immunized
birds became photorefractory but the rate of gonadal regression was markedly
slowed, and the photo-induced increase in prolactin was completely

suppressed in 50% of these birds. Molt was prevented in those birds in which
prolactin release was completely suppressed. In those VIP-immunized birds
in which the photo-induced increase in prolactin was inhibited but not
completely suppressed, gonadal regression was delayed, but molt occurred as
normal. The same was true for prolactin-immunized birds. There were no
significant differences in concentrations of plasma thyroxine between
treatment and control groups, indicating that the effects of immunization on
gonadal regression were not mediated by the induction of hypothyroidism.
Thus, in European starlings the associated increase in prolactin accelerates
gonadal regression during the onset of photorefractoriness but does not itself
cause photorefractoriness. In addition, the increase in prolactin associated
with photorefractoriness is required for the induction of the postnuptial molt
(Dawson and Sharp 1998). Following on from this, Dawson et al. (2002)
demonstrated that VIP as measured by radioimmunoassay did not change
over a photoperiod-induced cycle in European starlings. Therefore,
photorefractoriness in terms of prolactin secretion is not similarly related to a
decrease in basal hypothalamic VIP.
An exhaustion of some component of the hypothalamo-hypophysial axis
(Nicholls and Storey 1976) is a third hypothesis. The idea that some
component of the hypothalamo-hypophysial axis has, once stimulated, a
limited period of activity and then requires a recovery period to “build up
reserves” does fit in many ways with the annual reproductive cycle of birds
that exhibit photorefractoriness. One suggestion was, broadly, that the
hypothalamic GnRH-synthetic cells have a constant rate of GnRH synthesis
and that it is only the secretion rate that fluctuates during the annual change
in photoperiod (Nicholls and Storey 1976). The proposal is that secretion is
only permitted under long days (but some “leaks” out when the
neurosecretory GnRH cells are “full”—permitting photosensitivity), and that
under long days, secretion occurs at a greater rate than synthesis. Thus, the
rapid secretion of GnRH allows photorefractoriness to occur. A time lag is
subsequently needed (under short days, so that there is no secretion) for
GnRH reserves to “ build up” prior to exposure to long days in the spring.
They can then be released in sufficient quantities to elicit a reproductive
response.
The above hypothesis can be seen to tally with the responses shown by
starlings to long days when in a relatively photorefractory state - i.e. when
photorefractory birds are put into short days and after total
photorefractoriness has been lost, but before full photosensitivity has been
acquired. What is seen is that photorefractoriness is induced more rapidly
than in fully photosensitive birds, so it could be construed that GnRH levels
have not had time to build up to their maximum. Also, the fact that the rate of
onset of photorefractoriness is proportional to the duration of the prevailing
photoperiod (Hamner 1971; Dawson and Goldsmith 1983) could be assumed
to agree with this hypothesis. But since it has been shown that changes in
hypothalamic content of GnRH occur after gonadal regression has been

initiated, it seems improbable that the exhaustion hypothesis is tenable
(Nicholls et al. 1988). In addition, there is the fact that thyroidectomy permits
sexual maturity to be maintained, perhaps indefinitely on long days
(Wieselthier and van Tienhoven 1972; Goldsmith and Nicholls 1984b;
Dawson et al. 1985). This appears to demonstrate that without a higher neural
“switching” process occurring, GnRH continues to be synthesized and
secreted at a high rate during appropriate photoperiodic stimulation. Other
experiments involving the use of exogenous thyroxine or a thyroid-dependent
neurotrophin to mimic the effect of long days in reducing hypothalamic GnRH
content (Boulakoud and Goldsmith 1991) also dispute the “exhaustion”
hypothesis (Bentley et al. 1997a,b). Other experiments in which GnRH-I was
injected into photorefractory male Zonotrichia l. gambelii resulted in a dramatic,
but short-lived, increase in LH also argues against the exhaustion hypothesis
(Wingfield et al. 1979). Similar results were obtained following injection of the
excitatory amino acid agonist NMDA injected into photorefractory
Zonotrichia l. gambelii (Meddle et al. 1999).
The state of photorefractoriness can also be “broken”, or dissipated, by the
removal of circulating thyroid hormones—i.e. thyroidectomy. This technique
has shown that both the initiation and maintenance of photorefractoriness are
dependent upon the presence of thyroxine (Wieselthier and van Tienhoven
1972; Goldsmith and Nicholls 1984b). Termination of the breeding condition
by thyroidectomy has also been observed in other vertebrates—e.g. tree
sparrows (Wilson and Reinert 1993; 1995a,b), sheep (Moenter et al. 1991; Dahl
et al. 1994; Parkinson and Follett 1994; Parkinson et al. 1995) and red deer (Shi
and Barrell 1992).
Although the presence of thyroxine is required, and plasma thyroxine
levels rise markedly upon a starling’s transfer from short to long days
(Dawson 1984), and also in quail (Sharp and Klandorf 1981), it is likely that
thyroid hormones are not the cause of photorefractoriness. It appears that
thyroxine is a permissive factor allowing the mechanisms that cause the
reproductive system to “switch off” under long days to function, and may be
involved in the perception of day length (Dawson 1989a,b; Bentley et al.
1997a,b). Similar suggestions have been made for the termination of breeding
in sheep (Dahl et al. 1995). It has not been clear as to whether the long day-
induced rise in circulating thyroxine is a necessary precursor of the
photorefractory response, or if this rise is associated with other long day
responses, such as increased metabolic rate. Importantly, thyroidectomy does
not affect the daily or circadian pattern of circulating melatonin
concentrations (Dawson and King 1994).
At another level, it has been shown that in photostimulated Zonotrichia l.
gambelii, glial cell process do not interpose between axon terminals and
capillaries of the portal system in the median eminence, but they do in
photorefractory birds (see Fig. 5.11, from Bern et al. 1966; Farner et al. 1967;
Mikami et al. 1978) The same appears to be true for Japanese quail (Yamamura
et al. 2004).
5.7 A NEW LEVEL OF CONTROL OF AVIAN REPRODUCTION:

NOVEL PEPTIDES
5.7.1 A Third Type of GnRH
A recent study (Bentley et al. 2004) presented evidence for the presence of an
immunoreactive third GnRH in songbirds that is clearly hypophysiotropic
and has gonadotropin-releasing capabilities. This third GnRH, immunoreac-
tive (ir)-lamprey GnRH-III, possibly has multiple functions, as suggested by
its widespread distribution. In addition, ir-lamprey GnRH-III is present in
abundance in telencephalic areas, including the hippocampal formation and
the song control system. In no vertebrate has a GnRH been localized in these
“higher” control regions before, although fragments of mammalian GnRH
have been detected in primate forebrain, and their functions are unknown
(Terasawa et al. 2001). In fact, we are aware of only two studies that have
investigated the GnRH system concurrently with the oscine song control sys-
tem (MacDougall-Shackleton et al. 2001; Marsh et al. 2002), most likely because
the distribution of ir-chicken GnRH-I and -II are so distant from telencephalic
areas (Juss et al. 1992; Millam et al. 1993). The finding that a third GnRH is
likely involved in regulation of reproductive function in songbird species has
implications for the way in which we envisage the avian brain processes
environmental cues and transduces them into endocrine signals. We will
understand this putative peptide more when it is isolated and sequenced from
the songbird brain.
5.7.2 Gonadotropin-Inhibitory Hormone (GnIH)

Since the molluscan cardioexcitatory neuropeptide Phe-Met-Arg-Phe-NH2
(FMRFamide) was found in the ganglia of the venus clam (Price and
Greenberg 1977), neuropeptides that possess the RFamide motif at their C-
termini (i.e., RFamide peptides) have been characterized in various
invertebrates (see Tsutsui and Ukena 2006 for review). Subsequently, many
immunohistochemical studies that used the antiserum against FMRFamide
suggested that vertebrate nervous systems possess some unknown
neuropeptides similar to FMRFamide (Raffa 1988; Rastogi et al. 2001).
Immunohistochemical findings indicated that some of the FMRFamide-like
immunoreactive neurons projected to the hypothalamic region close to the
pituitary gland, and thus were expected to play an important role in the
regulation of pituitary function (see Tsutsui and Ukena 2006 for review).
Tsutsui et al. (2000) therefore looked for a novel RFamide peptide in the avian
brain.
Amino acid sequence analysis of a novel isolated substance revealed the
following sequence: Ser(62)-Ile(252)-Lys(233)-Pro(226)-Ser(38)-Ala(194)-
Tyr(173)-Leu(148)-Pro(104)-Leu(108)-Arg(45)-Phe(52) with the detected
amount (pmol) of each amino acid indicated in parentheses. The isolated
native peptide in the quail brian was confirmed as a 12 amino acid sequence
(SIKPSAYLPLRFamide) with RFamide at the C-terminus (Tsutsui et al. 2000).
This neuropeptide had not been previously reported in vertebrates, although
Fig. 5.11 A. and B. Posterior median eminence (PME) with a primary capillary
(PCp) of a photostimulated male white-crowned sparrow, Zonotrichia leucophrys
gambelii. The capillary is separated from the median eminence by two layers of
basement membrane (Bm) and is covered by perivascular cells (PVC) and/or
pericytes (Pc). Note that glial cells (Gc) do not interpose between axons with
neurosecretory material and the basement membranes and endothelial cells (En).
From Mikami et al. (1970) Zeitschrift für Zellforschung 106: 155-174, Springer-
verlag, Berlin, Fig. 3; see also Bern et al. 1966. Zeitschrift für Zellforschung 69: 198-
227, Springer-verlag, Berlin, Fig. 13. C. Electron micrograph of the ventral region of
the posterior median eminence of a photorefractory male white-crowned sparrow.
Axons contain small vesicles (Ve) and a few dense granules (Gr). A layer of glial
processes (Gl) is interposed between the axons and the basement membrane
region (Bm) of the portal capillary. An erythrocyte is at lower left. Mi = mitochondria.
From Farner, D. S., Wilson, F. E. and Oksche, A. (1967). Pp. 529-582. In L. Martini
and W. F. Ganong (eds.), Neuroendocrinology, vol. 2. Academic Press, New York,
Fig. 10.
the C-terminal LPLRFamide was identical to chicken pentapeptide

LPLRFamide peptide (Dockray et al. 1983). Subsequently, the isolated novel
peptide was shown to be located in the quail hypothalamo-hypophysial
system and to decrease gonadotropin release from cultured anterior pituitary
in a dose-dependent manner (Tsutsui et al. 2000). This novel RFamide peptide
was therefore named as gonadotropin-inhibitory hormone (GnIH) (Tsutsui et
al. 2000).
Using immunohistochemistry, clusters of distinct GnIH-immunoreactive
(-ir) neurons were found in the paraventricular nucleus (PVN) in the
hypothalamus. In addition to the PVN, some scattered small cells were
immunoreactive in the septal area (Tsutsui et al. 2000; Ukena et al. 2003;
Ubuka et al. 2003). In contrast to the highly-localized clusters of cell bodies,
GnIH-ir nerve fibers were widely distributed in the diencephalic and
mesencephalic regions. Dense networks of immunoreactive fibers were found
in the ventral paleostriatum, septal area, preoptic area, hypothalamus, and
optic tectum. The most prominent fibers were seen in the median eminence
(ME) of the hypothalamus, and in the dorsal motor nucleus of the vagus in the
medulla oblongata.
We further investigated GnIH localization in the brain of seasonally-
breeding songbird species (Bentley et al. 2003; Osugi et al. 2004). Dense
populations of GnIH-ir neurons were also found in the PVN of Song
sparrows, House sparrows, White-winged crossbills (Loxia leucoptera), Pine
siskins (Carduelis pinus), Redpolls (Carduelis flammea), Rufous-collared
sparrows (Zonotrichia capensis), Rufous-winged sparrow (Aimophila carpalis)
and Gambel’s white-crowned sparrows. The PVN was the only location
where immunoreactive neurons were located, regardless of sex or species
(data only published for four species in this list: Bentley et al. 2003; Osugi et al.
2004; Deviche et al. 2006). Thus the presence of GnIH in the PVN appears to be
a conserved property among several families and at least two orders of birds
(Galliformes and Passeriformes). In addition to the dense population of GnIH-
ir neurons within the hypothalamus of all the avian species studied so far,
there were extensive networks of branching beaded fibers emanating from
those cells, presumably transporting GnIH. Some of the fibers extended to
terminals in the ME, consistent with a role for GnIH in pituitary gonadotropin
regulation. In house sparrows, song sparrows and Gambel’s white-crowned
sparrows, other fibers extended through the brain caudally at least as far as
the brainstem and possibly into the spinal cord, consistent with multiple
regulatory roles for GnIH (see Fig. 5.12).
Tsutsui’s group further examined the precursor polypeptide for GnIH and
localization of its transcript, which would provide key information as to the
regulation of the mature GnIH peptide, along with confirmation of brain
area(s) that synthesize this novel peptide. A cDNA that encoded the GnIH
precursor polypeptide was identified in the quail brain by a combination of 3'
and 5' rapid amplification of cDNA ends (3'/5' RACE) (Satake et al. 2001). The
deduced GnIH precursor consisted of 173 amino acid residues that encoded
one GnIH and two putative GnIH-related peptide (GnIH-RP-1 and GnIH -RP-
2) sequences that included -LPXRF (X = L or Q) at their C-termini. All of these
peptide sequences were flanked by a glycine C-terminal amidation signal and
a single basic amino acid on each end as an endoproteolytic site.
In collaboration with Tsutsui’s group, we also cloned a cDNA that encoded
GnIH in the brain of Gambel’s white-crowned sparrow (Osugi et al. 2004). The
deduced sparrow GnIH precursor also consisted of 173 amino acid residues,
encoding one sparrow GnIH and two sparrow GnIH-related peptides
(sparrow GnIH-RP-1 and GnIH-RP-2) that included -LPXRFamide (X = L or
Q) at their C-termini. Although the homology of sparrow and quail GnIH
precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-
Fig. 5.12 A. Sagittal section from female house sparrow, Passer domesticus,
brain with dense GnIH immunoreactivity in the PVN. B. Higher magnification of the
PVN of male house sparrow brain in sagittal section showing individual GnIH-ir
neurons and fibers emanating from the cell bodies. C. Sagittal section showing
GnIH-ir fibers in the fascisculus longitudinalis medialis (FLM) of the brainstem of a
female house sparrow. Arrows indicate GnIH-ir fibers. Cb= cerebellum. D. Higher
magnifications of C. Scale bars, 100 mm. Original.
RP-1 and GnIH-RP-2 were all identical in two species (Satake et al. 2001;
Osugi et al. 2004). Subsequently, a cDNA encoding GnIH and GnIH-RPs was
also reported in the chicken from a gene database (see Tsutsui et al. 2005 for
review). The chicken LPLRFamide peptide (Dockray et al. 1983) is considered
to be a fragment of GnIH and GnIH-RP-1) (see Tsutsui et al. 2005 for review).
In situ hybridization further revealed the cellular localization of GnIH
mRNA solely in the PVN of quail and sparrow hypothalami (Ukena et al.
2003; Osugi et al. 2004). As already discussed, immunohistochemical analysis
using the quail and sparrow also showed that quail and sparrow GnIH-ir cell
bodies and terminals were localized in the PVN and ME, respectively. Thus
only the PVN expresses GnIH and, in birds, the immunoreactive peptide
found in fibers in multiple brain areas appears to originate from the PVN only
(Ukena et al. 2003; Osugi et al. 2004).
5.7.3 Relative Distributions of GnIH and GnRH

The cell bodies for each population of neurons containing chicken GnRH-I
(cGnRH-I), GnIH or cGnRH-II are in discrete locations. The preoptic area
(POA) contains cGnRH-I-ir neurons, the PVN contains GnIH-ir neurons and
the cGnRH-II neurons are located in the midbrain (Millam et al. 1993). Thus
we can confidently distinguish between two forms of GnRH based upon their
location in the brain and their appearance. Closer inspection of each area
indicates close proximity of GnIH fibers to the cGnRH-I neurons and fibers in
the POA in songbirds. The PVN also contains cGnRH-I fibers which pass
directly through and in close proximity to the population of GnIH neurons
and fibers as they project to the ME. GnIH-ir fibers are also in close proximity
to cGnRH-II neurons in the midbrain (Bentley et al. 2003). Further, confocal
microscopy indicates that the cGnRH-I and GnIH peptides are located within
the same 0.2 micron optical plane and possibly in contact with one another,
although electron microscopy will be necessary to determine contact
conclusively. Contact appears to occur between GnIH fibers and GnRH-I and
-II neurons, and between GnIH and GnRH-I fibers in the ME (Bentley et al.
2003).
Taken together, there is potential for GnIH to influence the GnRH system at
the neuron and fiber terminal levels. Furthermore, when song sparrows were
subjected to a simulated annual cycle of changing photoperiod, GnIH-ir
neuron area was significantly greater at the onset of photorefractoriness (long
days) when compared to photosensitive (short days) or photostimulated (long
days) birds. Thus there is potential for dynamic interactions of GnIH and
GnRH peptides in different reproductive states and neuroanatomical
locations. It is clear that further study is needed to elucidate the seasonal
dynamics of GnIH synthesis and release and its relation to seasonal changes
in GnRH in songbirds.
5.7.4 Actions of GnIH on Gonadotropin Synthesis and

Release in Galliformes
Using cultured quail anterior pituitaries, GnIH significantly inhibited LH
release, after 100-min incubation (Tsutsui et al. 2000). The inhibitory effect on
LH release was dose-dependent and its threshold concentration ranged
between 10–9 and 10–8 M. There was no effect of GnIH on prolactin release.
Addition of a physiological dose (10–7 M) of GnIH to short-term (120 min)
cultures of diced pituitary glands from adult cockerels suppressed common a
and FSH b subunit mRNAs, with no effect on LH b subunit mRNA (Ciccone et
al. 2004). The suppressive effect of GnIH on gonadotropin mRNA was
associated with an inhibition of both LH and FSH release in the adult chicken
(Ciccone et al. 2004). When administered intraperitoneally to quail in vivo via
osmotic pumps, GnIH significantly reduced gonadotropin common a and LH
b subunit mRNAs in a dose-dependent manner as well as reducing plasma
LH (Ubuka et al. 2006).
To elucidate the mode of action of GnIH, a novel G protein-coupled receptor
for GnIH was identified in quail (Yin et al. 2005). The identified GnIH receptor
was expressed in the pituitary and specifically bound to GnIH in a
concentration-dependent manner (Yin et al. 2005). Southern blotting analysis
of reverse-transcriptase-mediated PCR products revealed the expression of

GnIH receptor mRNA in the pituitary and several brain regions including the
hypothalamus in the quail (Yin et al., 2005). These results indicate that GnIH
acts directly on the pituitary via GnIH receptor to inhibit gonadotropin
synthesis and release. GnIH may also act on the hypothalamus to inhibit the
activity of GnRH neurons.
To clarify the functional significance of GnIH and its potential role as a key
neuropeptide involved in avian reproduction, GnIH actions on gonadal
development and maintenance were investigated in the male quail (Ubuka et
al. 2006). Continuous administration of GnIH to mature quail via osmotic
pumps decreased plasma testosterone concentrations (Ubuka et al. 2006).
Interestingly, administration of GnIH to mature quail induced testicular
apoptosis and decreased spermatogenic activity in the testis (Ubuka et al.
2006). GnIH administration to immature quail also suppressed normal
testicular growth and rise in plasma testosterone concentrations (Ubuka et al.
2006). These results indicate that GnIH inhibits gonadal development and
maintenance through the decrease in gonadotropin synthesis and release.
GnIH may participate not only in neuroendocrine function but also in
behavioral and autonomic mechanisms. Because GnIH neurons are localized
in the PVN, with their fibers visible in multiple brain locations including the
ME and brainstem, the action of GnIH on feeding behavior was further
investigated in chicks (Tachibana et al. 2005). Intracerebroventricular (i.c.v.)
injection of GnIH stimulated food intake in chicks. The chicken pentapeptide
LPLRFamide, a degraded C-terminus of GnIH did not stimulate feeding
thereby demonstrating the importance of the N-terminus of GnIH for the
orexigenic effect. Anti-GnIH antiserum suppressed appetite induced by
fasting. These data suggest that GnIH acts as an endogenous orexigenic factor
in the brain of chicks. Food restriction induces not only an increase of appetite
but also a reduction of the HPG axis activity (Richard-Yris et al. 1987). These
changes also occurred after GnIH treatment, since GnIH stimulated feeding
behaviour (Tachibana et al. 2005) and inhibited the HPG axis (Tsutsui et al.
2000; Osugi et al. 2004; Ciccone et al. 2004; Ubuka et al. 2006).
5.7.5 Seasonal Dynamics and Functional Significance of

GnIH in Passerines
Once we had identified the White-crowned sparrow GnIH precursor, we were
able to synthesize putative White-crowned sparrow GnIH (“sparrow” GnIH)
with the deduced amino acid sequence SIKPFSNLPLRFamide. We used this
peptide for in vivo analysis on gonadotropin release in field-caught White-
crowned sparrows during their breeding season in northern Alaska. Birds
that were injected with GnIH had lower plasma LH at 2 min than the saline-
injected group (Osugi et al. 2004). In contrast, saline-injected birds had high
plasma LH (approx 5 ng/ml) 2 min post-injection. This difference was short-
lived and had dissipated by 10 min.
As already mentioned, the distribution of GnIH in the avian brain suggests

that it has not only hypophysiotropic actions but also unknown behavioral
actions. GnIH fibers are present in the ME, and are in apparent contact with
chicken GnRH (cGnRH) -I and -II neurons and fibers. In birds, cGnRH-I
regulates pituitary gonadotropin release, whereas cGnRH-II is known to
enhance copulation solicitation in estradiol-primed females exposed to male
song (Maney et al. 1997). In a recent study (Bentley et al. 2006), we determined
the effects of GnIH administered centrally to female white-crowned sparrows.
A physiological intracerebroventricular (i.c.v.) dose of GnIH rapidly reduced
circulating LH and inhibited copulation solicitation, without affecting
locomotor activity. Using rhodaminated GnIH delivered i.c.v., putative GnIH
binding sites were seen in the ME close to GnRH-I fiber terminals, and in the
midbrain on or close to GnRH-II neurons. These data demonstrate direct
effects of GnIH upon reproductive physiology and behavior, possibly via
separate actions on two forms of GnRH.
Taken together, our results indicate that, despite amino acid sequence
differences, sparrow GnIH and quail GnIH (see Osugi et al. 2004) have similar
inhibitory effects on the reproductive axis in wild sparrow species. Overall,
GnIH appears to be a modulator of gonadotropin release in vivo as well as in
vitro. Taking the immunohistochemical data into consideration as well, there
is potential for GnIH to act on the reproductive axis at the level of the GnRH
neurons, the ME, and the pituitary, and thus possibly over different time-
frames. Furthermore, the wide distribution of GnIH terminal fields coupled
with the fact that GnIH can influence reproductive behavior implies that there
are as yet unknown actions of GnIH or GnIH-related peptides upon
physiology and behavior.
5.7.6 Regulation of GnIH Expression in the Brain

Until now, a regulatory mechanism(s) governing GnIH expression has
remained unclear. We have already mentioned that although many bird
species are photoperiodic, a dogma has existed that birds do not use seasonal
changes in melatonin secretion to time their reproductive effort, and a role for
melatonin in birds has remained enigmatic (Wilson 1991; Juss et al. 1993).
Despite the accepted dogma, there is strong evidence that melatonin is
involved in regulation of several seasonal processes, including gonadal
activity and gonadotropin secretion (Ohta et al. 1989; Bentley et al. 1999;
Bentley and Ball 2000; Bentley 2001; Guyomarc’h et al. 2001; Rozenboim et al.
2002). In light of these reports and considering GnIH’s inhibitory effects on
gonadotropin secretion (Tsutsui et al. 2000; Osugi et al. 2004), we investigated
the action of melatonin on GnIH expression in the quail brain (Ubuka et al.
2005). Pinealectomy combined with orbital enucleation (Px+Ex) decreased the
expression of GnIH precursor mRNA and the mature peptide GnIH in the
diencephalon including the PVN and ME. Melatonin administration to Px+Ex
birds caused a dose-dependent increase in expression of GnIH precursor
mRNA and production of mature peptide. The expression of GnIH was
photoperiodically controlled and increased under short day photoperiods
(Ubuka et al. 2005), when the duration of melatonin secretion increases

(Cockrem and Follett 1985; Kumar and Follett 1993). Finally, Mel1c, a
melatonin receptor subtype was expressed in GnIH-ir neurons in the PVN
(Ubuka et al. 2005). Thus melatonin appears to act directly on GnIH neurons
via its receptor to induce GnIH expression.
5.8 CONCLUSIONS
In conclusion we hope we have demonstrated, at least in part, how complex
are the mechanisms that control avian reproduction. Readers of this chapter
should also bear in mind that the recent discoveries of new hormones in this
field highlight that although a great deal is already known about neural
integration of environmental information, there is a great deal still waiting to
be discovered. This latter comment could easily be applied to other vertebrate
classes.
New tools at our disposal, such as those in molecular biology and
proteomics will undoubtedly hasten our discovery of novel peptides and
control mechanisms. However, the avian Class is so diverse, and their
reproductive systems and strategies so varied that future avian biologists will
undoubtedly be puzzling over some of the same questions that we have raised
in this chapter. Comparative studies on different species in different
vertebrate classes, such as those described in this chapter will enable us to
gain a better understanding vertebrate reproductive biology as a whole. For
example, studies on birds have uncovered several “firsts” in reproductive
biology; the discoveries of GnRH-II and GnIH are two prime examples. Both
of these neuropeptides appear to be relevant to reproduction in all vertebrates
studied, including humans. Another “first” is the dramatic adult
neurogenesis and neuroplasticity in the song control system of songbirds
(Nottebohm 1981). Again, this paved the way for exciting insights into adult
neuroplasticity in mammals, once believed not to exist.
5.9 ACKNOWLEDGMENTS
Preparation of this review was supported by grants IBN-0317141 and OPP-
9911333, from the National Science Foundation and by R01 MH65974-01 from
the National Institutes of Health and by 15207007, 16086206 and 18107002
from the Ministry of Education, Science and Culture, Japan.

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CHAPTER
6
Ovarian Dynamics and
Follicle Development
A.L. Johnson and Dori C. Woods
6.1 INTRODUCTION
The avian ovary represents a truly dynamic organ system capable of fostering
the annual development of one or more broods of viable eggs, then undergoing
nearly complete regression followed by eventual recrudescence (see also
Chapter 4 and Volume 6B, Chapter 13). The seasonal initiation of ovarian
recrudesence may be driven by endogenous circannual rhythmicity and
synchronized by environmental cues, the most important of which is
photoperiod and to a more limited extent, light intensity (Gwinner 2003). Such
cues are translated into neuroendocrine and endocrine signals, the primary
factors being hypothalamic gonadotropin releasing hormone (GnRH) and
pituitary gonadotropins, respectively. Subsequent events, which include
nesting, follicle maturation and ovulation, are generally initiated by factors
other than light (e.g., rainfall, temperature, food availability, male behaviors).
A unique morphological and functional aspect of the reproductively active
avian ovary, as compared to the mammalian counterpart, is that follicles at all
stages of development, from resting primordial and primary follicles to the
fully differentiated preovulatory stage, exist simultaneously during egg-
laying. As a consequence, the sequential selection of one undifferentiated
follicle into the final rapid growth stage of development provides for ovulation
of an oocyte from a fully differentiated follicle on an approximate daily basis
(the interval between ovulations is species-dependent). The ovarian follicular
hierarchy is a reflection of oviparity, and is a feature held in common with
avian predecessors, the reptiles and apparently some dinosaurs (Sato et al.
2005).
Department of Biological Sciences, The University of Notre Dame, Notre Dame, IN 46556,
USA
While there are some excellent field studies documenting seasonal changes
in reproductive hormones and comparing ovarian dynamics among free
ranging species (e.g., see review Wingfield and Farner 1993), the majority of
information pertaining to cellular and molecular mechanisms regulating
follicle growth and differentiation has been derived primarily from
domesticated birds. This is largely due to the quantity and ready availability
of tissues required for detailed studies, but nevertheless leaves open the
question as to what extent models of ovarian organization and function
developed from genetically-manipulated avian models maintained under
well-controlled environmental and nutritional conditions directly pertain to
wild birds. Accordingly, the following represents a discussion of the avian
ovary literature from a comparative and integrative perspective. A primary
objective is to highlight some of the many unresolved questions that can assist
both field and bench biologists in developing more refined working models of
ovarian function to better understand the remarkably diverse and successful
reproductive strategies attributed to avian species.
6.2 OVARY MORPHOLOGY AND DEVELOPMENT

The ovary is loosely attached to the peritoneal cavity by the mesovarian
ligament (the hilus) at the cephalic end of the kidney, and is suspended from
the dorsal body wall by a peritoneal fold, the mesovarium. As in mammals,
the avian ovary is covered by a single layer of surface epithelium that displays
characteristics associated with the potential for rapid proliferation, and
conversely, cell death by apoptosis (Chalana and Guraya 1979). These
accessory tissues accommodate the annual growth and regression of the
ovary, and in particular, the rapid growth of preovulatory follicles followed
by reabsorption of the postovulatory follicle.
Unlike the mammalian ovary, where developing follicles largely remain
embedded within the cortex, avian follicles beyond the primordial stage of
development protrude from ovarian stromal tissue much like grapes in a
bunch (Figs. 6.1-6.3). This pendulate architecture enables large amounts of
yolk to be incorporated within the follicle during the rapid growth phase. The
most prominent features of a reproductively active ovary are the developing
follicles that serve to: 1) incorporate liver-derived yolk within the oocyte; 2)
provide structural support for growth of the large oocyte (especially compared
to that in therian mammals); 3) synthesize and secrete innumerable paracrine
and autocrine factors that maintain oocyte viability and promote follicle
differentiation (including steroidogenesis), or alternatively promote apoptotic
cell death; and 4) release the oocyte in a timely fashion at ovulation.
The potential for annual nutritional investment required for follicle growth
and maintenance during development is illustrated by the 32 to 43% increase
in body mass of the Common eider (Somateria mollissima) from prior to ovarian
recrudescence compared to that during peak seasonal reproductive activity.
In a few instances, an important consequence of this significant investment
Ovarian Dynamics and Follicle Development "#
Fig. 6.1 Left ovary of the domestic hen (Gallus gallus) illustrating the hierarchal
structure of ovarian follicles. Under environmentally controlled conditions the laying
hen will continue to lay eggs on a daily basis for a year or longer with a typical
clutch size of 6 to 20+ eggs. F1-5 represent preovulatory follicles 12 to 40 mm in
diameter) that have been selected to enter the rapid growth phase of development.
SYF, small yellow prehierarchal follicles (6 to 8 mm diameter); SWF, small white
prehierarchal follicles (1 to 5 mm). POF, postovulatory follicle. Not readily visible
are primordial and primary follicles (<1 mm). Reprinted from Johnson, A.L. 1990.
Critical Reviews in Poultry Biology 2: 319-346. Fig. 1.
towards ovarian growth during the reproductive season may be temporary

flightlessness and increased risk of predation (Guillemette and Ouellet 2005).
The ovary itself increases in weight by greater than 100-fold from the
beginning of recrudescence to the peak of lay, and virtually all of this weight
represents the growth of yolk-filled follicles (Gilbert 1979).
6.2.1 Development and Asymmetry

The left and right ovaries are present during the early embryonic stages of
presumably all birds, but in many species only a functional left ovary is
maintained post-hatch. By comparison, some species commonly maintain both
gonads (e.g., a number of falcons, eagles, vultures, and the kiwi [Apteryx]; Fig.
6.3), yet these functional ovaries may be asymmetrical in size (the left
Fig. 6.2 Ovary from the Gentoo penguin (Pygoscelis papua) at an early and late
stage of the breeding cycle. Clutch size in the gentoo is typically two eggs, laid with
an interval of 3 to 4 days. A. Prehierarchal (non-vitellogenic) follicles prior to follicle
selection. B. Preovulatory follicle, with prehierarchal follicles destined to become
atretic. Reprinted from Valencia, J. and Leyton, V. 1992. Gonadal cycles of
Pygoscelis penguins of the South Shetland Islands. Pp. 198-207. In W.C. Hamlett
(ed.), Reproductive Biology of South American Vertebrates. Springer-Verlag, New
York, Fig. 14-3.
Ovarian Dynamics and Follicle Development "%
characteristically being the larger). It has been reported that in various birds,
including sparrows, gulls, doves and pigeons, a small percentage of
individual specimens may maintain two active ovaries through adulthood
(Kinsky 1971).
The left ovary consists of intermingling medullary and cortical tissues (the
ovarian stroma), and this supportive tissue is abundantly supplied with
blood vessels. Primordial and early developing primary follicles are clustered
in distinct masses of cortical tissue. The undeveloped right ovary is composed
mainly of medullary tissue with small patches of cortex, and as such,
resembles a testis. Removal of the dominant left ovary or exposure to
endocrine disruptors (particularly estrogenic compounds) during early
embryo development can result in the development of a testis or ovotestis, both
of which may be capable of producing spermatozoa (Gilbert 1979; Yoshimura
and Fujita 2005).
In birds, the female represents the heterogametic sex, with sex
chromosomes designated as ZW, while the male is homogametic (ZZ).
Although the basic mechanism for genetic sex determination in birds is still
unknown, there is evidence that candidate regulatory genes reside on both the
W and Z chromosomes (Smith and Sinclair 2004) (see also Chapter 25). For
instance, a functional W chromosome is required for regulating aromatase
enzyme expression during early embryogenesis (day 5 to 6 of incubation in
Gallus domesticus). Activity of this enzyme results in the synthesis of estrogen,
and is prerequisite for the development of a functional left ovary (Bruggeman
et al. 2002). The effects of estrogen are mediated by estrogen receptors, which
are selectively expressed on the left, but not right, gonad in females by day 7.
A phenotypic sex reversal in genetic males is affected by treatment, in ovo,
with estrogen, while administration of an aromatase inhibitor to genetic
females before day 7 will promote the differentiation of testes.
6.2.2 Primordial Germ Cells, Migration and Proliferation

During Early Embryogenesis
Primordial Germ Cells (PGCs) originate in the epiblast within the central zone
of the area pellucida at the early somite stage of chicken embryo development.
Following their translocation to the germinal crescent region, the number of
PGCs has been estimated at 200-250 (Tsunekawa et al. 2000). Avian PGCs
migrate from the germinal crescent to blood vessels by diapedesis and are
proposed to exit ovarian blood vessels in response to chemotactic signals from
the germinal ridge (Kuwana et al. 1986). This route of PGC transport distinctly
differs from that of amphibians and mammals, where PGCs reach the germinal
ridge via an extravascular migration. In the incubating pheasant (Phasianus
colchicus) embryo the number of circulating PGCs is maximal by 64 h of
development (somite number, 21-23), and dramatically decreases thereafter.
This decline in circulating PGCs is directly correlated with an increase in the
number of germ cells populating the embryonic ovary (Kim et al. 2005). Once
PGCs take up residence within the germinal ridge of the ovary, they become
Fig. 6.3 Paired ovaries from the adult North Island brown kiwi (Apteryx mantelli).
Although the left ovary is normally larger and contains a greater number of
developing follicles than the right, the number and size of follicles in both ovaries is
often similar early in the breeding season. Clutch size in the kiwi is typically one
egg, but a second and occasionally a third egg may be laid after intervals of about
25 days. LO, left ovary; LOV, left oviduct; RO, right ovary. Reprinted from Kinsky F.C.
1971. Journal of Ornithology 112: 334-357, Fig. 1, with permission.
Ovarian Dynamics and Follicle Development "'
mitotically active oogonia. Although specific factors implicated in PGC

survival, chemotaxis and proliferation have, to date, been insufficiently
studied in any avian species, a recent report has directly implicated the
chemokine, stromal cell-derived factor-1 (SDF-1), in the latter stages of PGC
migration (Stebler et al. 2004).
In teleost, amphibian and some reptilian species that are capable of
spawning hundreds to millions of ova within a reproductive season, the
ovarian oogonia constitute self-renewing stem cells that continue to proliferate
post-hatch and post-puberty. By comparison, in avian and mammalian
species that produce considerably fewer eggs, the oogonia divide a finite
number of times and produce the maximum available number of egg precursor
cells by the time of hatch or birth. The resulting cells progress through the first
meiotic prophase to the diplotene stage, at which point they are called
primary oocytes, and are maintained in an arrested state until puberty. The
final germ cell number is apparently dependent upon the local hormonal
environment, as follicle stimulating hormone (FSH) and estradiol treatments
during embryonic development have been reported to delay meiotic arrest and
increase the overall size of the oocyte pool (Xie et al. 2004). Resumption of
meiosis and completion of the first meiotic division occurs only shortly before
the fully matured ovarian follicle is destined to ovulate.
The total number of primary oocytes within the domestic chick ovary has
been estimated to increase from some 28,000 on the ninth day of incubation to
680,000 on the seventeenth day, then abruptly decrease to 480,000 by the time
of hatching or shortly thereafter. Only a small fraction of these primary
oocytes (200-500) subsequently develops to the preovulatory stage within the
lifespan of most domesticated species, and considerably fewer mature to this
stage in wild species. Ultimately, the fate for the vast majority of these
potential eggs, both prior and subsequent to hatch, is death via apoptosis (see
section 6.3.4).
Current dogma dictates that by the time of, or shortly after, hatch, no
additional number of germ cells can be attained or generated by the avian
ovary. A recent publication, however, has challenged this concept in
mammals by providing evidence that female germ line stem cells also take up
residence in the bone marrow, and that these stem cells continually migrate to
the ovary via the circulatory system over much of the female’s reproductive
lifespan (Johnson, Bagley et al. 2005). Should this concept of germ cell renewal
prove true in the avian ovary, there would exist numerous, significant
implications towards the potential for enhancing the propagation of
threatened and endangered species.
6.2.3 Sexual Maturation

Many species of wild birds hatch their eggs in spring or early summer, and
the offspring attain adult body size within a few weeks. In many respects, the
ovary of such fully-grown juveniles appears comparable in size and morphol-
ogy to that of photorefractory adults (Williams et al. 1987). Nevertheless, these
offspring do not attain sexual maturity until the following breeding season
presumably because their photoperiodic responsiveness is comparable to the
photorefractory state of adult birds. Note that opportunistic breeders represent
an exception to this paradigm (see section 6.2.5). By comparison, some larger,
long-lived birds, including albatrosses, condors and penguins, may take 5 to
10 years to reach sexual maturity.
6.2.4 Seasonal Reproduction

The onset of each reproductive season in free-range birds can typically be
separated into two distinct phases of ovarian growth, each with different
environmental cues controlling their onset (Jacobs and Wingfield 2000). In
photoperiodic species such as the sparrow (Zonotrichia), the first, or
proximate, phase is initiated by a lengthening photoperiod that increases
hypothalamic secretion of GnRH, followed by increases in circulating levels
of FSH. The initial growth of the ovary culminates after approximately six
weeks, when follicles reach a size of 2 to 3 mm in diameter but are as yet
undifferentiated (‘sub-functional’). Female sparrows will generally not
progress to the second phase of rapid growth and final differentiation until
appropriate supplementary information is provided. While the source of this
second set of supplementary cues is dependent upon species and latitude at
which it breeds, such ultimate factors likely include: the availability of food,
suitable nesting conditions, and interactions with a male. For example,
exposure to male song alone has been reported to augment the rate of follicle
development in White-crowned sparrows (Zonotrichia leucophrys) (Morton et
al. 1985).
During the reproductive season, avian species will develop and ovulate a
characteristic number of eggs (a clutch) prior to the initiation of incubation.
While there is the tendency for larger birds to lay fewer eggs, the evolutionary
forces that dictate clutch size remain elusive. For instance, clutch size is often
smaller in tropical and sub-tropical birds compared to those in temperate
northern climates, despite the prediction that abundant food availability in
tropical regions should provide for optimal egg laying. A recent study by
Martin et al. (2000) attempted to reconcile clutch size as a function of food
availability compared to the risk of predation. Their data support the
hypothesis that higher rates of nest predation in subtropical regions serve as
a constraint to the rate at which parent birds can provide food to the nestlings,
thereby limiting the number of young they can feed, and by implication, clutch
size. Alternative, and not necessarily mutually exclusive, hypotheses
implicate physiological constraints (e.g., large clutches place more
physiological strain on females than small clutches), plus seasonal and
habitat constraints as factors regulating clutch size. In lesser snow geese
(Anser caerulescens), it has been proposed that the clutch size can be adjusted to
the female’s nutrient reserve plane at a time just before or at the onset of laying.
This adjustment can occur by the reabsorption of the smallest developing
follicles, and assures adequate development of the remaining follicles plus a
Ovarian Dynamics and Follicle Development #
favorable nutrient plane for the nesting female. The loss of potential offspring
is offset by a higher probability of successful fledging (Hamann et al. 1986).
Many species will produce a single clutch per season (determinant layers;
e.g., white pelican, Pelecanus erythrorrhynchos, blue-winged teal, Anas discors,
barn swallow, Hirundo rustica). Others may produce more than one clutch
and/or extend laying within a clutch, particularly if the clutch is destroyed or
the first eggs of the clutch are removed (indeterminant layers; e.g., European
starling, Sturnis vulgaris, song sparrow, Melospiza melodia, common flicker,
Colaptes auratus, barn owl, Tyto alba). There is evidence that the extension of a
clutch in indeterminant breeders can be accomplished by the rescue of small
preovulatory follicles from atresia, rather than the selection of additional
follicles into the preovulatory hierarchy (Challenger et al. 2001). Finally,
species that invest a prolonged period of time to raise their young may not
breed every year (e.g., king penguin, Aptenodytes patogonica; some species of
albatrosses, Diomedeidae). This strategy is typically associated with
advanced age of sexual maturity (albatrosses as late as 10 years of age) and
longevity of adult survival (Jouventin and Dobson 2002).
6.2.5 Opportunistic Breeders

Free-ranging birds that live in areas where environmental changes are cyclical
and highly predictable typically exhibit regular phases of annual ovarian
recrudescence, regression and refractoriness. By comparison, those species
that live in regions where environmental changes are less predictable or
entirely unpredictable, lack appropriate proximal factors to cue ovarian
recrudescence in a timely fashion. Accordingly, such species must
opportunistically respond to ultimate factors as rapidly as possible to insure
the coincidence of fertility with optimal breeding and rearing conditions.
One example of an opportunistic breeder is the Zebra finch (Taeniopygia
(=Poephila) guttata) that has adapted to the semi-arid and arid regions of
Australia. Female finches are reported to undergo ovarian growth beginning
shortly after hatch and continuing throughout the first 100 days. Thereafter,
the developing ovarian follicles enter an indefinite resting stage where,
following an appropriate ultimate cue (e.g., rainfall), selected follicles can enter
the rapid growth phase and be ovulated within one to two weeks (Sossinka
1980). In contrast to photoperiodic species (McNaughton et al. 1992), the
precocious ovarian development observed in the zebra finch following hatch
is attributed to the absence of any detectable refractory period.
Moreover, the White-winged crossbill (Loxia leucoptera) inhabits an Alaskan
region where its primary food source, conifer seeds, can vary in quantity from
year to year. Results from endocrinological studies (including the seasonal
content of hypothalamic GnRH) support the view that these birds have the
potential to respond to ultimate factors and to breed throughout the year
depending upon food availability, except for the period of fall molt when
elevated levels of prolactin presumably suppress ovarian activity (Deviche
and Sharp 2001).
These two examples further illustrate the adaptiveness of birds to extreme

and variable environmental conditions, but also raise important questions as
to how the ovary maintains an appropriate milieu to support long-term
prehierarchal follicle viability (e.g., prevent cellular apoptosis and consequent
atresia over a period from weeks to months), and how the presence of ultimate
factors becomes translated into appropriate cellular signals to initiate follicle
selection into the rapid growth phase. Such questions can be appropriately
addressed only in free-ranging birds, and the mechanisms will likely be
species-dependent.
6.2.6 Reproductive Aging and Ovarian Senescence

Reproductive aging can be defined as any decline in reproductive
performance, and includes a decrease in fitness of the offspring (Saino et al.
2002). By comparison, ovarian senescence more specifically constitutes a
significant decline or loss of the primary follicle reserve within the ovary,
and/or the significant loss of oocyte quality (capacity for fertilization).
Reproductive senescence is often preceded by a gradual decline in egg
production. Associated with this decline in the domestic hen is an overall
increase in atresia specifically in follicles within the slow growth phase, and
a decreased rate of follicle selection into the preovulatory hierarchy
(Waddington et al. 1985).
Holmes et al. (2003) have described female reproductive aging in birds
according to three paradigms. The first is characterized by a short life span
with relatively rapid declines in fertility (e.g., many Galliformes, including
domesticated species). A second consists of moderately slow aging birds with
a long life span (greater than 10 years) where the slow decline in reproductive
success correlates closely with increased mortality (e.g., passerine songbirds,
including the Great tit, Parus major, and small raptors like the European
sparrowhawk, Accipiter nisus). The third model is characterized by long
life spans, slow aging, and negligible reproductive declines in either sex.
For instance, neither the Common tern (Sterna hirundo; Nisbet et al. 1999)
nor the Nazca boobie (Sula granti; Anderson and Apanius 2003) appears
to show an appreciable decline in reproductive effort even after 20 years
of reproductive activity. Some terns and gulls have been reported to
actually increase the number of offspring they successfully fledge with
advancing age, though this may be more related to post-hatch survival
and parental behaviors than to enhanced ovarian fecundity. These
proposed models, though not yet rigorously tested, raise important questions
regarding the role of the ovarian environment in promoting long-term germ
cell survival and viability. Unfortunately, there appears to be no published
information on primary oocyte reserves with aging in species which have
differing rates of reproductive longevity, nor of cellular mechanisms that
might account for the apparent variability in the onset of ovarian senescence
among avian species.
Ovarian Dynamics and Follicle Development #!
6.2.7 Vascularization and Nervous Innervation of the Ovary

The entire process of follicle growth can be conveniently divided into: 1) the
organization of primordial follicles and the early growth of primary follicles,
the latter of which may persist for years; 2) the recruitment of primary follicles
into the slow growth phase (the prehierarchal or previtellogenic stage) lasting
weeks to perhaps months; and 3) the selection and rapid growth of
preovulatory follicles which occurs during the final days preceding ovulation.
The continued viability and development of ovarian follicles within a
breeding season is critically dependent upon endocrine factors (e.g.,
gonadotropins) circulated by the blood, paracrine and autocrine factors
produced within the ovary itself, plus neurochemical and neurohumoral
factors expressed by the nervous system.
Blood supply to the avian ovary generally originates from the renal artery
and exits the ovary directly into the caudal vena cava. Prior to the initiation of
growth, primary follicles remain embedded within the highly vascular
stromal tissue and receive blood from adjacent capillaries. To service growing
follicles that begin to extrude from the stromal tissue, the ovarian artery
supplies two to four smaller arteries that enter the follicle via the follicular
stalk (or pedicle). While small arteries and arterioles extend throughout the
follicle theca layers, there is no direct blood supply to the inner granulosa cell
layer (Fig. 6.4). Not unexpectedly, blood flow is proportionately greatest to the
most mature follicles within the preovulatory hierarchy. In most species, an
avascular stigma region forms where the follicle fuses with the surrounding
surface epithelium, and serves as the site of rupture at ovulation.
Lymphocytes and macrophages circulate throughout ovarian stromal tissue
and the follicle theca layer (Barua and Yoshimura 1999), and these cells
represent a potential source of cytokines that can influence follicle viability
(see section 6.3.4.2). Immune competence of tissues within growing and
postovulatory follicles is implied by the reported expression of major
histocompatibility complex (MHC) molecules within both the theca and
granulosa layers of follicles throughout development (Subedi and Yoshimura
2005).
The ovary is well innervated by both adrenergic and cholinergic fibers.
Nerve fibers enter through the pedicle and radiate through the theca (but not
granulosa) layer. The number of neurons within the theca layer increases as a
follicle progressively matures. It is clear that such innervation provides a
variety of neurochemicals (e.g., catecholamines) and neurohumoral factors
(e.g., neurotropins, vasoactive intestinal peptide, substance P) to the ovary and
developing follicles. Such factors are proposed to function in diverse roles
such as regulation of steroidogenesis in the embryonic ovary (Muller-
Marschhausen et al. 1988), the organization and recruitment of primary
follicles (Gilbert 1979), and the growth and differentiation of growing follicles
in the adult (Johnson et al. 1994; Jensen and Johnson 2001). More generally, it
is likely that ovarian innervation plays an integral role in the seasonal
CMYK
Fig. 6.4 Organization of domestic hen ovarian follicles during development.

Primary oocytes enclosed by the vitelline membrane become organized into a
primordial follicle (up to ~80 m diameter) following the recruitment of presumptive
granulosa cells, and the perivitelline membrane is subsequently formed by
granulosa cells. The initiation of primordial follicle growth to the primary follicle
stage is associated with the formation of the theca layer (from mesenchymal cells),
CMYK
CMYK
which is separated from the granulosa layer by the basal lamina. Primary follicles
range in size from .08 to 1 mm in diameter. Further growth to the prehierarchal
follicle stage (1-8 mm) entails the accumulation of lipoprotein-rich, white yolk, plus
the differentiation of the theca into interna and externa layers. Vasculature and
nervous innervation reaches the follicle through the pedicle and radiates through
the theca layer. Following selection into the preovulatory hierarchy, preovulatory
follicles rapidly grow from 9 mm to 40 mm over the course of days. Granulosa cells
from preovulatory follicles facilitate the uptake of large amounts of vitellogenin and
very low density lipoprotein, except within the germinal disc region. Ovulation of the
largest preovulatory follicle eventually occurs at the region of the comparatively
avascular stigma region. See text for additional details.
recrudescence of the ovary. Of interest is one report that functional

innervation persists in the regressed right ovary of the hen (Ohmori et al.
1994). While the physiological significance of such innervation has not been
established, it may contribute to the generation of testicular tissue following
abnormal loss of the functional left ovary during early development.
6.3 STEROIDOGENESIS AND CELLULAR MECHANISMS

MEDIATING FOLLICLE GROWTH AND DIFFERENTIATION
Unfortunately, the majority of information pertaining to cellular and molecu-
lar mechanisms regulating follicle growth, selection and final differentiation
is derived from a limited number of domesticated species (e.g., Gallus gallus,
Coturnix japonica, Meleagris gallopavo). Nevertheless, such studies have begun
to provide an understanding of ovarian processes common to the vertebrate
CMYK
Ovarian Dynamics and Follicle Development ##
archosaur and synapsid lineages, plus those unique to the oviparous bird. It
is emphasized, however, that the working models presented will undoubtedly
require modifications to accommodate both reproductive seasonality under
free-ranging conditions, and the diversity of reproductive strategies among
birds. Additional, detailed discussions of circulating hormonal profiles
relative to the ovulatory cycle, cell signaling, steroidogenesis, and follicle
selection can be found elsewhere (Wingfield and Farner 1993; Johnson 1990,
2000; Woods and Johnson 2005).
6.3.1 Primordial and Primary Follicles

Beginning shortly after hatch, primary oocytes become organized into
primordial follicles following the recruitment of a single somatic (presumptive
granulosa) cell layer (Fig. 6.4); oocytes that fail to become enclosed die via
apoptosis. Granulosa cells of primordial follicles are cuboidal, densely packed
and coupled via tight junctions. Such multifunctional cells have been
implicated in a number of critical processes during follicle growth and
differentiation, including meiotic arrest, perivitelline membrane formation,
gonadotropin-induced hormone production, mediation of yolk protein
(vitellogenin) uptake, and transfer of maternal RNAs to the egg. Primordial
follicles do not increase in size appreciably and have the potential to survive
in this arrested state of development for years.
The transition to a primary follicle represents the stage when primordial
follicles are recruited from the resting state to begin slow growth. While a
variety of growth factors have been implicated in initiating (e.g., stem cell
factor, basic fibroblast growth factor) or inhibiting (anti-Müllerian hormone)
follicle recruitment in mammals (Skinner 2005), these have apparently not
been investigated in the avian ovary. Mesenchymal cells are subsequently
recruited by one or more factors presumably secreted from granulosa cells
(possibly including stem cell factor), to form a peripheral theca layer. The
granulosa and theca layers are separated by an acellular basement membrane
(basal lamina) that effectively isolates the granulosa layer from receiving a
direct supply of blood and nervous tissue (Fig. 6.4). The morphology of the
basal lamina and its relationship to the granulosa layer have recently been
described by Asem et al. (2000). The single layer of theca will eventually
differentiate into morphologically and functionally distinct theca interna and
externa layers. While primary follicle growth is proposed to occur
independent of gonadotropins, follicle viability remains dependent upon the
support of both gonadotropin and growth factors (Johnson et al. 1996b). This
earliest growth phase can progress over several years, and in the turkey the
overall growth is from approximately 80 mm to 1 mm in diameter, primarily
via an increase in the size of the oocyte without an increase in the germinal
vesicle (Carlson et al. 1996).
Primordial follicles and early developing primary follicles remain
embedded within the ovarian stromal tissue, and thus lack a surrounding
epithelial cell layer. The collective stromal tissue containing such follicles
expresses both FSH and luteinizing hormone (LH) receptors, and is

steroidogenically active when challenged with LH, in vitro, as short term
incubations produce progestin, androstenedione and estradiol (Levorse and
Johnson 1994). Moreover, isolated primary follicles (<1 mm) produce both
androgen (dehydroepiandrosterone and androstenedione) and estradiol in
response to LH treatment (Robinson et al. 1988). While the amount of
gonadotropin-induced steroid production at this stage of development is not
particularly impressive on a per follicle basis, the cumulative steroid
production is considered physiologically significant based upon the large
total number of such follicles present within the ovary. The expression of
gonadotropin receptors or steroidogenic enzymes within the seasonally
regressed ovary has apparently not been evaluated in any species, however it
is predicted that resting ovarian follicles remain competent to produce some
level of steroids, even during the nonbreeding season.
6.3.2 Prehierarchal Follicles

A second, comparatively more rapid phase of growth lasting for a period of
weeks to perhaps months occurs as previtellogenic (1-5 mm diameter
prehierarchal follicles) follicles begin to incorporate small amounts of a
lipoprotein-rich, white yolk. More follicles than are ultimately required to
establish the preovulatory hierarchy are recruited into this growth phase, thus
the fate of many prehierarchal follicles is atresia (see section 6.3.4). Although
the exact rate of atresia among all prehierarchal follicles has not been
precisely estimated, it is assumed that it is rather high, perhaps up to 20%
(Gilbert et al. 1983). Conditions that can promote atresia at this stage include
inadequate growth factor and gonadotropin support of survival, and genetic
or morphological defects.
As the follicle continues to grow it protrudes from the surface of the stromal
tissue and becomes enveloped by the ovarian epithelium. Internally, an
acellular perivitelline layer (homologous to the zona pellucida of mammals)
forms from the granulosa cells and appears as electron-dense granules. The
granulosa cells themselves are densely packed and form projections that
extend into the oocyte plasma (vitelline) membrane. The theca has now
differentiated into morphologically and functionally distinct externa and
interna layers. By the end of this prehierarchal phase, follicles in Gallus and
Zonotrichia reach the size of 6 to 8 mm and 2 to 3 mm, respectively. In free-
ranging birds, the termination of this slow growth phase corresponds to the
‘sub-functional’ level of development as described by Jacobs and Wingfield
(2000). Much of the lipoprotein-rich yolk incorporated during the early phase
of prehierarchal follicle growth may transverse the granulosa cell layer either
by transcytosis or, perhaps more importantly, by a paracellular transport
route. Nevertheless, the amount of yolk transported is limited at this stage of
development by the tight junctions (and a major tight junction protein,
occludin) that serve to connect adjacent granulosa cells and restrict
paracellular transport (Schuster et al. 2004). Occludin expression is regulated
Ovarian Dynamics and Follicle Development #%
by the stimulatory effects of FSH and the transforming growth factor-b (TGFb)
superfamily member, activin. Eventually, the levels of occludin associated
with tight junctions gradually decrease such that progressively greater
amounts of yolk, including the lipid-rich yellow yolk, transverse the
granulosa cell layer to reach the oocyte. Thus, 6 to 8 mm prehierarchal follicles
begin to assume a yellow color (often referred to as small yellow follicles). A
single receptor type has been implicated in the uptake of the two major yolk
precursors, very low-density lipoprotein (VLDL) and vitellogenin (VTG), by
the oocyte (Schneider 1996). This oocyte-specific VTG/VLDL receptor is
initially localized within the central portion of the cell in slow growing
follicles, and rapid yolk uptake occurs only following its redistribution to the
oocyte vitelline membrane (Shen et al. 1993).
6.3.2.1 Granulosa and theca cells
The theca interna of prehierarchal follicles becomes both innervated and
vascularized, and initiates pregnenolone and androgen (largely
androstenedione) production. Structural support of the follicle is provided by
sheets of collagen fibers and fibronection plus smooth muscle located within
the theca externa, which gives this layer its stratified appearance (Fig. 6.4).
The externa layer contains groups of cells selectively expressing aromatase
activity, and represents the sole site within the follicle for the conversion of
androgens to estradiol (Nitta et al. 1991). Collectively, the theca layers express
comparatively high levels of both LH receptor (Johnson et al. 1996a) and FSH
receptor (You et al. 1996) mRNA. While treatment of whole prehirearchal
follicles or isolated theca layers with either LH or FSH, in vitro, promotes
secretion of progestin, androgen and estrogen, LH is comparatively more
potent (Robinson et al.,1988; Kowalski et al. 1991).
In contrast, granulosa cells from prehierarchal follicles are incapable of
synthesizing significant levels of the progesterone precursor, pregnenolone,
due to the absence of cytochrome P450 side-chain cleavage (P450scc) enzyme
activity. In addition, these cells lack detectable levels of the cholesterol
transport protein, steroidogenic acute regulatory (StAR) protein, required for
the transfer of cytoplasmic cholesterol from the outer to the inner
mitochondrial membrane (the site of P450scc activity), plus cytochrome P450
17a-hydroxylase (P450-17a-OH) required for androgen production. Granulosa
cells at this stage of development express FSH receptor (but not readily
detectable LH receptor) mRNA and produce the second messenger, cyclic
adenosine monophosphate (cAMP), in response to FSH treatment, in vitro.
Because StAR, P450scc and P450-17a-OH expression are primarily dependent
upon the cAMP/protein kinase A cell signaling pathway, the absence of
steroidogenesis in prehierarchal follicle granulosa cells, in vivo, has been
proposed to be the result of tonic inhibitory cell signaling at a site distal to
cAMP production (Woods et al. 2005). Nevertheless, it is important to note that
treatment of such granulosa cells with FSH, in vitro, initiates StAR, P450scc
and P450-17a-OH expression plus production of modest amounts of
progesterone and androstenedione (Li and Johnson 1993; Johnson et al.
2002b). Finally, granulosa cells from prehierarchal follicles demonstrate the

capacity for proliferation (Tilly et al. 1992), and this is promoted by one or
more members of the epidermal growth factor (EGF) family, FSH, and possibly
the TGFb superfamily member, growth differentiation factor 9 (GDF9). EGF
ligands represent paracine/autocrine factors produced within the oocyte, the
granulosa or theca layers of the follicle or ovarian stromal tissue, whereas
GDF9 is produced specifically by the oocyte (Yao and Bahr 2001; Johnson,
Dickens et al. 2005; Woods et al. 2005).
With regard to free-ranging species, plasma levels of estrogen in Zonotrichia
are observed to increase shortly after the females arrive at the breeding area
(Wingfield and Farner 1993). This increase in estrogen production occurs at a
time when the ovary presumably contains primarily prehierarchal follicles
(e.g., prior to when granulosa cells become steroidogenically active).
Accordingly, the capacity of the theca layer from prehierarchal follicles to
produce estrogen no doubt represents a mechanism to provide for the
stimulation of VTG/VLDL synthesis required for the imminent rapid growth
phase.
6.3.2.2 Follicle selection into the preovulatory hierarchy
Follicle selection represents the process that ultimately determines the
characteristic number of eggs to be ovulated (clutch size) by any given species.
A prehierarchal follicle selected into the preovulatory hierarchy immediately
begins the process of growth and final differentiation, and this is reflected by
both the ability of the follicle to rapidly incorporate yellow yolk, and of the
granulosa layer to initiate increasingly large (mg) amounts of progesterone
production. While the ultimate signal(s) and cellular events that provide a
single follicle the selective advantage to initiate rapid growth and final
differentiation have yet to be described in birds (or for that matter, any
vertebrate), the consequent differentiation that occurs within the granulosa
layer within the hen follicle immediately following selection has been the
subject of recent investigations.
Among the endocrine, paracrine and autocrine factors that have been
implicated in directly initiating steroidogenesis and/or enhancing
gonadotropin (FSH and LH) receptor expression in granulosa cells from
prehierarchal follicles are included FSH and the TGFb superfamily members,
TGFb and activin. Both TGFb and activin signal via type I plus type II receptor-
mediated phosphorylation of the Smad2 regulatory protein, and a primary
action in granulosa cells is to enhance FSH receptor expression (Johnson et al.
2004). The actions of activin, but not TGFb, can be modulated by the presence
of an activin-binding protein, follistatin.
However, as noted above, evidence supports the proposal that tonic
inhibition of FSH-mediated StAR, P450scc, and P45017a-OH expression, in
vivo, renders granulosa cells from prehierarchal follicles steroidogenically
incompetent. Such inhibitory signaling is also proposed to prevent any
increase in FSH receptor number and to preclude premature LH receptor
expression, thus maintaining all but one prehierarchal follicle in an
Ovarian Dynamics and Follicle Development #'
undifferentiated state despite the continued, daily exposure of all follicles to

circulating concentrations of FSH.
In fact, FSH-induced P450scc and StAR expression plus progesterone
production, in vitro, is completely blocked by EGF family ligands (including
EGF, transforming growth factor-a and betacellulin) signaling via the mitogen
activated protein kinase (MAPK)/extracellular regulated kinase (Erk)
pathway. Moreover, MAPK/Erk signaling blocks TGFb- and activin-induced
receptor signaling by preventing the phosphorylation required for Smad
signaling. Significantly, inhibition of Erk signaling using the selective MAP
kinase inhibitor, U0126, not only promotes a synergistic enhancement of StAR
expression and progesterone production compared to FSH treatment alone,
but also promotes greatly enhanced expression of the FSH receptor plus the
initiation of LH receptor expression (Woods and Johnson 2005). Accordingly,
a model depicting events initiated within the granulosa layer immediately
following the selection of a prehierarchal follicle into the preovulatory
hierarchy is presented in Fig. 6.5.
6.3.3 Preovulatory Follicles, Ovulation and Postovulatory Follicles
The preovulatory phase is the shortest, and perhaps most characteristic of the
avian ovary, and culminates with ovulation. In general, its duration tends to
be associated with species size, weight of the egg and precociousness of the
newly hatched bird. For instance, the rapid-growth phase typically lasts
between 6 and 11 days in the domestic hen and barn owl, approximately
12 days in the Canada goose (Branta canadensis) and up to 16 days in some
penguins, but only 5 to 7 days in the Ring-necked dove (Streptopelia capicola)
and Japanese quail (Coturnix japonica) (Gilbert 1979). By comparison, the
Brown kiwi (Apteryx mantelli) has one of the largest egg-to-body ratios and the
most yolk as a percentage of total egg weight (61%) among birds. Accordingly,
the rapid growth phase is estimated to occur over a period of some 15 to
17 days and results with an egg size of some 80+ mm. Combined with a
comparatively long, 70- to 90- day incubation period (conducted exclusively
by the male), these adaptive characteristics enable the kiwi embryo to hatch at
an advanced state of development (Jensen and Durrant 2006).
Considerably more work has been conducted to characterize cellular
mechanisms mediating rapid growth (e.g., yolk incorporation) and final
differentiation (e.g., steroidogenesis) in preovulatory follicles compared to
those follicles prior to selection, and only an updated summary will be
provided here.
6.3.3.1 Preovulatory follicles
Rapid growth of preovulatory follicles occurs largely due to the enhanced
capacity for VTG and VLDL uptake by receptor-mediated endocytosis. A
second receptor, with preference for binding LDL, is localized to granulosa
cells and the theca externa layer in preovulatory follicles, and selective
expression of this LDL receptor is associated with supplying LDL-derived
cholesterol as precursor for steroid production (Hummel et al. 2003). While the
CMYK

CMYK
CMYK
Fig. 6.5 Proposed model for cellular events mediating the initiation of
steroidogenesis and gonadotropin receptor expression in granulosa cells
immediately subsequent to follicle selection. A. Both type I and type II TGFb plus
Fig. 6.5 Contd. ...

CMYK
Ovarian Dynamics and Follicle Development $
majority of the oocyte surface is actively engaged in the uptake of yellow yolk,
the 2 to 3 mm diameter germinal disc (the portion of oocyte containing the
nucleus, most cellular organelles and the largest amount of cytoplasm)
remains visible as a white plaque due to the comparatively lower expression
of VTG/VLDL receptors. The comparative reduction of yolk incorporation at
this site presumably facilitates the migration of pronuclei and the process of
fertilization. As rapid follicle growth proceeds, granulosa cells distal to the
germinal disc undergo remodeling to become squamous in appearance and
less densely packed (Fig. 6.4) due to a reduction of cell-to-cell connections
(tight junctions). This latter change facilitates the paracellular transport of
some 2 g of yellow yolk per day into domestic hen preovulatory follicles.
Accordingly, follicle mass increases from approximately 0.15 g in 6 to 8 mm
prehierarchal follicles to some 12 to 14 g just prior to ovulation.
Increased production of the yolk precursors, VTG and VLDL, by the liver
occurs in response to increasing estrogen concentrations produced by the
developing follicles. The total energetic costs of yolk formation can account for
40% to 50% of the bird’s daily energy budget, and the extra energy required
results in a 13% to 41% increase in basal metabolic rate in passerines, to more
than a 200% increase in some waterfowl (Meijer and Drent 1999). Given that
such an investment of protein synthesis and the production of yolk precursors
are energetically expensive, it can be predicted that the supply of yolk
precursors should closely match the demand for vitellogenin uptake by
preovulatory follicles. In fact, plasma levels of vitellogenin in female zebra
Fig. 6.5 Contd. ...

activin receptors are expressed within the granulosa cell layer. While the autocrine/
paracrine factor, TGFb, can bind to its respective receptors, the bioactivity of activin
is modulated by follistatin (produced within granulosa cells). It is propoed that
active induction of FSH receptor (FSH-R) expression by TGFb (and possibly activin)
is precluded in all but one prehierarchal follicle per day by one or more EGF family
ligand binding to one or more ErbB receptor and the tonic activation of MAPK/Erk
signaling. The sites of MAPK/Erk-mediated tonic inhibition include the absence of
Protein Kinase A/cAMP signaling pathway both prior and subsequent to cAMP
formation, and the prevention of Smad2 phosphorylation (Smad2-P). The absence
of phosphorylation prevents transport of regulatory (r-) Smad2 to the nucleus via
the co-regulatory Smad, co-Smad4. B. Following selection, a transient attenuation
of MAPK signaling enables TGFb (and activin) signaling via Smad, and results in
enhanced FSH-R expression (1). Elevated FSH-R expression subsequently
facilitates FSH-induced LH-R expression (2), P450scc and StAR expression, and
as a consequence, progesterone production (3). Mechanisms proposed to
attenuate MAPK/Erk signaling, in vivo, in the single follicle selected into the
preovulatory hierarchy per day include the rapid upregulation of Erk-specific
phosphatases. For additional details, see text and Woods and Johnson (2005);
Woods et al. (2005). Reprinted, with modifications, from Woods, D.C. and Johnson,
A.L. 2005. Biology of Reproduction 72: 643-650, Fig. 9.
finches (Taeniopygia guttata) are undetectable in non-breeders as well as

following termination of a six-egg clutch. In contrast, circulating vitellogenin
is dramatically increased at the onset of the rapid yolk development stage,
highest at the one- and three-egg stage of the clutch (in excess of 1.4 mg
vitellogenin per ml plasma), and significantly decreased by the fifth-egg stage
in advance of clutch completion (Salvante and Williams 2002). This ability to
precisely regulate yolk precursor availability according to the changing mass
of developing follicles as a clutch progresses has similarly been documented
for the European starling (Challenger et al. 2001).
In addition to yolk, a developing follicle accumulates both maternal
messenger RNA (mRNA) and hormones, factors that have been proposed to
influence oocyte and early zygote viability plus embryo development. In
preovulatory follicles of the Japanese quail, two distinct pools of maternally-
derived mRNA have been described. One pool exists within the oocyte
germinal disc region, while a second, larger pool is localized in the
cytoplasmic layer surrounding the yolk (Malewska and Olszanska 1999). The
amount of RNA in the germinal disc region prior to fertilization varies little
from that in the blastoderm at oviposition, but RNA content at the vitelline
membrane is found to decrease by 80% from fertilization to oviposition.
Although the exact timing of genome activation in the avian embryo is not
known, it is proposed to occur subsequent to ovulation and the early cleavage
stages but prior to oviposition. A variety of transcripts, presumably of
maternal origin, have been identified in the unfertilized blastodisc, including
those encoding pro- and anti-apoptotic proteins (Muscarella et al. 1998). In
particular, the expression and translation of maternally-derived anti-
apoptotic genes prior to the initiation of embryonic transcription may provide
enhanced resistance of the early zygote to environmental stressors, such as
fluctuations in temperatures that can occur during the laying of a clutch and
the incubation period.
Similarly, there is considerable information regarding yolk steroid and
thyroid hormones of maternal origin. Exposure to such hormones during
embryo development has been associated with the programming of
reproductive organs, early somatic growth, development of the brain, sexual
behavior and immune function, even long after fledging (Groothuis et al. 2005).
For example, in the Japanese quail the yolk content of thyroxine (T3) and
triiodothyronine (T4) has been positively associated with accelerated cartilage
growth and differentiation in the developing embryo (Wilson and McNabb
1997), while levels of yolk corticosterone have been negatively associated with
the growth rate of the growing chick (Hayward and Wingfield 2004). The
incorporation of maternally-derived progesterone, testosterone and estradiol
has been described within the yolk of Dark-eyed junco (Junco hyemalis) and
Red-winged blackbird (Agelaius phoeniceus) eggs (Lipar et al. 1999). Patterns of
steroid deposition conform to the concentric rings of yolk incorporated by the
follicle on a daily basis, with concentrations of progesterone highest in the
most peripheral layers, estradiol concentrations highest near the center of the
Ovarian Dynamics and Follicle Development $!
yolk, and testosterone concentrations highest in the intermediate layers. This

pattern of steroid deposition largely reflects the temporal changes in steroid
production by preovulatory follicles during development (Bahr et al. 1983) and
may result in the embryo being exposed to varying levels of hormones during
embryonic development. Nevertheless, despite the potential adaptive value of
this non-genetic mechanism of inheritance, a critical question is whether the
biased allocation of hormones and other factors into the yolk of developing
follicles represents a flexible maternal strategy to influence the phenotype of
offspring, or alternatively, a passive consequence of maternal physiology at
the time of follicle development.
6.3.3.2 Preovulatory follicle granulosa and theca cells
A significant change in gonadotropin receptor expression occurs in the
granulosa layer during the transition of a selected prehierarchal follicle into
preovulatory stage of development. Granulosa cells from preovulatory follicles
demonstrate predominant expression of LH receptor, while FSH receptor
mRNA declines to low, but still detectable, levels. Progesterone production is
controlled by LH, and LH-induced cAMP formation increases dramatically as
a follicle approaches the time of ovulation. Fully potentiated production of
progesterone, androgens and estradiol by preovulatory follicles requires the
participation of granulosa cells combined with cells located in both the theca
interna and externa layers (Nitta et al. 1991).
The portion of the granulosa layer distal to the germinal disc is the
predominant source of progesterone that serves as the primary steroid
involved in potentiating the preovulatory surge of LH that precedes and
subsequently induces ovulation. In addition, granulosa-derived progesterone
serves as precursor for androstenedione, and testosterone, synthesis by the
theca layer, and to a lesser extent by granulosa cells. Steroid production by the
theca layer is also regulated almost exclusively by LH, despite the continued
expression of FSH receptor mRNA.
Significantly, LH receptor mRNA levels and LH-induced cAMP plus
androstenedione formation dramatically declines specifically within the theca
from the largest preovulatory follicle (Johnson et al. 1996a; Marrone and
Hertelendy 1985). At the same time, granulosa cells from the largest
preovulatory follicle become capable of producing considerably greater
amounts of progesterone, both in vivo and in vitro, compared to those from the
second largest follicle. This enhanced progesterone production is associated
with significantly increased mitochondrial activity and/or number of
mitochondria together with increased levels of P450scc mRNA, but is not
directly related to enhanced LH-induced StAR protein expression (Dive et al.
1992; Johnson et al. 2002b). Presumably, the combination of these events
ensures maximal progesterone secretion from the largest preovulatory follicle
at the time of the preovulatory LH surge.
It is important to note, however, that granulosa cells differ not only in
morphology relative to their proximity to the germinal disc (see section
6.3.3.1), but also phenotype. Preovulatory follicle granulosa cells localized

adjacent to the germinal disc region (animal pole) remain mitotically active,
whereas those cells distal to the germinal disc (vegetal pole) become non-
mitotic (Perry et al. 1978; Tilly et al. 1992). Moreover, cells at the vegetal pole
express comparatively less EGF (ErbB1 and ErbB4) receptor mRNA but more
LH receptor mRNA and produce more progesterone in response to LH
compared to those at the animal pole (Tischkau et al. 1997; Yao and Bahr
2001). Finally, germinal disc-derived factors (including one or more EGF
family members) can stimulate granulosa proliferation and inhibit
progesterone production, in vitro (Tischkau and Bahr 1996). Accordingly,
these findings are consistent with the model proposed for the initiation of
granulosa cell differentiation following follicle selection (see section 6.3.3.2),
in that granulosa cells are maintained in a proliferative and undifferentiated
state until released from tonic inhibition. It is further speculated that
processes leading to the eventual differentiation of granulosa cells within the
vegetal pole are those described for prehierarchal follicles following selection
(Fig. 6.5).
Finally, granulosa and theca cells produce additional endocrine/
paracrine/autocrine factors whose functions in follicle development are
currently being investigated. For instance, the TGFb superfamily members,
inhibins (in particular, inhibin A) are produced by granulosa cells from
preovulatory (particularly the largest) follicles (Johnson, Brooks et al. 2005),
and are proposed to negatively modulate pituitary FSH secretion. However, a
negative relationship between plasma levels of inhibin A and FSH during the
ovulatory cycle has yet to be conclusively demonstrated. Inhibins may also
competitively antagonize the actions of activin by its association with
betaglycan and the activin type II receptor (Knight et al. 2005).
6.3.3.3 Ovulation and postovulatory follicle
Final maturation of the oocyte and ovulation of the largest preovulatory
follicle is triggered primarily by a surge of pituitary-derived LH that, in
domesticated birds (e.g., Gallus gallus, Coturnix japonica, Meleagris gallopavo)
precedes ovulation by 4 to 6 hours. The initiation of germinal vesicle
breakdown occurs coincident with peak concentrations of circulating LH, and
condensation of chromatin, extrusion of the first polar body, and formation of
the second maturation spindle are completed 2.5 to 1 h prior to ovulation. At
the same time, interdigitations and the remaining tight junctions between
granulosa cell cytoplasmic projections and the oocyte plasma membrane
dissociate, and a perivitelline space develops as a result of fluid accumulation
(Yoshimura et al. 1993). Significantly, such changes are not induced within
the second largest preovulatory follicle. Although the cellular mechanisms
providing for this selectivity are not yet clear, it is reasonable to propose a role
for the local, high concentrations of progesterone produced within the largest
preovulatory follicle.
At the time of ovulation, rupture of the follicle occurs along the stigma by a
combination of factors: 1) activation of proteases (including collagenase) that
Ovarian Dynamics and Follicle Development $#
reduce tensile strength in the follicle wall; 2) increased intrafollicular pressure

due to increased blood flow to the follicle; 3) induction of smooth muscle
contractions in the follicle wall; 4) a weakening of granulosa cell support
following the retreat of microvilli from the surface of the oocyte; and 5) a
selective loss of cells within the stigma region via apoptosis. The structure
remaining after release of the oocyte is the postovulatory follicle (POF), and
contains both granulosa and theca tissues. This structure is not homologous
to the mammalian corpus luteum in that the POF remains steroidogenically
active for, at most, a few days following ovulation. Cell death via apoptosis is
initiated shortly after ovulation, and the POF is largely reabsorbed within
several days. Regression of the POF may be further aided by the presence of
immunocompetent cells expressing the MHC class II antigen (Barua et al.
2001). Importantly, the most recent POF of the domestic hen exerts a direct or
indirect influence on the oviduct, as removal of the largest (but not next most
recent) POF is associated with delayed oviposition of the egg derived from that
follicle. This rapid loss of functional postovulatory tissue presumably insures
that a direct influence on oviposition is limited to that egg present within the
reproductive tract. The POF, like the mammalian corpus luteum, reportedly
expresses relaxin (Brackett et al. 1997), yet its role in oviposition, if any, has
yet to be described.
6.3.4 Follicle Atresia

Vertebrate follicle atresia represents the death of an organized follicle at any
time during development beginning with the primordial stage through the
ovulatory stage. Atresia is generally considered a normal physiological
process by which surplus or non-viable growing follicles are rapidly
reabsorbed by a non-inflammatory process, though it can also be abruptly
induced by environmental or physiological perturbations. It is significant to
note, however, that among vertebrate species the overall proportion of ovarian
follicles lost by atresia, as well as the time during follicle development at
which atresia can occur, differs according to reproductive strategy. In
particular, those animals that produce thousands to perhaps millions of
mature gametes within a reproductive cycle (spawners; e.g. fish) show a very
low overall incidence of atresia, and most of the oogonia produced during the
lifespan of the female are ovulated as mature oocytes. By comparison, those
groups that produce a considerably more limited number of offspring (many
reptiles, birds, mammals) demonstrate a proportionately higher incidence of
atresia. Furthermore, within a normal reproductive cycle a major difference
between reptiles and birds compared to mammals is that in the former
species, atresia occurs prior to selection into the preovulatory hierarchy,
whereas in mammals atresia of subdominant follicles occurs following
selection of the dominant follicle(s) (Rothchild 2003). The following sections
will first address stages of the avian reproductive cycle when follicle atresia
can occur, then some cellular mechanisms mediating this process.
6.3.4.1 Avian follicle atresia

In birds, the loss of follicles by atresia can occur any time during the active
breeding season, at the termination of breeding (following the onset of
photorefractoriness or at the start of incubation), and during seasonal molt
(Erpino 1973), and the overall rate of atresia has been observed to increase
with advancing age (Waddington et al. 1985). Under optimal breeding
conditions atresia rarely occurs in preovulatory follicles, but can be induced
at this final stage of development by inappropriate environmental conditions
(e.g., during forced molt induced by food deprivation and/or reduction in
photoperiod) and a decline in circulating gonadotropins. By contrast, follicle
atresia frequently occurs in prehierarchal follicles (Gilbert et al. 1983). Thus, it
has been proposed that a transition from atresia-susceptibility to atresia-
resistance is initiated coincident with follicle selection into the preovulatory
hierarchy (Johnson 2003).
A variety of factors contributes to the survival of preovulatory follicles. It is
clear that the integrity of the germinal disc region is required for follicle
viability given that its destruction results in the rapid onset of atresia in the
affected follicle (Yoshimura and Bahr 1995; Yao et al. 1998). The critical
supportive factors produced by the oocyte have not been unequivocally
identified, but likely include one or more growth factor(s), including those
from the EGF family (Yao and Bahr 2001). Continued support by circulating
gonadotropins is a second factor, as hypophysectomy leads to the loss of all
follicles within the hierarchy. Conversely, in vivo treatment of chicken or quail
hens with FSH or equine chorionic gonadotropin (eCG) decreases the rate of
atresia and increases the overall number of prehierarchal and preovulatory
follicles.
Interestingly, Challenger et al. (2001) provide evidence that the clutch size
in starlings is increased, not by the selection of additional prehierarchal
follicles, but by rescue of the smallest, most recently selected preovulatory
follicles from atresia. This suggests that in at least some indeterminant layers
(including the barn owl, Durant et al. 2004, and Eurasian kestrel, Meijer et al.
1989), there are more follicles selected into the preovulatory hierarchy than
will normally be fully developed and ovulated. In the event that eggs are lost
during the early portion of the clutch, a prolonged or increased secretion of
survival factors (e.g., FSH and LH from the pituitary and/or growth factors
from ovarian tissues) may serve to support the continued growth of
supplemental follicles.
The comparatively high incidence of atresia that occurs in prehierarchal
follicles that have yet to enter the rapid growth phase represents a
reproductive strategy that maximizes the availability of healthy prehierarchal
follicles readily available for selection into the preovulatory hierarchy, yet
minimizes the loss of energetically expensive maternal resources that would
otherwise be invested in extensive VTG/VLDL deposition. There is evidence
that non-growing follicles embedded within cortical tissue of the mature
ovary also undergo atresia, but the overall incidence has not been reported.
Ovarian Dynamics and Follicle Development $%
Underlying causes of follicle loss at this early stage of development include

genetic or meiotic anomalies.
6.3.4.2 Apoptosis as modulator of ovarian function and as a proximal
cause of follicle atresia
It has been recognized for some time that apoptosis is a proximal cause of
follicle atresia in mammals and birds (Tilly et al. 1991). In particular,
apoptosis is observed first in isolated granulosa cells, and subsequently
progresses throughout the granulosa layer. This rapid progression of
apoptosis is facilitated by an increase in the number of gap junctions that
provide for cell-to-cell communication (Krysko et al. 2004) and the transfer of
apoptosis-inducing factors. Signals initiating granulosa cell apoptosis can
variably originate from the oocyte, from the granulosa layer itself, or from
tissues peripheral to the basement membrane (theca or circulatory system). As
atresia continues, apoptosis eventually progresses throughout the theca
layers. Additionally, during the late stages of embryogenesis and the early
post-hatch period, there are data to suggest that apoptosis occurs in ovarian
cortical follicles, and that the incidence of apoptosis decreases to negligible
levels by day 14 post-hatch (Yoshimura and Nishikori 2004). These latter
observations can explain the decline in available germ cell number during the
latter stages of embryo development (see section 6.2.2). Finally, the
reabsorption of postovulatory follicle tissues also occurs via apoptosis, and is
initiated within the first 24 h after ovulation (Tilly et al. 1991). Accordingly,
apoptosis represents a fundamental process by which the avian ovary
accomplishes: 1) the regression of the right ovary early in ontogeny; 2) a
reduction of germ cell number by the time of hatch; 3) the selection of a limited
number of the most viable follicles for ovulation; 4) the rapid elimination of
postovulatory tissues as a clutch progresses; and 5) the reduction of ovarian
mass that accompanies the non-breeding season.
There are several reviews detailing cellular mechanisms involved in the
regulation of apoptosis specifically in the avian ovary (Johnson 2003; Johnson
and Bridgham 2002), and only an updated summary will be provided here.
Apoptosis involves a complex and well-coordinated sequence of intracellular
signaling events that results in the elimination of affected cells without the
initiation of an inflammatory reaction. The entire process can be divided into
phases of initiation, execution and termination, with the first of these two
phases dependent upon the activation of one or more cysteine proteases
(caspases). Terminal endpoints include membrane blebbing, cell shrinkage,
the formation of membrane-enclosed vesicles (apoptotic bodies), proteolytic
cleavage of numerous structural plus functional proteins, and ultimately
DNA fragmentation (oligonucleosome formation).
In the domestic hen, the comparatively high incidence of follicle atresia in
prehierarchal follicles, in vivo, correlates with susceptibility of the granulosa
cell layer to undergo apoptosis as observed, in vitro (Johnson et al. 1996b). In
light of this observation, and because apoptosis in granulosa cells represents
the earliest event described during the onset of atresia, most studies of the
ovary have focused primarily on this cell type.
The progression and amplification of the apoptotic process in granulosa
cells is initiated by either intrinsic or extrinsic pathways (Fig. 6.6). Activation
of an intrinsic pathway can occur by a variety of factors, including cellular
(e.g., oxidative) stress, and/or the withdrawal of growth factor support. Either
of these stimuli may be initiated by the lack of adequate food or the
withdrawal of growth factor and/or gonadotropin support, as typically
occurs with photorefractoriness. At the cellular level, such stimuli cause
perturbations in mitochondrial function that result in the release of
mitochondrial proteins, including cytochrome C, into the cytoplasm. Cytosolic
cytochrome C (cytC) promotes the formation of an apoptosome complex
consisting of Apoptosis Protease Activating Factor-1 (Apaf-1) and the initiator
caspase, caspase-9. In turn, activated caspase-9 activates the executioner
caspase, caspase-3, a primary mediator of the terminal endpoints mentioned
above, together with the proteolysis of many structural and catalytic proteins
(e.g., Poly-(ADP-ribose)-polymerase, PARP).
By comparison, an extrinsic pathway can be activated by a variety of
cytokines that bind to one or more receptors containing intracellular death
domains (DD) (Fig. 6.6). Such receptors belong to an evolutionarily conserved
family of death receptors known as the Tumor Necrosis Factor Receptor
Superfamily (TNFRSF) (Bridgham et al. 2003). Ligand-activated death
receptors recruit cytoplasmic adaptor proteins (e.g., Fas-Associated Death
Domain, FADD) by the homodimerization of death domains (DD). In turn,
FADD recruits and activates an alternative initiator caspase, caspase-8,
through the homodimerization of their respective death effector domains
(DED). Activated caspase-8 cleaves the cytoplasmic protein, BH3 Interacting
Domain (Bid) death agonist, that promotes release of mitochondrial cytC, as
well as activation of the executioner caspase, caspase-3. From this point, the
downstream events converge with those described for the intrinsic pathway.
No less than five DD-containing receptors from this death receptor
superfamily are expressed by hen granulosa cells: TNFRSF1 (TNF receptor
type 1); TNFRSF6 (Fas); TNFRSF10B (DR5); TNFRSF16 (p75 Nerve Growth
Factor receptor); and TNFRSF23 (Bridgham and Johnson 2004). Several known
cytokines are produced in a paracrine or autocrine fashion by one or more cell
types within the ovary, and these include Tumor Necrosis Factor a (TNFa;
binds to TNFRSF1), TNF-Related Apoptosis Inducing Ligand (TRAIL; binds
to TNFRSF10B), and Fas Ligand (TNFRSF6). Moreover, potential death-
inducing ligands can be trafficked to the follicle via nervous innervation (e.g.,
NGF; binds to TNFRSF16) or by immune cells from the vasculature (e.g., TNFa,
TRAIL). Intuitively, the finding that no less than five death domain-containing
receptors are expressed within this cell type implies that multiple ligands may
regulate cell viability under varying environmental and physiological
conditions. To date, TNFa has been demonstrated to promote hen granulosa
cell apoptosis, in vitro, in granulosa collected from atresia-susceptible
CMYK
Ovarian Dynamics and Follicle Development $'

CMYK
CMYK
Fig. 6.6 Simplified model of pro- and anti-apoptotic pathways in hen granulosa
cells. Apoptotic cell death can be initiated by either an intrinsic or extrinsic pathway,
both of which converge at the activation of caspase-3. Activation of this executioner
enzyme is irreversible, and results in the cleavage of numerous structural and
functional proteins (e.g., PARP) and the internucleosomal cleavage of genomic
DNA. Cell death pathways are opposed by anti-apoptotic proteins that protect
mitochondrial membrane integrity (Bcl-x, Bcl-2), block caspase activity (IAPs) or
prevent signaling via death receptors (the adaptor protein, FLIP). Expression of
these anti-apoptotic proteins is regulated by gonadotropins (FSH and LH) and
locally produced growth factors (IGF-I and EGF family ligands) via cell survival
signaling pathways. The absence of sufficient anti-apoptotic protein expression
(dotted green lines) is proposed to tip the balance in favor of activating pro-
apoptotic pathways (solid red lines). See text for further details. Abbreviations:
aCasp.-3, -7, -8, activated caspases; Apaf-1, Apoptosis Protease Activating Factor-1;
Bid and tBid, intact or truncated BH3 Interacting Domain death agonist; cytC,
cytochrome C; DD, death domain; DED, death effector domain; EGF, epidermal
growth factor; FADD, Fas-Associated Death Domain; FLIP, Flice-like inhibitory
protein; FSH, follicle-stimulating hormone; Inhibitor of Apoptosis Protein, IAP; IGF-I,
insulin-like growth factor I; LH, luteinizing hormone; PARP, Poly-(ADP-ribose)-
polymerase; PKA/cAMP, protein kinase A/cyclic AMP signaling pathway; PKB/Akt,
protein kinase B/Akt signaling pathway. Each of the proteins and cell signaling
pathways depicted has been characterized in cultured hen granulosa cells. For a
more complete description with citations, see Bridgham et al. 2003; Johnson 2003
and Johnson and Bridgham 2002.
CMYK
prehierarchal follicles (Witty et al. 1996). Studies are ongoing to elucidate the
ability of additional cytokines (e.g., TRAIL and Fas) to promote granulosa cell
apoptosis, and the conditions under which this can occur.
The finding that cellular components of both the intrinsic and extrinsic cell
death pathways are constitutively expressed, clearly indicates that
mechanisms exist to counteract their actions. Not surprisingly, granulosa
cells express a variety of anti-apoptotic proteins that prevent initiator and
effector caspase activity (Inhibitor of Apoptosis Proteins, IAPs) or prevent
mitochondrial membrane perturbations (Bcl-2, Bcl-x). In addition, a decoy
adaptor protein that mimics caspase-8 but lacks caspase activity (Flice-Like
Inhibitory Protein, FLIP) can block death receptor activity by preventing the
recruitment of caspase-8 (Fig. 6.6). In fact, immediately subsequent to follicle
selection, granulosa cells from atresia-resistant preovulatory follicles have
been demonstrated to express significantly higher levels of Bcl-x, Bcl-2 and
IAP compared to granulosa from atresia-susceptible prehierarchal follicles
(Johnson et al. 1997, 1998, 1999). Furthermore, the activation of cell survival
signaling pathways, such as the protein kinase A pathway, enhances
expression of IAP and Bcl-x protein in hen granulosa cells while PKB
signaling promotes cell cycle progression and survivin expression (Johnson et
al. 2002a). To a large extent, it is by tipping the balance in favor of such anti-
apoptotic mechanisms that the gonadotropins (FSH and LH) and locally
produced growth factors (EGF family members, insulin-like growth factor-I)
are proposed to support granulosa cell, and by implication follicle, viability
throughout the preovulatory stage of development.
6.3.5 Avian Clock Genes and Reproduction

Finally, it is clear from the above discussion that circannual and circadian
rhythmicity is a fundamental component of seasonal reproduction. A central
site of seasonal (periodic) time measurement in birds has been localized to the
mediobasal hypothalamus (MBH), and lesions within the avian MBH can
block photoperiodic-induced increases in gonadotropin secretion and
gonadal growth. Such photoperiodic responsiveness is mediated at the
molecular level by oscillations in the expression of clock genes, and each of
the known avian clock genes required to retain a steady state photoinducible
phase have now been localized to the MBH (e.g., Clock, Per, Bmal, Cry, E4bp4)
(Yasuo et al. 2003). Significantly, circadian clock genes are also expressed in
the ovary of the rat, and their expression is influenced by the preovulatory LH
surge (Karman and Tischkau 2006). Accordingly, it is predicted that each of
the phases within the avian reproductive cycle (seasonality, follicle
recruitment, follicle selection, and ovulation-oviposition cycles) can be
explained at a molecular level by the ebb and flow expression of clock genes.
A challenge for the future is to expand our knowledge of molecular clocks to
the avian ovary in an effort to elucidate the means by which seasonal and
daily fluctuations of environmental cues (e.g., photoperiod) are ultimately
translated and/or modified at the level of the ovary to influence follicle
recruitment, selection and the timing of ovulation.
Ovarian Dynamics and Follicle Development %
6.4 ACKNOWLEDGMENTS
The authors thank Drs. Jamie T. Bridgham and Tom Jensen for their many
contributions to portions of the data discussed herein, and Morgan Haugen
for help with the preparation and editing of the manuscript. We acknowledge
the National Science Foundation (IBN31185, IOB45949) and the National
Institutes of Health (HD-36095) for recent research support.

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n n
CHAPTER
7
Spermatogenesis and
Testicular Cycles
Tom A. Aire
7.1 INTRODUCTION
The seminiferous epithelium in sexually mature and active testis is made up
of germ cells at varying levels of development, and Sertoli cells, which are the
only somatic, non-dividing cells of the epithelium. Sertoli cells have been
described in some detail in Chapter 2. Sertoli cells have multiple functions,
including, but not limited to, contributing significantly to the formation of the
blood-testis barrier by means of the so-called Sertoli-Sertoli junctional
complexes (Dym and Fawcett 1970; Fawcett et al. 1970; Dym 1973), providing
anchorage and nutrition for, as well as regulation of, germ cells during
development (Morris et al. 1987; Jégou 1991; Skinner et al. 1991; Vogl et al.
1991). The germ cells usually develop, and grow older as they move from the
basement membrane of the seminiferous epithelium toward the tubular lumen.
Thus, the most primitive or immature germ cells lie on the basement
membrane and the mature germ cells, the spermatozoa, line the lumen of the
seminiferous tubule. Germ cells develop in close association with one another
because as they divide they maintain close linkage through intercellular
bridges which are the result of incomplete cytoplasmic divisions (Fawcett
1961).
7.2 SPERMATOCYTOGENESIS
7.2.1 Spermatogonia
Spermatogonia are the most immature or primitive germ cells that divide to
produce the cells which eventually differentiate into spermatozoa in the
seminiferous epithelium of physiologically active testes. Spermatogonia divide
Department of Anatomy and Physiology, Faculty of Veterinary Science, University of
Pretoria, Onderstepoort, Republic of South Africa. E-mail: tom.aire@up.ac.za
repeatedly by mitosis to produce spermatocytes, as well as preserving (by self-

replication) a store of stem cells that continue the process of spermatogenesis.
The replacement of these dividing stem cells has received considerable
attention in mammals (Regaud 1901; Roosen-Runge and Giesel 1950; Clermont
and Leblond 1953) but to a much lesser extent in birds (Marchand 1977; Lin
and Jones 1992).
The existence of several types of spermatogonia in the seminiferous
epithelium was first recognized by Regaud (1901) in Rattus norvegicus, the
laboratory rat. Several authors subsequently reported varying numbers of
generations of these cells in different mammalian species. Ortavant (1959)
describes three successive generations of spermatogonia in mammals: type A
whose nucleus is dust-like, as first described by Regaud (1901); type B whose
nucleus exhibits crust-like chromatin attachments to the inner part of the
nuclear membrane. The third generation of spermatogonia, the intermediate
spermatogonium, has also been described in several mammalian species (see
Courot et al. 1970). Several successive generations of type A spermatogonia
have been described subsequently in a number of species of mammals (Courot
et al. 1970; Clermont and Bustos-Obregon 1968; Huckins 1971a,b,c; Oakberg
1971a,b; Lok et al. 1982; Hadley and Dym 1983). Thus, Clermont and Bustos-
Obregon (1968) have recognized, in R. norvegicus, five successive generations
of type A spermatogonia (A 0, A1, A2, A3 and A4) and Ekstedt et al. (1986) three
types of A (A1, A2, A3) in the bull, Bos taurus. This large array of spermatogonia
notwithstanding, the type A0, regarded as the stem cell (Clermont and Bustos-
Obregon 1968), but disputed by Courot et al. (1970), is further subdivided into
types Asingle, Apaired, and Aaligned in Rattus norvegicus (Huckins 1971a,b,c).
Considerable difficulty has been encountered in defining spermatogonial
morphology and pattern of division in Homo sapiens, other primates and in
birds, possibly due to the heterogeneous cellular associations that are
characteristic of the seminiferous epithelia of these species of animals. Unlike
in the laboratory and farm animals, three generations of spermatogonia, viz.,
type A spermatogonia, made up of two divisions, dark type A (Ad) and pale
type A (Ap), and type B spermatogonia, have been described by several
investigators in primates (Clermont 1963; Clermont and Antar 1973;
Choudhury and Steinberger 1976; Shulze 1979; Hadley and Dym 1983).
The nature of spermatogonia and their renewal is even more problematic
and controversial in birds, not only because of the heterogeneous cellular
associations in the seminiferous epithelium of these animals, but also because
the earlier studies were performed in paraffin-embedded tissues. Zlotnik
(1947), Kumaran and Turner (1949) and Sharma et al. (1956) in Gallus gallus
domesticus (the domestic fowl—henceforth referred to as the rooster), Clermont
(1958) in the Mallard drake (Anas platyrhynchos) and Marchand (1977) in
Muscovy duck (Cairina moschata) have identified a single type of
spermatogonium, while Lake (1956) describes types 1 and 2 spermatogonia in
the rooster. Gupta (1955) describes two types of spermatogonia (primary and
secondary) in an unspecified species of duck, and Aire et al. (1980) describe
Spermatogenesis and Testicular Cycles &
two types, A and B, in Guineafowl (Numida meleagris), while Yamamoto et al.

(1967) identify three types (A, Intermediate and B) in Japanese quail (Coturnix
japonica). By using plastic/resin sections and electron microscopy, as well as
radiolabeling and autoradiography, Lin and Jones (1992) have shown that
there are four types of spermatogonia (dark type A [Ad], 2 pale type A [Ap1
and Ap2] and type B) in Japanese quail. However, according to Ortavant
(1959) “in spite of superficial differences the length of life of the various
spermatogonial generations is in constant relation to the cycle of the
seminiferous epithelium.”
7.2.2 Spermatocytes
Primary spermatocytes are the product of the last mitotic division of type B
spermatogonia, and are involved in meiotic division. As in mammals, there
are several generations of primary spermatocytes in the seminiferous
epithelium of birds because meiosis is an extended and prolonged process.
7.2.2.1 Primary spermatocytes
In mammals, six phases of the meiotic prophase of the primary spermatocyte
are recognized: a premeiotic interphase, leptotene, zygotene, pachytene,
diplotene and, as the last phase of the meiotic prophase, diakinesis (Ortavant
1959; Courot et al. 1970; Ekstedt et al. 1986). In birds, similar phases of the
meiotic prophase have been recognized, generally (Clermont 1958; Marchand
1977; Lin et al. 1990). However, Lin and Jones (1990), in a recent study,
describe eight different phases (preleptotene, leptotene, zygotene, pachytene,
diplotene, diakinesis, metaphase and anaphase) of primary spermatocytes
during the process of spermatogenesis, in Japanese quail. The metaphase,
anaphase and telophase are, however, known to occur rapidly (Ortavant
1959).
7.2.2.2 Secondary spermatocytes
Secondary spermatocytes are the two products of the first meiotic division of a
primary spermatocyte. The secondary spermatocyte is rarely seen in the
seminiferous epithelium because it has a very short life span. The nuclei of
these cells are not as large as those of mid- to late primary spermatocytes, but
are larger than those of round spermatids which they produce by the second
meiotic division. The secondary spermatocyte has been described in members
of the Anatidae (Clermont 1958; Marchand 1977), in the rooster (Zlotnik 1947),
Guineafowl (Aire et al. 1980) and Japanese quail (Yamamoto 1967; Lin et al.
1990). The nucleus of this cell exhibits a number of chromatin aggregations
and a distinct nuclear membrane. Each secondary spermatocyte divides, in
the last meiotic division, to produce two haploid round spermatids.
7.2.2.3 Round spermatids
These cells, containing a haploid complement of chromosomes, represent the
phase of differentiation in which relatively small cells containing round
nuclei evolve, in a complex and delicate structural transformation, into highly

elongated, motile, itinerant cells, the spermatozoa. This unique process in
cytodifferentiation is called spermiogenesis or spermateliosis, the former term
being more commonly used.
7.3 SPERMIOGENESIS
7.3.1 Spermiogenesis in Non-passerine Birds
The complex structural evolution of the spermatozoon from a round cell, with
the loss of certain organelles, even as new ones are formed, has intrigued
investigators. During this process, and relative to the young, round spermatid,
the mature, highly elongated spermatid loses over one hundred times its
volume (McIntosh and Porter 1967) or 97% (Sprando and Russel 1988), while
the volume of the nucleus is reduced from 110 cubic micrometres to 2 cubic
micrometres (McIntosh and Porter 1967) or by 96% (Sprando and Russel
1988). The spermatid also radically changes its shape and evolves a number
of morphologically elaborate organelles, including the acrosome, the midpiece
and the flagellum (Lin et al. 1997). Biochemical changes that are controlled by
genes which are active only in spermatids and in the process of
spermiogenesis (Oko 1995), including the elaboration of new and unique
structural elements of the spermatozoon, also take place, and are, indeed,
mostly responsible for the visible morphological alterations expressed
variably in the spermatid.
Spermiogenesis is an integral and important part of spermatogenesis in
animals, the study and understanding of which phenomenon is essential for
a critical evaluation of the form and function of the seminiferous epithelium,
in health and disease. Spermiogenesis has been studied and reported
extensively in mammals (Leblond and Clermont 1952a,b; Clermont and
Leblond 1955; Courot et al. 1970; Fawcett et al. 1971; Setchell 1978; Ekstedt et
al. 1986; Plöen and Courtens 1986), and to some extent, also in birds, although
most of the reports are fragmentary (Zlotnik 1947; Leblond and Clermont
1952a,b; Clermont and Leblond 1955; Sotelo and Trujillo-Cenóz 1958; Nagano
1962; McIntosh and Porter 1967; Yamamoto et al. 1967; Courot et al. 1970;
Fawcett et al. 1971; Mattei et al. 1972; Tingari 1973; Humphreys 1975;
Okamura and Nishiyama 1976; Gunawardana and Scott 1977; Marchand et
al. 1977; Yasuzumi and Yamaguchi 1977; Ekstedt et al. 1986; Plöen and
Courtens 1986; Aire et al. 1980; Baccetti et al. 1980; Kondo et al. 1988; Sprando
and Russel 1988; Soley 1992; Góes and Dolder 2002; Aire 2003; Jamieson and
Tripepi 2006). Most of these studies are, understandably, on the rooster (e.g.
Zlotnik 1947; Nagano 1962; McIntosh and Porter 1967; Tingari 1973;
Okamura and Nishiyama 1976; Gunawardana and Scott 1977), and a few on
other species of birds, including passerines and the Paleognathae (e.g. Sotelo
and Trujillo-Cenóz 1958; Aire et al. 1980; Kondo et al. 1988; Lin and Jones
1993; Góes and Dolder 2002; Aire 2003). The advent of electron microscopy,
using both thick and ultra-thin plastic sections, has greatly facilitated the
Spermatogenesis and Testicular Cycles &!
study of spermiogenesis in birds whose acrosome is quite small and thin, and
therefore only discernible with difficulty in paraffin sections, PAS-stained or
otherwise. The study of spermatid development is more profitably based on
one or both of the classical systems enunciated by Leblond and Clermont
(1952a) or by Roosen-Runge and Giesel (1950) and Ortavant (1954). Leblond
and Clermont’s system is based on acrosome development, sub-divided into
four phases: the Golgi phase, cap phase, acrosome phase and maturation
phase, while that of Roosen-Runge and Giesel (1950) rests on evolving nuclear
changes during spermiogenesis. There are few reports in which the full
process of spermiogenesis has been detailed in birds, rather than fragmentary,
segmental studies, e.g. of the acrosome or nucleus. In this regard complete
accounts of spermiogenesis at the ultrastructural level have been reported in
only the rooster (Nagano 1962; Gunawardana and Scott 1977), Rhea (Phillips
and Asa 1989), Japanese quail (Yamamoto et al. 1967; Lin and Jones 1993), the
turkey (Meleagris gallopavo) (Aire 2003) and House sparrow (Passer domesticus)
(Góes and Dolder 2002). Several reports dealing with only segments of the
developing spermatid have shed considerable light on spermiogenesis in
some species of birds. These will be referred to where appropriate in this
review.
In those studies in which the step-wise system was used, 12 steps of
spermiogenesis have been reported in Japanese quail (Lin and Jones 1993) and
Turkey (Aire 2003), and 6 steps in House sparrow (Góes and Dolder 2002). In
this review, both systems (involving acrosomal as well as nuclear
morphogenetic processes, according to Clermont and Perey 1957, and Roosen-
Runge and Giesel 1950, respectively) will be combined in the so-called “step-
wise” changes in spermatid morphogenesis, using spermiogenesis in Turkey
(Aire 2003), as a model. Specific features in spermiogenesis of oscine passerine
birds will also be highlighted separately. Only relevant, important and
contentious features of spermiogenesis will be described and discussed, as
necessary and convenient in each step, on account of space limitation.
Step 1 spermatid. The youngest spermatids that have just emerged from the
second meiotic division, and along with the step 11 spermatids, line the
subluminal and luminal border of the epithelium, respectively. The oval
nuclei of the round spermatids contain scattered chromatin aggregations in
the karyoplasm or adhering to the nuclear membrane (Fig. 7.1A). A few
proacrosomal granules may be seen in the large Golgi complex during the late
phase of this step. The diplosome, comprising the proximal and distal
centrioles articulated at right angle to each other, lies free in the cell cytoplasm.
Step 2 spermatid. The nuclear chromatin begins to de-condense and appear
uniformly distributed in the nucleoplasm. A large, single acrosomal granule
appears in the Golgi complex (Fig. 7.1B). The distal centriole of the diplosome
makes contact with the cell cytoplasm, at which junction, an ill-defined
annulus occurs. The flagellum grows from this junction.
Fig. 7.1 Meleagris gallopavo. A. Step 1 spermatid, along with step 11 spermatid.
S, Sertoli cell; clumps of chromatin (arrowheads) in the nucleus of step 1 spermatid;
Fig. 7.1 Contd. ...

Spermatogenesis and Testicular Cycles &#
Step 3 spermatid. The nucleus remains spherical in shape, but chromatin de-
condensation has advanced into a finely granular matrix, with only a few,
small clumps of chromatin present (Fig. 7.2A). The Golgi complex becomes
inconspicuous, and the acrosomal granule lies very close to, or just makes
contact with the nucleus, and thickening of the nuclear membrane commences
at the site of contact. The diplosome nearly makes contact with the nucleus,
close to the developing acrosomal and Golgi complex.
Step 4 spermatid. The chromatin of the nearly spherical nucleus continues to
de-condense and becomes uniformly granulofilamentous (Fig. 7.2B). The
homogeneously dense acrosomal granule becomes slightly elongated and
forms the acrosome which invaginates into the nucleus; the nuclear membrane
becomes thickened at the contact site. The nucleus becomes eccentric within
the cytoplasm, and the acrosome abuts on the adjacent Sertoli cell (Fig. 7.2
inset). Sections of microtubules, in small groups, lie close to the nuclear
membrane, especially in the rostral portion of the nucleus. The diplosome
attaches obliquely at an indentation of the nucleus, by its proximal centriole.
The long axis of the distal centriole remains perpendicular to that of the
proximal centriole.
Unlike in mammals in which the acrosomal granule/vesicle contains a
concentrated granule (Burgos and Fawcett 1955; Clermont and Leblond 1955;
Plöen and Courtens 1986) or a clear vesicle/vacuole in Tammar wallaby
(Macropus eugenii) (Lin et al. 1997), the acrosome of birds arises from the Golgi
complex as a homogeneously dense membrane-bound granule (Nagano 1962;
de Reviers 1971; Okamura and Nishiyama 1976; Gunawardana and Scott
1977; Lin and Jones 1993; Soley 1996; Aire 2003).
Step 5 spermatid. The nucleus, now pear-shaped, contains uniform, finely
granular nucleoplasm (Fig. 7.3A). The acrosome elongates further and the
central part of the thickened nuclear membrane, at the contact site with the
acrosome, invaginates into the nucleoplasm (Fig. 7.3, inset). There is an
increased amount of smooth endoplasmic reticulum (SER), sparsely granular
endoplasmic reticulum (SGER), as well as lysosomes in the cytoplasm.
The invaginated precursor of the endonuclear canal has been reported in
the rooster, the Budgerigar (Melopsittacus undulatus), Turkey (Melaeagris
gallopavo), Japanese quail and Ostrich (Struthio camelus) (Nagano 1962;
Fig. 7.1 Contd. ...

a large Golgi complex (G) and mitochondrial aggregates (M) around it; numerous
profiles of SER and a few small lysosomes (L) occur in the cell. Inset: the centriolar
complex (C) in the cytoplasm. B. Step 2 spermatid nuclear chromatin is de-
condensing. A large proacrosomal granule (A) is leaving the Golgi complex (G).
Some microtubules (arrowheads) are not arranged in any specific manner in the
cell. Inset: the diplosome (D) and the fibrous sheath (F) of the developing tail. Bar:
A = 2 µm, B = 1 µm. All figures on spermiogenesis (Figs. 7.1 to 7.6) are those of
the turkey, Meleagris gallopavo, and are taken from Aire, T. A. 2003 British Poultry
Science 44: 674-682, with the kind permission of British Poultry Science Ltd.
Fig. 7.2 Meleagris gallopavo. A. Step 3 spermatid nucleus (N) de-condenses

further, and the diplosome (D) associated with lamellae of the Golgi complex (G)
lies close to the nuclear membrane. Step 12 spermatid (arrowhead) occurs along
Fig. 7.2 Contd. ...
Spermatogenesis and Testicular Cycles &%
Humphreys 1975a; Gunawardana and Scott 1977; Baccetti et al. 1980; Saita et
al. 1980; Soley 1996; Aire 2003). An osmiophilic content of this invagination is
the precursor of the perforatorium. However, Lin and Jones (1993) have
observed that the precursor of the perforatorium, in Japanese quail, is an
intranuclear granule.
Step 6 spermatid. The spermatid nucleus is finely granular, elongated and
slightly wavy in profile (Fig. 7.3B). The elongating, dense, acrosome occupies
only the central one-third of the rostral surface of the nucleus, and its rostral
tip abuts on an adjacent Sertoli cell. Cross-sections of profiles of microtubules
of the circular manchette (CM) appear sporadically along the length of the
nucleus, in no regular pattern. A CM is absent in some orders (see Chapter 8).
Mitochondria migrate to the caudal part of the cytoplasm, and are still round
or oval in shape, and not organized in any special fashion. The endonuclear
canal is well formed and contains the developing perforatorium (Fig. 7.3B
inset).
Although the step 6 spermatid nucleus is elongated and slightly wavy in
profile in the turkey (Aire 2003) as in the rooster (Gunawardana and Scott
1977) and Japanese quail (Lin and Jones 1993), that of Guineafowl (Aire et al.
1980), Ostrich (Soley 1997) and Crested tinamou (Eudromia elegans elegans),
Tinamou (Asa et al. 1986) appears ‘spiral’ or irregular in shape due to
differential subnucleolemmal chromatin condensation, and concomitant
constriction of the nucleus. Light microscopical observations in the rooster
(Zlotnik 1947) and in certain members of the Anseridae (Gupta 1955; Sharma
et al. 1956) indicate that the spermatid nucleus, during the elongating phase,
is coiled within the cell cytoplasm, apparently to ensure that the elongating
nucleus is accommodated in the fixed volume of cytoplasm. This
developmental feature needs clarification.
The development of the circular manchette (CM) seems to follow a similar
pattern in all non-passerine birds investigated (McIntosh and Porter 1967;
Okamura and Nishiyama 1976; Gunawardana and Scott 1977; Xia et al. 1986;
in the rooster; Humphreys 1975a, in Budgerigar, Fawcett et al. 1971 in a
Columba sp.; Phillips and Asa 1989 in the rhea (Rhea americana albisceus); Lin
and Jones 1993 in Japanese quail (Coturnix japonica) and Aire 2003 in the
turkey), but it is not developed to the same degree in all non-passerine birds.
Fig. 7.2 Contd. ...

with step 3. Inset: The acrosomal granule (A) makes contact with the nuclear
envelope which is thickened at the contact site (open arrow). B. Step 4 spermatid.
The chromatin has become finely granulofilamentous in appearance. The
acrosomal granule (A) invaginates further into the nucleus (N) at a thickened part
of the nuclear envelope (arrow). The diplosome (D) attaches to the nucleus at the
implantation fossa of the nucleus. Inset: The acrosomal granule lies close to the
cell membrane. S, Sertoli cell; arrowhead, microtubules running circularly around
the nucleus. Bars: A = 1 µm, Inset = 2 µm; B = 1 µm, Inset = 2 µm.
Fig. 7.3 Meleagris gallopavo. A. Step 5 spermatid is pear-shaped. Nuclear

chromatin is uniformly and finely granulofilamentous. Acrosomal granule (A)
Fig. 7.3 Contd. ...

Spermatogenesis and Testicular Cycles &'
Whereas the CM is substantially well developed in Ostrich (Soley 1997), it is

poorly developed in Columba sp. (Fawcett et al. 1971; Mattei et al. 1972) and the
Cuckoo (Crotophaga ani) (Saita et al. 1982) but does not develop in the Swift
(Apus apus) and the Nightjar (Caprimulgus europaeus) which have only a
longitudinal manchette (Tripepi et al. 1991; Jamieson and Tripepi 2006; see
Chapter 8). The function or role of the CM in spermiogenesis is controversial
and not clearly understood. McIntosh and Porter (1967), Okamura and
Nishiyama (1976), Gunawardana and Scott (1977), Lin and Jones (1993) and
Soley (1997) are of the opinion that the manchette has an important function
in nuclear shaping, but Fawcett et al. (1971), Asa and Phillips (1988), Phillips
(1970, 1974, 1976) are emphatic that the CM plays no role in nuclear shaping
in the pigeon spermatid. Myles and Hepler (1982) exhort that the role of the
manchette in nuclear morphogenesis, acting singly or in combination with
other cell components, must await precise determination.
Step 7 spermatid. The elongating acrosome is as wide as the spermatid
nucleus (Fig. 7.4A). The nucleus is slimmer than in step 6, slightly curved, and
its tapering rostral end projects into the subacrosomal concavity. The fine,
granular, nuclear chromatin condenses to become granulofilamentous, and
deeply-stained. The cell cytoplasm extends caudally from the distal border of
the acrosome, which now projects into a deep crypt of an adjacent Sertoli cell
cytoplasm. The CM is well established as a layer of microtubules that aligns
closely with the external surface of the elongated nucleus, extending distally
from the caudal border of the acrosome to the region of the rostral end of the
distal centriole. The spermatid tail is well formed (Fig. 7.4B). Mitochondria
begin to elongate, and display longitudinal cristae, as they continue to migrate
into the cytoplasm, much of which is displaced caudally.
The development of the flagellum of the spermatozoon is generally similar
in mammals (Fawcett and Phillips 1969; Yasuzumi et al. 1972; Irons and
Clermont 1982a,b; Clermont et al. 1990) and non-passerine birds (Nagano
1962; McIntosh and Porter 1967; Mattei et al. 1972; Tingari 1973; Okamura and
Nishiyama 1976; Maretta 1977; Gunawardana and Scott 1977; Phillips and
Asa 1989; Soley 1994; Aire 2003). In all the birds, the centriolar pair, lying at
right angle to each other, is closely associated with the Golgi apparatus, and,
initially, lies mid-way between the cell membrane and the nucleus. The
Fig. 7.3 Contd. ...

elongates and is laterally compressed. Arrowhead, obliquely sectioned
endonuclear cavity containing the perforatorium; Inset: Arrowheads, thickened,
granular nuclear membrane invaginates at its centre; arrow, dense granule in the
developing perforatorium. A, acrosome. B. Step 6 spermatid has an elongated
nucleus (N); arrowheads, microtubules of developing circular manchette; A,
acrosome lies rostral to most of cell cytoplasm; Arrows, ‘shoulder’ of the nucleus.
Inset: the endonuclear canal contains the developing perforatorium. Bars: A = 1
µm, Inset: 2 µm; B (including inset) = 1 µm.
Fig. 7.4 Meleagris gallopavo. A. Step 7 spermatid shows granular nuclear

chromatin beginning to condense and become more electron-lucent than in step
6. The circular manchette (arrowhead) is established, extending from the nuclear
Fig. 7.4 Contd. ...
Spermatogenesis and Testicular Cycles '
diplosome thereafter migrates gradually towards the nucleus, to which it

eventually attaches, usually obliquely, at the portion of the nucleus destined
to become the caudal pole.
The avian proximal and distal centrioles differ in length, with the distal
one being slightly to much longer than the proximal centriole (see Chapter 8),
unlike in mammals in which both centrioles, if persistent, are of similar length.
Also, the distal centriole persists without much modification in birds, but
disintegrates during flagellar formation in mammals (Fawcett and Phillips
1969; Phillips 1974). Thus, in non-passerine birds, the distal centriole forms
the foundation upon which the midpiece of the spermatozoon is built. It
produces the central pair of the axonemal microtubules at the base of this
centriole, determines the length of the midpiece and, according to Phillips and
Asa (1989), allows the spermatozoon to form a midpiece without moving the
annulus, relative to the distal centriole, as occurs in mammals (Phillips 1974).
The axonemal microtubules, generally, extend from the proximal centriole
caudad, into the flagellum, in mammals (Fawcett and Phillips 1969; Gordon
1972; Fawcett 1975).
Although in all birds studied, the proximal and distal centrioles lie
perpendicular to each other, in both the spermatid (e.g. Nagano 1962;
Marchand 1977; Phillips and Asa 1989; Lin and Jones 1993; Soley 1994; Aire
2003) and mature spermatozoon (Tingari 1973; Phillips and Asa 1989;
Thurston et al. 1982; Thurston and Hess 1987), those of Guineafowl initially
lie at right angle to each other, but the angle between them gradually becomes
obtuse as the centriolar complex inserts in a deep vault in the nucleus of the
spermatid (Aire and Soley 2003). Both centrioles subsequently become in-line
aligned so that the junction between them is hardly discernible in
longitudinal sections. Guineafowl therefore does not lack the proximal
centriole, as conjectured by Thurston et al. (1982). However, this type of
centriolar alignment is uncommon, and found mainly in invertebrate
organisms (Afzelius 1979).
The attachment of the tail to the nucleus is simpler in birds than in
mammals. In the latter, an electron-dense basal plate or capitulum inserts
between the proximal centriole and the implantation fossa. In birds, there is
Fig. 7.4 Contd. ...

‘hump’ (arrows) of the cell cytoplasm to the centriolar region, caudally. A, the
acrosome is as wide as the nucleus. B. Lysosomes (L) are numerous; P, proximal
centriole; D, distal centriole; F, fibrous sheath; arrow, annulus. C. Step 8 spermatid.
Inset: the acrosome elongates and is pointed rostrally; C, collar of SER of the
Sertoli cell surrounds the acrosome. In the main micrograph, the white arrow
shows the dense coarse, round or rod-shaped granules; thick arrow, chromatoid
body. D. Circular (arrowheads) and longitudinal (arrows) manchette occur
concurrently at a later stage of this step of spermiogenesis; The central part of the
nucleus is largely devoid of these dense granules. Bars: A, B = 1 µm, C, D = 2 µm,
Inset of C = 1 µm.
no capitulum, although in the ostrich a thin layer of dense material, in place

of the capitulum, occurs (Soley 1994). The cross-striated connecting piece
found in mammals is also absent in birds, although a structure akin to it
develops in early spermatids; this merges to form the non-segmented columns
found in mature spermatozoa in Ostrich (Soley 1994).
Step 8 spermatid. The diameter of the nucleus further reduces. The nuclear
chromatin begins to condense into coarse, round or rod-shaped granules that
are immersed in the granulofilamentous matrix of the karyoplasm (Fig. 7.4C).
A chromatoid body is seen in the proximal region of the cell. Other dense,
amorphous aggregations, of unknown function, may be seen along the entire
length of the centriolar complex. A significant, rarely observed feature of the
spermatid during this step is the concurrent occurrence of both the CM and
longitudinal manchette (LM), with profiles of the latter being lateral to the
former, which appears to be patchy in distribution, at this stage (Fig. 7.4D).
The conversion of the CM to the LM is very transient.
The chromatoid body has been described in male germ cells in several
mammals (Sud 1961; Fawcett et al. 1970; Susi and Clermont 1970; Fawcett
1971; Bawa 1975; Söderstrom 1978; Thorne-Tjömsland et al. 1988), but there is
still some controversy concerning its origin, precise structure and function.
This structure “is an irregularly shaped, dense mass of fine fibrillar material
generally found near the acrosomal vesicle and Golgi complex of early
spermatids” (Fawcett 1971). It is typically reticular in structure in sections.
The origin and structure of the chromatoid body has been studied in mammals
(Sud 1961; Eddy 1970; Fawcett 1971; Bawa 1975). It is apparently formed by
an aggregation of filamentous material that is abundant in spermatocytes.
There are very few reports, indeed, on the chromatoid body in birds. Gupta
(1955) has described the presence of this structure in unfixed seminiferous
tubules of the drake. The chromatoid body has been demonstrated in
spermatids of Rhea by Phillips and Asa (1989), while Soley (1994) and Aire
(2003) have described it in the spermatids of the ostrich and the turkey,
respectively. Guineafowl spermatids also possess chromatoid bodies (Soley
and Aire unpublished). According to Fawcett (1971) the chromatoid body
migrates caudally, disperses in the process, and ultimately disappears, but its
fate in the avian spermatid is not known. The role of this structure in
spermiogenesis is not clearly understood. It is considered to play roles in the
development of the connecting piece in mammals (Fawcett 1971) and in the
transportation of ribonucleoproteins to the structures in the neck region of the
developing spermatid (Paniagua et al. 1986) or maturation of the nuclear
chromatin of the spermatid (Sud 1961), but the latter view has been
discounted by Eddy (1970) who failed to demonstrate RNA presence in the
chromatoid body, nor its involvement in the elaboration of the connecting
piece, in various laboratory animals.
The presence of a dual set of manchette microtubules has now been
established in mammals and non-passerine birds. The CM appears to be
Spermatogenesis and Testicular Cycles '!
poorly developed in a pigeon (Columba sp.) (Fawcett et al. 1971; Mattei et al.
1972) and cuckoo (Crotophaga ani) (Saita et al. 1982), but very well developed
in the ostrich, in which it consists of a double set of tubules that are linked
together (Soley 1997). The transition to the LM in the elongated spermatids of
animals has also provoked some controversy. There appears to be a
transition, of quite brief duration, between the disappearance or
reorganization of the CM and the establishment of the LM. Both sets of
manchette microtubules occur concurrently, for apparently a fleeting period
only, in the African collared dove (Streptopelia roseogrisea) (Mattei et al. 1972),
Rhea (Phillips and Asa 1989), Ostrich (Soley 1997) and Turkey (Aire 2003),
during which period the microtubules of the CM are probably rearranged to
become the LM (Phillips and Asa 1989). Although Okamura and Nishiyama
(1976) consider the transition to be abrupt in the rooster, an illustration by
Gunawardana and Scott (1977), in the same species of bird, indicates a
concurrent presence of both sets of manchette microtubules, as has been
observed in Rhea (Phillips and Asa 1989) and Turkey (Aire 2003) (see further
examples in Chapter 8).
Step 9 spermatid. The LM is fully established and extends, beyond the
midpiece and annulus, into the trailing cell cytoplasm, caudally (Fig. 7.5A).
Step 10 spermatid. The nucleus elongates further in a gentle curve, and with
a reduced diameter compared to the preceding spermatids (Fig. 7.5B,C,D). The
acrosome is well formed and houses the perforatorium in its subacrosomal
space. The coarse nuclear chromatin granules become more electron-dense
and more compactly packed together than in spermatid step 9. Mitochondria
continue to elongate and increase the density of their matrix.
Step 11 spermatid. The nuclear chromatin granules become large, highly
electron-dense and compact (Fig. 7.5E,F,G). The spermatid is cylindrical, and
maintains the gentle curvature. The acrosome is lanceolate and accommodates
a perforatorium that extends from the depth of the endonuclear canal to close
to the apex of the acrosome. The tapering end of the nucleus remains
protuberant into the subacrosomal space. The LM is still present and is
surrounded distally by elongated mitochondria in the caudal part of the
trailing cytoplasm. During the latter part of this step, the LM begins to break
up patchily. Glycogen aggregations may be seen in the cytoplasm (Fig. 7.5G).
The acrosome and the perforatorium have, together, been regarded as the
acrosome complex (Baccetti 1979). The perforatorium is a fibrous structure
consisting of parallel bundles of filaments (Baccetti 1979) composed of actin
(Campanela et al. 1979; Baccetti et al. 1980). The structure of this complex in
birds has been described by several authors, in the rooster (Lake et al. 1968;
Bakst and Howarth 1975; Thurston and Hess 1987), the turkey (Thurston et al.
1982), Guineafowl (Thurston et al. 1982; Thurston and Hess 1987), Mallard
drake (Maretta 1975), Rhea (Phillips and Asa 1989), Crested tinamou
(Eudromia elegans elegans) (Asa et al. 1986) and Ostrich (Soley 1993; Baccetti et
al. 1991) (see also Chapter 8). However, there are only a few reports on the
Fig. 7.5 Meleagris gallopavo. A. Step 9 spermatids. The LM is fully established,

with the disappearance of the CM. Straight arrows, dense amorphous material; A,
annulus; F, fibrous sheath. Step 10 spermatids (B, C and D): B. and C, the
Fig. 7.5 Contd. ...

Spermatogenesis and Testicular Cycles '#
development of the complex in birds, and these are specifically in the rooster
(Nagano 1962; Tingari 1973; Okamura and Nishiyama 1976; Gunawardana
and Scott 1977; Baccetti et al. 1980), Budgerigar (Humphreys 1975a), Japanese
quail (Saita et al. 1980; Lin and Jones 1993), Guineafowl (Aire 2003) and
Ostrich (Soley 1996).
In all reports, a similar process of development exists in non-passerine
birds. From about Step 6 of spermiogenesis (in Turkey, Aire 2003), the crater in
the nucleus formed by and lodging the acrosomal granule, flattens out, and
the rostral end of the nucleus becomes convex, once again. Concurrently, the
caudal part of the elongating acrosome, at the interface with the nucleus,
begins to invaginate, thus, beginning the formation of the subacrosomal space.
As this space deepens, the developing rostral end of the perforatorium is
pulled along or pushes into the space. The perforatorium is thought to assist
in elongating and supporting the acrosome, during its development (Baccetti
et al. 1980) but it is known to project after disruption of the acrosome vesicle in
the acrosome reaction of some vertebrates, e.g. Lamprey and Sturgeon (see
Jamieson 1991). The fully developed perforatorium is embedded in the
endonuclear canal and projects into the deep and ample subacrosomal space,
in most birds, extending to just beneath the rostral end of the acrosome
(Nagano 1962; Tingari 1973; Gunawardana and Scott 1977; Baccetti et al.
1980; Soley 1996; Aire 2003). The perforatorium is absent in passerine birds,
vide infra, and in some non-passerines (see Chapter 8).
Whereas the rostral tip of the nucleus tapers slightly and projects for a
short distance into the subacrosomal space, thus forming the intra-acrosomal
portion of the nucleus, in most birds (Lake et al. 1968; Bakst and Howarth
1975; Maretta 1975; Thurston and Hess 1987), the intra-acrosomal portion of
the nucleus is extremely long and occupies almost all of the subacrosomal
space, bearing the equally long perforatorium in its extensive endonuclear
canal in Struthio camelus (Soley 1996). It is noteworthy that the rostral tip of the
nucleus of Budgerigar (Humphreys 1975; Jamieson et al. 1995), white-naped
Crane (Phillips et al. 1987), cockatiel, and peach-faced lovebird (Jamieson et al.
1995) does not project into the subacrosomal space, as in other birds, but
makes direct, en face contact with the caudal rim of the acrosome. In these
birds, therefore, the sperm nucleus has no intra-acrosomal portion because the
Fig. 7.5 Contd. ...

acrosome is highly elongated and compressed laterally, D. the central part of the
nucleus is devoid of chromatin granules; arrowheads, LM; arrows, numerous
multivesicular bodies; L, lysosomes; M, mitochondria with longitudinal cristae.
Step 11 spermatids (E, F and G); E, chromatin granules are dense and compacted
in the nucleus, F, the acrosome (A) is lanceolate, P, perforatorium surrounded by
fuzzy material in the subacrosomal space; arrows, LM. G, transverse section of
spermatid showing the principal piece of the spermatid; M, mitochondria
surrounding the LM (arrow); G, glycogen granules. Bars for all figures = 1 µm.
Fig. 7.6 Meleagris gallopavo. Step 12 spermatids. A. The mitochondria (M) form a
sheath around the midpiece, and the spermatid continues to withdraw from its
Fig. 7.6 Contd. ...

Spermatogenesis and Testicular Cycles '%
acrosome does not overlap it. It is also interesting to note that the nucleus of
the mature avian spermatid and spermatozoon consists of compact chromatin
granules, and not of condensed, homogeneous chromatin, found in insect and
mammalian spermatozoa (Okamura and Nishiyama 1976; Phillips and Asa
1989; Thurston et al. 1982), though it approaches homogeneity in Apus apus
(Jamieson and Tripepi 2006).
Step 12 spermatid. The LM disappears, allowing the elongated mitochondria
to form a helical sheath closely around the midpiece (Fig. 7.6A). The evolved
mature spermatid moves away from most of the redundant cell cytoplasm
which still contains glycogen accumulations (Fig. 7.6B), and profiles of the
endoplasmic reticulum as well as multivesicular bodies. The mature
spermatid attains the luminal surface of the seminiferous tubule, and is held
in place by only slips of Sertoli cell cytoplasm. It is ready for spermiation (Fig.
7.6C). The released spermatozoon possesses no cytoplasmic droplet, as
formed in mammals.
The dissolution or disassembly of the LM during the early phase of Step 12
of spermiogenesis in Turkey (Aire 2003) appears to make way for the
elongated, dense mitochondria, that have aggregated to surround, sometimes,
at least, helically, the proximal axoneme as the mitochondrial sheath. A
similar observation has been made in mammals (Courot et al. 1970; Phillips
1974), Rhea (Phillips and Asa 1989) and Ostrich (Soley 1994). A departure
from this developmental process is in Japanese quail, in which the
mitochondrial sheath has already begun to form in Step 10 spermatid, even
when the CM is still in place, and is complete before the LM disappears in the
early phase of Step 12 spermatids (Lin and Jones 1993). The mitochondrial
sheath is also in place when the LM is still well developed, in the absence of
a CM, in Caprimulgus europaeus (see Chapter 8). It is not known whether the
mitochondria move through the curtain of manchette or migrate into this
curtain through its distal end, in this species.
7.3.2 Spermiogenesis in Passerine Birds

The passerine birds constitute a large group among birds, and although they
are more derived than, for example, members of the Galliformes,
Columbiformes, Anseriformes or Struthioniformes and other ratites, the study
of spermatogenesis, and its related phenomena, in this group of birds has
lagged behind those of non-passerine birds. They have been the most common
birds, from the time man started studying this class of animals. There are only
Fig. 7.6 Contd. ...

own redundant, electron-dense cytoplasm (C); N, nucleus of spermatid. B.
Transverse sections of step 12 spermatids: the LM disappears and mitochondria
(M) then align themselves around the midpiece; glycogen granules (Gl) are still
present in the cytoplasm. C. Late phase of step 12, showing a well established
mitochondrial sheath. Slips of Sertoli cell cytoplasm (arrows) hold on to the
spermatid tenuously, as the spermatid is ready for spermiation. Bars: A = 4 µm;
B = 2 µm and C = 1 µm.
Fig. 7.7 Diagram of the 6 steps of spermiogenesis of the domestic sparrow,

Passer domesticus. Two views (A: dorso-ventral view and B: side-view), show
Fig. 7.7 Contd. ...

Spermatogenesis and Testicular Cycles ''
a few reports on the male gamete and its morphogenesis in passerine birds,
such as House sparrow (Passer domesticus) (Yasuzumi 1956; Sotello and
Trujillo-Cenóz 1958; Góes and Dolder 2002), Lovebird or Bengalese finch
(Lonchura striata var. domestica) (Fawcett et al. 1971; Yasuzumi and Sugioka
1971; Kondo et al. 1988) and Zebra finch (Taeniopygia (=Poephila) guttata)
(Nicander 1970). For accounts of mature spermatozoa, see Chapter 8.
During the early and late acrosome phase in oscine birds, e.g. House
sparrow and Lovebird (Bengalese) finch (Lonchura), the acrosomal vesicle, in
Step 2 spermatids, contains a matrix of low and medium density and lodges
in a nuclear cavity or depression (Fig. 7.7), without forming a cap (Góes and
Dolder 2002), as in non-passerines. As the acrosome elongates, the matrix
differentiates into an outer low-density zone as well as a central higher
density zone, and assumes a zigzag course, with sharp angulations of its
membrane projecting alternately on both sides (Fawcett et al. 1971). The
perforatorium is absent. In early spermatids, the nuclear chromatin is loosely
arranged (Góes and Dolder 2002) or condenses particularly into fine granules
(Kondo et al. 1988). Further condensation of the nuclear chromatin forms
denser coarse granules. There is no agreement on the appearance of
microtubules around the nucleus of Lonchura spermatids (Fawcett et al. 1971;
Kondo et al. 1988). However, microtubules appear very early around the
spherical nucleus in a cluster, extending from the rostral to the post-nuclear
region (Kondo et al. 1988), but they are only evident in intermediate and late
stages of differentiation in Step 3 spermatids (Góes and Dolder 2002) when
the nucleus has already assumed a helical form (Nicander 1970; Fawcett et al.
1971; Yasuzumi and Sugioka 1971; Góes and Dolder 2002).
The bundle of microtubules describes a helical course on the outer surface
of the gyres of the nucleus and the developing flagellum (Fig. 7.7). The
mitochondria, located predominantly in the postnuclear region, begin to fuse
together, forming a long strand alongside the axoneme. The helical
microtubular bundle around the nucleus does not extend rostrally beyond the
basal turn (in the region of the base of the acrosome) of the helix, indicating
that the spiral shape of the elongated acrosome may not be influenced by the
microtubules (Fawcett et al. 1971) but rather by an intrinsic mechanism yet to
be understood. Following the establishment of the microtubular helix, during
the mid-maturation phase, the long strand of mitochondria winds round the
axoneme helically, such that it lies between the axoneme and the microtubule
bundle (Fawcett et al. 1971; Kondo et al. 1988; Góes and Dolder 2002). The
Fig. 7.7 Contd. ...

different sectioning planes, have been diagrammed for steps 2, 3 and 4, so as to
include all organelles. A, acrosome; ax, axoneme; ca, centriolar adjunct; df, dense
fibers; ER, endoplasmic reticulum; G, Golgi complex; ig, pro-acrosomal internal
granule; ms, mitochondrial sheath; mt, microtubules; Pa, pro-acrosome; sr,
spiraling ridges of the acrosome. From Góes, R. M. and Dolder, H. 2002 Tissue
and Cell 34: 273-282. Reproduced with permission of Elsevier Science Ltd.
bundle of microtubules, the homologue of the manchette in mammals and

non-passerine birds (Fawcett et al. 1971), makes shallow indentations on the
outer surfaces or ridges of the helical nucleus.
During the late maturation phase of the spermatid, acrosomal condensation
and elongation is completed, and the cell contains 2.5 gyres in Lonchura striata
(Kondo et al. 1988). The bundles of microtubules are arranged in rows that
alternate with cisternae of smooth endoplasmic reticulum (Fawcett et al. 1971;
Kondo et al. 1988; see also Chapter 8). The role of the latter is unknown. It is
generally agreed that the microtubular bundles are transient, being lost before
spermiation of mature spermatids in Lovebird (Bengalese) finch (L. striata var.
domestica) (Kondo et al. 1988; Fawcett et al. 1971) or shed as the spermatozoa
pass through the ductus deferens in Zebra finch (Nicander 1970) and House
sparrow (Passer domesticus) (Góes and Dolder 2002), although Yasuzumi and
Sugioka (1971) regard them to be a permanent feature involved in motility in
the spermatozoa of L. striata.
The development of the flagellum of the passerine bird has received even
less attention than acrosomal and nuclear morphogenesis. Centriolar complex
development in passerine birds is generally similar to that in mammals and
non-passerines (Sotelo and Trujillo-Cenóz 1956), but whereas Góes and
Dolder (2002) consider that the proximal centriole lodges in an implantation
fossa of the nucleus while the distal centriole extends, caudally, to the cell
membrane and forms an annulus at the contact junction, Sotelo and Trujillo-
Cenóz (1956) state that one of the centriolar pair in House sparrow
disappears, unaccounted for. Nicander (1970) also states that passerine birds
possess only one modified centriole, and that both a segmented pericentriolar
material as well as the nine identical coarse peripheral tail fibers anchor the
neck to the base of the head. A similar striated structure, akin to the
mammalian connecting piece, has been shown in micrographs of Lonchura
striata spermatozoa, published by Fawcett et al. (1971). For further details of
mature sperm, see Chapter 8.
7.4 SPERMIATION
Spermiation has been reported in only two birds, the rooster (Sprando and
Russel 1988) and Japanese quail (Lin and Jones 1993). The process is similar
in both species of birds, belonging to the Galliformes. During the process of
spermiation, the spermatid cytoplasm tends to condense and become more
electron-dense relative to the cytoplasm of other germ cells, as well as the
Sertoli cells (Sprando and Russel 1988). Residual bodies, light-staining in
birds, but highly condensed in mammals, form late in spermiogenesis, near
the time of sperm release (Sprando and Russel 1988). They are phagocytised
by Sertoli cells (Sprando and Russel 1988; Lin and Jones 1993). The
tubulobulbar complex has not been observed in birds. Further studies on
spermiation, involving other orders of birds are necessary for a complete
picture of this phenomenon in this large class of animals. Unlike in mammals
Spermatogenesis and Testicular Cycles !
(Fawcett and Phillips 1969), there are no reports of cytoplasmic droplets in

newly released spermatozoa of birds (Lake 1981; Sprando and Russel 1988)
except in those of Ostrich (Aire and Soley 2000). This indicates that the loss of
the excess cytoplasm in the region of the head of the spermatid in the ostrich
is probably similar to that described for the Galliformes (Sprando and Russel
1980; Lin and Jones 1993), but unlike in mammals where it is from the
junction between the head and neck of the spermatid. The study of the
formation of the cytoplasmic droplet in the spermatozoon, and its loss in the
post-testicular spermatozoon of Ostrich is in progress in our laboratory.
7.5 KINETICS OF SPERMATOGENESIS IN BIRDS

In most mammals, cross-sections of seminiferous tubules contain varying
generations of germ cells arranged in a definite, successive, repeatable order,
from the basement membrane to the lumen of the tubule (von Ebner 1871;
Regaud 1901). It has been established that each generation of germ cells,
resulting from stem cell divisions, are all linked by cytoplasm bridges (Dym
and Fawcett 1971) to form a mass of syncytial cells that develop in synchrony.
According to Perey et al. (1961) each generation of germ cells is at exactly the
same step of development, and normally associated with other generations in
a manner that is predictable and constant for the species. Thus, “groups of
spermatids at a given step of development are always associated with the
groups of spermatocytes and spermatogonia” (Perey et al. 1961), as well as the
same group of Sertoli cells which provide support, nutrition and regulatory
mechanisms for their development (Dym and Fawcett 1970; Morris et al.
1987a; Jégou 1991; Skinner et al. 1991). This constant grouping of the same
generations of cells, arising from the same spermatogonium in a segment of
the seminiferous epithelium is known as a cellular association. Leblond and
Clermont (1952a,b) identified 14 consistent cellular associations in the
seminiferous epithelium of Rattus norvegicus. The identification or
classification of cellular associations has been facilitated by the use of specific
morphological changes in developing spermatids. Cellular associations occur
in an orderly synchronized, successive series in any given area of the
seminiferous tubule, such that, in Rattus norvegicus, the 14 associations,
arranged in a succession, numbered I to XIV, constitute a cycle of the
seminiferous epithelium. Each cellular association occurs in a specific period in
the cycle, and is known as a stage of the cycle. In most mammals, each cross-
section of the seminiferous tubule consists of a single stage of the cycle of the
seminiferous epithelium. But in birds, as in primates (Roosen-Runge 1952;
Clermont 1963; Chowdhury and Marshall 1980; Dietrich et al. 1986; Johnson
et al. 1981), cross-sections of the seminiferous tubule display several different
(heterogeneous) cellular associations or stages of the seminiferous epithelium,
e.g. there are up to 6 cellular associations in the rooster (Courot et al. 1970), 10
in Japanese quail (Lin and Jones 1990) and 3 in primates (Schulze 1982;
Schulze and Rehder 1984; Dietrich et al. 1986; Schulze et al. 1986).
Although Gunawardana (1976) and Gunawardana and Scott (1977) used

the term “stage” in their studies of the the rooster, they were, in fact, referring
to steps of spermiogenesis. Certain constraints, such as heterogeneous, as well
as atypical cellular associations occupying small areas of the seminiferous
tubule (Table 7.1), unlike in non-primate mammals (Clermont 1963; Dietrich et
al. 1986; Aire et al. 1980), the lack of sensitivity of the PAS method for
classifying steps of spermiogenesis in birds and the use of paraffin-embedded
material, have confounded the study of various aspects of spermatogenesis in
birds (Clermont 1958; Yamamoto et al. 1967; de Reviers 1971; Aire et al. 1980).
In paraffin-embedded tissues, eight stages of the cycle of the seminiferous
epithelium were identified in each of Japanese quail (Yamamoto et al. 1967),
Mallard (Clermont 1958) and Guineafowl (Aire et al. 1980). However, the
introduction and use of electron microscopic facilities and processes have
made it possible for the identification of subtle aspects of avian
spermatogenesis, thus significantly enriching our knowledge (Lin and Jones
1990; Lin et al. 1990; Lin and Jones 1992). It is noteworthy that these studies
are the most complete and up-to-date evaluations of the seminiferous
epithelium in any avian species. This review will therefore draw heavily on
these reports. Lin et al. (1990) have been able to classify the seminiferous
epithelium of Coturnix into 10 stages (I to X), and 12 steps of spermiogenesis,
using acrosomal development (Leblond and Clermont 1952b) and nuclear
morphogenesis of the spermatids (Roosen-Runge and Giesel 1950; Ortavant
1954) as criteria, in plastic sections. Subsequently, and by means of
autoradiography and radiolabeling, Lin and Jones (1992) have also
determined the process of stem cell renewal, and spermatogonial proliferation
and differentiation into spermatocytes (Fig. 7.8). Figure 7.9 shows the cycle of
the seminiferous epithelium of Japanese quail, which includes
spermatogonial types, as well as other generations of germ cells and their
occurrences in the various, successive stages of the cycle of the seminiferous
epithelium. Furthermore, both the frequency and duration of the 10 stages of
Table 7.1 Area of the wall of a seminiferous tubule occupied by a cellular association of
the seminiferous epithelium and by a Sertoli cell, and the number of Sertoli cells in a cellular
association
Cellular associations Sertoli cells

2
Quail No. measured Area (mm ) No. measured Area (mm 2) No. per association
a
208 37 12760 ± 978 23 1625 ± 732 7.9 ± 1.9
269a 24 21291 ± 1812 21 1405 ± 455 15.2 ± 4.1
278a 31 19655 ± 1737 24 1133 ± 88 17.3 ± 2.9
Meanb - 17902 ± 2614 - 1388 ± 142 13.5 ± 2.8
Values are mean ± s.e.m. for 3 birds, with the s.e.m. calculated from the variance between astages
within a seminiferous tubule and banimals. From Lin, M. and Jones, R.C. 1990. Journal of Reproduction
and Fertility 90: 361-367, Table 1. © Society for Reproduction and Fertility (1990). Reproduced by
permission.
Spermatogenesis and Testicular Cycles !!
the cycle of the seminiferous epithelium (Tables 7.2 and 7.3) have been
determined for Japanese quail (Lin and Jones 1990). The duration of one cycle
of the seminiferous epithelium in Japanese quail is estimated to be 2.69 ± 0.08
days.
Compared to mammals, spermatogenesis in birds, which are generally very
promiscuous, is a very rapid process because they invest heavily in rapid
sperm production, since, unlike mammals, they do not store them for any
appreciable length of time. For example, whereas the testis weight relative to
body weight in Japanese quail is between 2.26 and 3.3% (Clulow and Jones,
1982; Aire 2005), that in R. norvegicus is 0.67% (Clulow and Jones 1982). It is
generally agreed that seminiferous tubular diameter, epithelial height,
testicular weight and spermatogenesis are positively related.
Table 7.2 Relative frequency (mean ± s.d.) and duration of the stages of the cycle of the
seminiferous epithelium in the Japanese quail
Stage
I II III IV V VI VII VIII IX X
Frequency (%) 11.9 14.8 24.1 10.3 8.2 6.4 9.4 5.5 3.8 5.4
±3.1 ±5.1 ±3.9 ±3.4 ±1.4 ±0.5 ±5.5 ±2.4 ±1.5 ±0.8
Duration (h)* 7.7 9.5 15.5 6.6 5.3 4.1 6.1 3.6 2.5 3.5
*Based on the estimate of 64.4 h for one cycle. From Lin, M., Jones, R. C. and Blackshaw, A. W.
1990. Journal of Reproduction and Fertility 88: 481-490., Table 1. © Society for Reproduction and
Fertility (1990). Reproduced by permission.
Table 7.3 Estimates of the duration of the seminiferous epithelium in the Japanese quail
Most advanced labeled cell

Time (h) after
injection of Cycle No. of cycles Duration of one cycle
[ 3H] thymidine stage* traversed
Cell type Days Hours
2 Leptotene primary
spermatocyte 2, IV - - -
26 Pachytene primary
spermatocyte 2, VII 0.345 2.90 69.6
spermatocyte 3, II 0.759 2.63 63.1
spermatocyte 3, V 1.187 2.53 60.7
98 Secondary
spermatocyte 3, X 1.493 2.68 64.3
Mean± s.e. 2.69±0.08 64.4±1.88
*No difference found among the animals within each group. From Lin, M., Jones, R. C. and Blackshaw,
A. W. 1990. Journal of Reproduction and Fertility 88: 481-490, Table 2. © Society for Reproduction and
Fertility (1990). Reproduced by permission.
Fig. 7.8 Diagram showing the mode of stem cell renewal and proliferation of
spermatogonia in the quail. Numeric superscripts show the number of germ cells
Fig. 7.8 Contd. ...

Spermatogenesis and Testicular Cycles !#
7.5.1 Wave of the Seminiferous Epithelium

The completion of a full series of cellular associations, between two successive
appearances of the same stage, along the length of the seminiferous tubule, is
known as the wave of the seminiferous epithelium. Thus, for Rattus norvegicus, the
wave of the seminiferous epithelium is “a series of adjacent segments, each of
which includes the 14 possible types, in addition to any segment which is
involved in modulation” (Perey et al. 1961). ‘Modulations’ are irregularities
caused by temporary and spatially limited reversion or inversion of the
numerical order in successive occurrences of stages in the cycle of the
seminiferous epithelium (Perey et al. 1961; Dietrich et al. 1986). In R. norvegicus,
the numbering of the stages in the wave decreases from the rete testis, into
which the loop of the seminiferous tubule opens, at both ends (Perey et al.
1961). Modulation appears to be a frequent occurrence in the waves, because
only 20% of the waves are unaffected, while up to 17% have more than three
modulations in R. norvegicus (Perey et al. 1961). The phenomenon of
modulation has not been reported in birds, apparently because of the
anastomotic nature of the seminiferous tubules in this class of animals (Lin
and Jones, 1990).
The occurrence of heterogeneous cellular associations in the cross-section
of the seminiferous tubule of birds and primates has impeded the study of the
kinetics of the seminiferous epithelium in these animals. That is why only a
few studies on the nature of the wave of the seminiferous epithelium in birds
and primates have been reported, in Japanese quail (Lin and Jones 1990), and
Homo sapiens (Schulze 1982; Schulze and Rheder 1984). Perey et al. (1961)
show that in most mammals, waves are not arranged spirally in the
seminiferous tubule. However, Lin and Jones (1990) have demonstrated a
spiraled helical arrangement of the waves of the seminiferous epithelium in
Japanese quail, as has been described for some primates (Schulze 1982;
Schulze and Rehder 1984; Dietrich et al. 1986; Schulze et al. 1986). This
arrangement in primates and the quail, as perhaps also in other birds, may
partly explain why there are heterogeneous cellular associations in the
epithelium of cross-sections of the seminiferous tubules in these animals, but
not in other mammals that lack this helical pattern of wave arrangement. The
crab-eating macaque, one of the primates exhibiting heterogeneous cellular
associations, displays frequent modulations in the wave of the seminiferous
epithelium (Dietrich et al. 1986). The branching pattern of seminiferous tubules
in birds has not made it possible to determine the presence and pattern of
modulations. In adult animals, there are no further migrations of primordial
Fig. 7.8 Contd. ...
in each generation. See Fig. 7.9 for time of occurrence of each cell type and division
during the cycle of seminiferous epithelium. From Lin, M. and Jones, R. C. 1992
Cell and Tissue Research 267: 591-601, Figure 12. Reproduced with the kind
permission of Springer Science and Business Media.
germ cells or gonocytes (which mature to become spermatogonia) into the

seminiferous tubules, neither do Sertoli cells undergo further mitotic divisions;
the numbers of Sertoli cells are therefore constant in the seminiferous
epithelium. The process of spermatogenesis commences with the mitotic
divisions of spermatogonia in the active testis. Spermatogenesis is a
continuous process in the fully stimulated and functionally active testis. The
continuation and sustenance of spermatogenesis is therefore contingent upon
the availability of stimulated spermatogonia whose successive mitotic
divisions will produce spermatocytes. The replacement of these dividing
spermatogonia has engaged the attention of reproductive biologists over the
years. According to Roosen-Runge (1962) the process of spermatogenesis in
mammals is cyclic, such that in any given area of the seminiferous tubule, a
new generation of germ cells begins to differentiate before the last generation
has completed development. These new generations initiate their development
at definite and periodic intervals, in fixed relation to preceding generations.
The nature and process of spermatogonial replacement and/or conversion to
primary spermatocytes has been examined in several mammals, although
reports are not without discrepancies, contradictions and controversy
(Roosen-Runge 1951; Leblond and Clermont 1952b; Oakberg 1956a; Clermont
1958; Clermont and Leblond 1959; Hochereau 1967; Clermont 1972; Clermont
and Antar 1973; Clermont and Hermo 1975; Roosen-Runge 1977; Huckins
1978; Setchell 1982). The methods of investigation have been part of the
problem, but it appears certain that, in all animals, “Cycle after cycle, the
primitive cells repeat the same behavior” (Roosen-Runge 1962), with regard to
the pattern and nature of spermatogonial differentiation and renewal. The
heterogeneous cellular associations, as well as the small areas occupied by
stages of the cycle of the seminiferous epithelium might have impeded studies
involving spermatogonia and their divisions in birds. Thus, there are only two
main reported studies on spermatogonial divisions, differentiations and
renewal in birds (Clermont 1958; Lin and Jones 1992).
Clermont (1958) employed colchicine as a method of evaluation of the
spermatogenic epithelium in Mallard, but the limitations of this method
include the absence of adequate knowledge of the structural features of all
possible types of spermatogonia. In addition, colchicine arrests mitotic
division at metaphase, and cells in such a state contributed to the counts. The
studies reported by Lin and Jones (1992), in order to overcome these
limitations, used autoradiography and radiolabeling to investigate the process
of spermatogonial differentiation and renewal, in Japanese quail.
In an 8-stage cycle of the seminiferous epithelium in Mallard, Clermont
(1958) observed and described three types of spermatogonia: type A (stem)
spermatogonium which divides during Stage V of the cycle of seminiferous
epithelium. The products of this division are two, non-identical, variably-
tracked cells: a new, resting type A (stem cell) spermatogonium and a
differentiating type B spermatogonium. The latter undergoes mitotic division,
during Stage VIII of the seminiferous epithelial cycle, to produce two type C
Spermatogenesis and Testicular Cycles !%
spermatogonia, which, on division during Stage III of the cycle, continue with
germ cell differentiation, into spermatids. In their study on Japanese quail, Lin
and Jones (1992) have described four types of spermatogonia in the
seminiferous tubules, as follows: a dark type A (Ad), 2 variants of pale type A
(Ap1 and Ap2), and a type B (Figs. 7.8 and 7.9).
By means of electron microscopy, the morphological features of each type of
spermatogonium have been described in detail, for the first time, in an avian
species (Lin and Jones 1992). The following brief review is taken, mainly, from
this work. Dark type A (Ad) spermatogonia lie on the basal lamina, are
elliptical in shape and stain densely (Fig. 7.10A). The eccentric nuclei are
small, ovoid and dark-staining, and the nucleoplasm contains uniformly
dispersed heterochromatin aggregations. The cytoplasmic organelles appear
sparse, and include a moderate abundance of mitochondria located
basolateral to the nucleus, and a few strands of RER. Type Ad spermatogonia
are regarded as the undifferentiated stem cells in Japanese quail, as (a) since
they are present in all stages of the cycle of the seminiferous epithelium (Lin
and Jones 1990), (b) they occupy and make greater contact with the inner
surface of the basal lamina than other spermatogonial types, (c) they form
discrete, solitary cells, and (d) they reflect radiolabeling less frequently than
the other spermatogonial types. The appropriateness and adequacy of this
method of identification of the types and profiles of spermatogonial division
is underscored by the observed number of spermatids (32), in each bundle
embedded in Sertoli cells of the quail.
The pale type Ap1 spermatogonia have the largest ovoid, spermatogonial
nuclei, which are light-staining, although they contain a few (3-4) large and
several smaller aggregations of chromatin (Fig. 7.10B). The cytoplasm is also
light-staining, and slightly endowed with organelles. However, type Ap2
abounds in numerous, basally aggregated, mitochondria and associated RER,
ribosomes, and an active Golgi complex (Fig. 7.11A). Cytoplasmic bridges
commonly link adjacent Ap2 spermatogonia. Type Ap1 occurs in Stages X, I
and III, while type Ap2 occurs during Stages III, IV, V and VI of the
seminiferous epithelium (Fig. 7.9).
The type B spermatogonia, obviously the last in the series to divide
mitotically, are often connected to their fellows by cytoplasmic bridges (Fig.
7.11B). Both their cytoplasm and nuclei are densely stained with toluidine
blue, in plastic sections. The nuclei are ovoid and display variably-shaped
clumps of chromatin attached to the inner surface of the nuclear membrane.
Multiple nucleoli (2-4) are scattered within the nucleoplasm. A prominent
Golgi complex is basally situated. Mitochondria lie on either side of the
nuclear poles. The cell lies on the basal lamina.
The type Ad spermatogonia are considered by Lin and Jones (1992) to be
the stem cells because they occur in all stages of the cycle of the seminiferous
epithelium, as well as being solitary cells and not showing radiolabeling as
frequently as the other types of spermatogonia. As a stem cell, the type Ad
spermatogonium duplicates itself, during Stage IX of the cycle, by producing,
Fig. 7.9 The cycle of the seminiferous epithelium in the Japanese quail showing associations of germ cells in the 10 stages of
the cycle. Ad, Dark type A spermatogonia; Ap1, pale type A1 spermatogonia; Ap2, pale type A2 spermatogonia; B, type B
spermatogonia; L, leptotene primary spermatocytes; Z, young primary spermatocytes in zygotene; P, pachytene primary
spermatocytes; Dp, diplotene primary spermatocytes; An, anaphase primary spermatocytes; II, secondary spermatocytes; 1-12,
Step 1 to Step 12 spermatids. From Lin, M. and Jones, R. C. 1992 Cell and Tissue Research 267: 591-601, Figure 13. Reproduced
with the kind permission of Springer Science and Business Media.
Spermatogenesis and Testicular Cycles !'
Fig. 7.10 Coturnix japonica. Spermatogonia. A. Spermatogonium type Ad lies on

the basal lamina, has a relatively small nucleus displaying small heterochromatin
aggregations; mitochondria (M) are sparse and admixed with short profiles of RER
(arrowheads). B. The spermatogonium type Ap1 also lies on the basal lamina,
and displays a relatively large, less heterochromatic nucleus than Ad type. At least,
two centrally located nucleoli occur in the nucleoplasm. Only sparse organelles
occur in the cytoplasm. Bars: 2 µm for both figures. Original.
Fig. 7.11 Coturnix japonica. A. Spermatogonium type Ap2 nucleus has clumps of
heterochromatin lying in the nucleoplasm or attached to the nuclear membrane. A
relative abundance of organelles, preponderantly mitochondria, occur in the
cytoplasm. B. Spermatogonium type B has a nucleus that is relatively euchromatic
and displays only a few clumps of heterochromatin as well as two to four nucleoli.
The organelle content is moderate. Arrowheads, a cytoplasmic bridge between
adjacent type B spermatogonium. Bars: 2 µm for both figures. Original.
mitotically, a replicate (type Ad) stem cell, and a type Ap1 spermatogonium
that differentiates to continue the proliferation and differentiational phases of
the process of spermatogenesis. Types Ap1 spermatogonia occur during Stages
X, I and II of the spermatogenetic cycle. Each type Ap1 spermatogonium
divides and produces two type Ap2 spermatogonia during Stage II of the cycle,
while the Ap2 spermatogonia, together, produce four type B spermatogonia in
Stage VI of the cycle. The last mitotic division by the spermatogonial series
occurs during Stage III of the cycle, when the type B spermatogonia, together,
produce eight primary spermatocytes. The meiotic segment of spermatogenesis
begins with primary spermatocytes that, eventually and ideally, produce
thirty-two spermatids. The small number of spermatogonial divisions (three)
in Japanese quail, compared, for example, with 6 to 7 in R. norvegicus (Hilscher
and Makoski 1968; Clermont and Bustos-Obregon 1968; Huckins 1971a,b;
Oakberg 1971a,b; Clermont and Hermo 1975) and 6 in Bos taurus (Hochereau
1967), is in accord with the small area occupied by stages of the cycle of the
seminiferous epithelium, and, also, with the number of Sertoli cells in each
stage, in the quail (Table 7.1). Theoretically, 32 spermatids arise from one stem
cell (Fig. 7.8), and this number was actually counted in a bundle of spermatids
embedded in Sertoli cells in the Japanese quail (Lin and Jones 1992). Germ cell
degeneration occurs frequently in mammalian seminiferous tubules (Roosen-
Runge 1962). Germ cell loss, of about 22%, has been reported in the rat
(Roosen-Runge 1958 cited by Roosen-Runge 1962) and in Mus musculus, the
mouse (Oakberg 1956). Whether all 32 spermatids are present all the time,
without loss of germ cells remains to be determined in birds. Numerous
multinucleated giant cell balls are not uncommonly seen in the seminiferous
epithelium of sexually mature and active ostriches (personal observations), as
well as in cycling sexually immature Ostrich (Madekurozwa et al. 2002). The
nature of these balls of cells has not been determined, but more studies on the
kinetics of spermatogenesis in birds, in general, need to be undertaken in
order that the process of spermatogenesis in this large class of animals may be
better understood.
Jones and Lin (1993) have calculated that Rattus norvegicus produces more
spermatids per stem cell than Japanese quail by about 128-fold, and that the
area occupied by a stage of the cycle of the seminiferous epithelium is 55 times
more than that of Japanese quail. This very low value of spermatids (32) per
stem cell in Japanese quail may be a consequence of the small area occupied
by a stage in the cycle, compared to R. norvegicus.
7.5.2 Duration of Spermatogenesis

The proper evaluation of the function of the testis in health and disease is
predicated upon a thorough understanding of the entire process of
spermatogenesis, and the factors controlling it. An important aspect of this
process is the life span of not only the individual cells but also their
relationships to other cell types in the process. Spermatogenesis begins with
the first spermatogonial division that initiates the spermatogenic series
(Ortavant 1959). Discrepancies in the estimation of the duration of
spermatogenesis arise, partly, from certain methods used in investigating this

process.
The duration of spermatogenesis has been studied in various mammalian
species, and according to Ortavant (1959) all germ cells that proceed in their
development do so at the same speed, and hence the spermatogenic cycle
seems to be a biological constant. The estimation of the duration of
spermatogenesis in birds has, as in other aspects of male reproductive
biology, also received scant attention. The employment of radiolabeling and
autoradiography has, however, considerably improved the determination of
this process. By means of radiolabeling and daily ejaculation methods, de
Reviers (1975) has estimated the duration of spermatogenesis in the rooster to
be between 12 and 13 d, which is the period elapsing between injection of
tritiated thymidine and the release of spermatozoa from the seminiferous
epithelium, and the presence of labeled spermatozoa in semen 13-14 d
following radiolabeling. Labeled spermatozoa are, similarly, present in semen
of the Barbary drake 12 d after [3H]thymidine injection (Marchand et al. 1977;
Marchand 1979). Lin et al. (1990) estimate that the duration of one cycle of the
seminiferous epithelium in Japanese quail is 2.69 ± 0.08 d. Therefore, Lin and
Jones (1992) estimate (Fig. 7.9), that the process of spermatogenesis in the same
species of bird lasts through 4.747 cycles or 12.77 d (i.e. 2.69 d ¥ 4.747 cycles),
from the first spermatogonial (type Ad) division during Stage IX of the cycle to
the release of spermatozoa from the seminiferous epithelium during Stage V.
The duration of spermatogenesis, therefore, is similar in all birds studied
thus far, and is found to be a much faster process than in mammals (25 days
in Boar, Sus scrofa: Swierstra, 1967; 29 day in Sheep, Ovis aries: Ortavant 1959;
32d in Rabbit (Oryctolagus cuniculus): Amman et al. 1965). Also worthy of note
is that the duration of spermatogenesis is constant, and independent of
season, although transit time for spermatozoa through the excurrent ducts
may vary with season (de Reviers 1975).
7.6 RESPONSE OF BIRDS TO REPRODUCTIVE DEMANDS

ON THE TESTIS
Birds are generally socially monogamous, but a high proportion engage in
extra-pair copulation during the short breeding season, especially in the
temperate zone region (see, among others, Volume 6B, Chapters 6 to 9). Gallus
domesticus has been known to mate up to 30 times a day (Penquite et al. 1930).
If more spermatozoa originate from one division of a stem cell in the rat, and
also in other mammals, than in birds, e.g. the quail, how do birds make up for
this deficiency in number of germ cells per stem cell, in their reproductive
process involving frequent copulations during the breeding season? It is
known that reproduction in most species of birds is seasonal or
discontinuous, an adaptation to a period environment (Wilson and Donham
1988). Most birds therefore have a short period of breeding, especially in the
higher latitude (Wingfield et al. 1997a), and many are polygynous. For birds,
reproduction is a very energy-demanding and resourced process. It therefore
needs precise timing to coincide with good environmental conditions (Lack

1968; Perrins 1970; Price et al. 1988; van Noordwijk et al. 1995; Wikelski et al.
2000). Several biological strategies have been adopted by birds to accomplish
their reproductive goals.
Birds, generally, have both large absolute and, in particular, relative testis
weights, as compared to mammals (Lake 1981; Clulow and Jones 1982), and
sperm production has a high positive correlation with testes mass (Møller
1989). Similarly, there is a strong positive relationship between relative
testicular size and relative sperm production, as well as between relative
testes size and relative size of sperm reserve in mammals (Møller 1989), a
situation that may be similar in birds (Møller 1991; Pitcher et al. 2005). It is
therefore expected, and it has been shown, that several birds, e.g. the quail,
have high absolute and relative testis weights, and hence a relatively large
sperm content in ejaculates (Clulow and Jones 1982). However, figures need
to be obtained for birds that are more volant than these relatively terrestrial
species. Several species of birds are polygynous and/or are engaged in
multiple pair and extra-pair copulations. Accordingly, relatively large testes
ensure that such birds can engage in frequent pair, and, in particular, extra-
pair copulations, in order to avoid being cuckolded (Birkhead 1998). Most
species of feral birds and several domestic birds are polygynous, and
therefore engage in intense sperm competition. In passerine birds, the
development of the seminal glomus, as a sperm-storage structure, is necessary
in birds that can not afford to bear the burden of very large testes sizes, and
accordingly, have low daily sperm production rates [e.g. 1.885 ¥ 106 day–1, or
35 ¥ 106 d–1 g–1 testis tissue for Zebra finch (Taeniopygia guttata)] (Birkhead et
al. 1995) compared with a non-passerine bird [92.5 ¥ 106 d–1 g–1 testis tissue
for Japanese quail] (Clulow and Jones 1982). The mass of this structure is
positively correlated with body mass and with the relative testis mass
(Birkhead 1998), and has been shown to attain maximum size in polyandrous
or promiscuous passerine birds that are engaged in intense sperm competition
(Birkhead et al. 1991). Sperm transport from the testis to the seminal glomus in
the Zebra finch occurs primarily at night (Birkhead, T. personal
communication), and the organ is considered to be a temporary overnight
storage depot for spermatozoa (Birkhead et al. 1994) at temperatures that are
lower than core body temperature (Wolfson 1954).
What is lost in the small number of spermatogonial divisions and therefore
sperm numbers per group of Sertoli cells (Lin and Jones 1993) is compensated
for by the relatively large testes size/mass in birds (see Japanese quail and
Rattus norvegicus, above (Clulow and Jones 1982). Thus, the daily sperm
production rate (106/g testis) is 92.5 for Coturnix and 23.7 for R. norvegicus
(Clulow and Jones 1982). Avian testes have high fluid content (Lake 1957; Aire
1979a; Clulow and Jones 1988) that flushes this large number of mostly
immotile spermatozoa rapidly through the seminiferous tubular lumina into
the excurrent duct system. The fluid absorptive properties and the
microenvironments of the various excurrent ducts of the testis are
indispensable for the maturation, viability and fertilizing ability of the post-
testicular spermatozoa that traverse them (Turner 1991; Hinton and Palladino
1995; Hess et al. 1997). The efferent ducts of birds are extremely well
developed, constituting a large volume proportion of between 35 and 62%
(Aire 1979b) of the epididymis, and absorb about 86% (Clulow and Jones
1988) of the copious flow of testicular fluid into the epididymis.
Referring to the reproductive organs of man, as probably also applicable to
most mammals, de Graaf (1668, cited by Joclyn and Setchell 1972), long ago,
observed as follows: “During the time the ingredients of the semen are
propelled through the very long ducts of the testicle, the semen is elaborated
in their cavities in such a way that what was watery and ash-like in the
testicles becomes milky and thick in the epididymides. It is clear that the ducts
of our tubules, epididymides and vasa deferentia are very long and were
shaped by a most discerning Nature primarily so that the seminal matter
might be better elaborated by long delay in transit”. It is also interesting and
noteworthy that, according to Orgebin-Crist (1998), irrespective of the species,
the length of the epididymis, or the rate of sperm production, the passage of
spermatozoa through the epididymis of different mammals lasts
approximately 10 days. The ‘long delay’ necessary for sperm maturation is
absent in birds, in which regard, Nature seems to have adopted new
strategies. It is established that whereas it takes spermatozoa of Japanese quail
only one day to traverse the entire length of the excurrent duct system, it takes
over 8 days, in the rat, to do the same (Clulow and Jones 1982). The rapid
transit time of spermatozoa through the excurrent ducts of the testis, in birds,
seems to be correlated with several structural and physiological modifications
in both the spermatozoa and the ducts themselves. These modifications or
peculiarities serve birds very well in sperm competition, which requires the
availability of a large number of ejaculates of viable spermatozoa per day.
In mammals, “there are dynamic interactions between the epididymal epi-
thelium, the microenvironment and the spermatozoa, interactions which
ensure optimal conditions for sperm maturation” (Hinton and Palladino
1995). On the other hand, spermatozoal motility in birds does not appear to
require the involvement of secretions or luminal microenvironment of the
ducts. Spermatozoa, obtained from the testis of the rooster have been shown to
exhibit motility, and are able to fertilize eggs (Munro 1938). Motility of avian
spermatozoa, however, increases cranio-caudally, as they traverse the excur-
rent ducts in non-passerine birds (Munro 1938; Bedford 1979) and Ostrich
(Aire and Ozegbe, unpublished observations), although they are only motile
in the seminal glomera in passerine birds (Bedford 1979). Avian spermatozoa
are transported rapidly, and, unlike in mammals, are not normally stored in
the excurrent ducts for any appreciable length of time, a requirement for sperm
competition by male birds. Therefore avian spermatozoa do not require andro-
gen-dependent prolongation of their viability in non-passerine birds, as is
required in mammals (Munro 1938; Bedford 1979). Passerine birds appear to
be different in this regard, because their spermatozoa may require androgen
for their viability since the existence and perhaps maintenance of the seminal
glomus is androgen-dependent (Bailey 1953).
Spermatogenesis and Testicular Cycles !#
Birds, generally, have adopted a relatively simple form of spermatozoal

maturation or modification along the reproductive tract. In mammals
exposure of the itinerant spermatozoa, as they pass through the various
microenvironments of the epididymis of mammals confers upon these cells
their motility capability and fertilizing ability (Bedford 1967; Turner 1991).
Further adaptations adopted by birds for rapid transport and maturity of
spermatozoa through the excurrent ducts include the relative non-reliance of
spermatozoa for proteins secreted by the duct epithelia for further
maturational processes, as well as not requiring capacitation in order to
fertilize ova (Howarth 1970, 1995). Although spermatozoa acquire about four
Wolffian duct proteins as they pass through the epididymal duct unit of the
rooster, Guineafowl and Japanese quail (Esponda and Bedford 1985; Morris et
al. 1987b), and only one androgen-dependent protein of 17kDa in Japanese
quail (Kidd 1982, cited by Jones 1998), the functions of these proteins, which
are order-specific in birds (Esponda and Bedford 1985; Morris et al. 1987), is
unknown. Although these and other proteins in the duct lumina may not be
necessary for promoting the fertilizing ability of avian testicular spermatozoa
(Howarth 1970, 1983), they do not have decapacitation ability on rooster
spermatozoa (Dukelow et al. 1967), and it has not been determined if the
proteins are necessary for sperm viability and/or fertility in the female
reproductive tract. The motility, viability, fertility and durability of avian
spermatozoa seem to be intrinsic, largely, rather than generated or sustained
by the series of microenvironments of the various duct lumina through which
they pass, rather rapidly. An intriguing phenomenon is the ability of the male
fowl to allocate spermatozoa both strategically and differentially to a number
of females in varying circumstances, such as female promiscuity, novelty, and
reproductive quality (Birkhead et al. 2003).
7.7 TESTICULAR CYCLES

Reproduction in birds is a cyclical phenomenon (Lofts and Murton 1973), and
this is an adaptation to a periodic environment, especially at middle and high
altitudes (Wilson and Donham 1988). The purpose of seasonal breeding is to
ensure that the mechanisms that govern gametogenesis are synchronized by
environmental stimuli so that the young are produced during the most
appropriate and favorable time of the year for their survival (Lofts and Murton
1973). In addition, in certain seasonally unpredictable environments,
organisms cannot anticipate and prepare for a regular yearly reproductive
period, and have therefore adopted strategies, within the reproductive cycle,
to produce, opportunistically, their young at any time of the year (Hau et al.
2004). Seasonal breeding also permits the sexually resting or inactive bird to
allocate vital resources to other organismal functions, such as build up of
flight muscles in migrating birds (Bairlein and Gwinner 1994; Gwinner 1996).
In all of these birds, therefore, testicular, and hence general reproductive
activity, undergo morphological and functional changes that are imposed on
them by hormonal changes which, in turn, are a result of the response of the
neuroendocrine system to environmental signals or cues.
With the approach of or during unfavorable environmental circumstances,

the reproductive activity is slowed down and/or halted. There is a carefully
integrated and balanced relationship between the bird and its environment
(Lofts and Murton 1966; Lofts et al. 1970), such that changes in the
environment are monitored by the neuroendocrine system of the animal. Thus,
environmental cues or stimuli exert influence on the neuroendocrine system,
with a view to causing appropriate response with regard to stimulating or
causing the cessation of gonadotropic release from the pituitary gland.
According to Wingfield et al. (1997b) the endocrine system is well conserved
in all vertebrates, and it is likely that any deviation from the general vertebrate
model may represent adaptations rather than phylogenetic constraints. The
degree of neuroendocrine activities is directly related to gonadal size and
function. Natural selection has favored the establishment of annual breeding
cycles, and an endogenous rhythm seems to fine-tune responses to
environmental cues (Lofts and Murton 1973; Marshall and Serventy 1958).
7.8 ENVIRONMENTAL EFFECTS ON REPRODUCTION

IN MALE BIRDS
It is now generally agreed that the degree of environmental predictability has
considerable and far-reaching implications on the reproductive physiology of
the organism (Wikelski et al. 2000) and, not the least, in birds. Thus, birds are
able to transduce seasonal or environmental cues into appropriate
physiological signals, via the neuroendocrine system to produce the various
phases of the reproductive cycle. There are several environmental and other
parameters that impact upon the reproductive cycle of birds, including
photoperiodism, temperature, food availability, nesting sites, environmental
perturbations, etc. According to Gwinner (1996) circannual rhythms are
synchronized with and modified by environmental factors in a complex way
but the endogenous mechanisms usually respond to environmental cues such
that an optimal adjustment to season and latitude is guaranteed. The effect of
each of these cues on the reproductive activity of the male bird will, hereunder,
be briefly examined:
7.8.1 Photoperiodism
There has been almost a deluge of reports on studies involving the influence
of photoperiodism in reproductive activities in birds since Rowan’s (1925)
first publication on this subject. This review will, in the circumstances, be
rigorously selective in presenting an up-to-date picture of this environmental
index as it affects male reproduction in birds.
Photoperiod is now widely known as a primary proximate factor that
controls annual rhythms in the biology of a considerable variety of middle
and high-latitude avian species (Wilson and Donham 1988; Gwinner 1996)
and a major environmental stimulus that synchronizes the breeding season in
some species of birds (Wolfson 1959; Marshall and Serventy 1956; Engels
1961). Long days stimulate LH secretion in intact males merely by reducing
Spermatogenesis and Testicular Cycles !%
the efficacy of testosterone negative feedback on the hypothalamo-

hypophyseal axis (Wilson 1985). Thus, the reduced sensitivity of the
hypothalamo-hypophyseal axis to testosterone enhances LH secretion, and
hence, its concomitant effect on spermatogenesis. Major increases in the levels
of LH and FSH are pre-requisites for testicular development as well as
recrudescence in resting organs. Periodic photoperiodism is, however,
supplemented, modified and fine-tuned by a variety of other factors
(Immelman 1971, 1973; Murton and Westwood 1977; Farner and Follet 1979;
Farner and Gwinner 1980; Wingfield and Farner 1980; Wingfield and Kenagy
1991; Wingfield et al. 1992; Gwinner 1996), which, together, bring about a
coherent organismal response.
Although day-length is still maximal and above the gonad-stimulating
threshold, during summer, the chronically stimulated gonads are no longer
responsive to it, and, indeed, normally commence regression, followed by
reproductive collapse (Wilson and Donham 1988). By mechanisms, yet
unknown, the hypothalamo-hypophyseal axis becomes insensitive to a
normally stimulating photoperiodic threshold. Consequently, the testes
regress in structure and function (Robinson and Follet 1982). This phase of
adequate, but progressively ‘ineffective’ and non-stimulating, day-length on
testicular function is known as “photorefractoriness” (Robinson and Follet
1982). Refractoriness, according to Robinson and Follet (1982), does not seem
to depend upon the activity of the hypothalamo-hypophyseal axis as it
develops in castrated or androgen-implanted birds. It is a necessary universal
phenomenon in birds because it prevents the animal from assuming a
dangerous physiological condition at an inappropriate season, e.g. for rearing
the young during food scarcity (Marshall 1961; Lofts 1962; Lofts and Coombs
1965; Lofts and Murton 1973), and providing a resting and regenerative
period in the reproductive cycle during which spermatogenesis, along with
other reproductive activities, is terminated so that the ‘exhausted’ gonad can
be rehabilitated (Marshall 1961) and rejuvenated.
In temperate zone birds, annual reproductive cycles have evolved in
response to proximate environmental information (especially photoperiod,
food situation, and nesting sites) that regulates breeding seasonally
(Wingfield 1980; Wingfield et al. 1992; Ball 1993; Nager and van Noordwijk
1995; Morton 2002). Even location and latitude (elevation) can influence
reproductive development (Perfito et al. 2004). In temperate zone birds,
testicular recrudescence begins in spring and reproduction terminates by
early summer, whereupon the testes rapidly regress and remain small
throughout the winter months (Lofts and Murton 1973). However, it was
thought that in the tropical zone, the changes in photoperiod were too small
to be used as seasonal cues by birds. Recent reports have begun to show that
most tropical birds exhibit a distinct seasonal pattern in life history
parameters such as breeding activity (Snow and Snow 1964; Fogden 1972;
Stiles 1980; Bell 1982; Dittami and Gwinner 1990; Tye 1991; Hau et al. 1998;
Wikelski et al. 2000). Hau et al. (1998) and Wikelski et al. (2000) have
demonstrated that tropical zone birds are extremely sensitive to variation in
photoperiod of as little as 17 minutes, and use it as an environmental cue, and

as an important characteristic of their natural tropical habitat. The ability of
these birds to transduce the photoperiodic changes into physiological signals,
fine-tuned by supplementary cues, is considered to be similar to the situation
in temperate zone birds (Wingfield 1980; Wingfield and Moore 1987).
An adequate level of environmental signal causes an elevation of LH levels
(Wikelski et al. 2000) in birds. This occurs as an adjustment of their
reproductive activity to differences in the availability, as well as quality, of
food (Hau et al. 2000). Ground doves (Columba squamata), in seasonal savannas
in Venezuela, while breeding, mostly, during the dry season, retain fully
functional testes independent of rainfall or longer photoperiod. It is significant
and note-worthy that a member of the Ratitae, the emu (Dromaius
novaehollandiae), differs from many, if not most, birds, in being a short-day
breeder in southwestern Australia (Malecki et al. 1998). This bird breeds
between May and August, during the winter period. The control of the
reproductive cycle of the emu appears to have an endogenous component that
uses seasonal change in photoperiod and rainfall as the critical
environmental factor in order to ensure breeding at the appropriate time of the
year (Malecki et al. 1998; Sharp et al. 2005). It appears that rainfall and food
availability have an overriding influence on increasing day-length in this
species.
It is of interest that testes sizes in tropical wild birds exhibit relatively small
seasonal fluctuations (Bosque et al. 2004), and that the relative testes sizes are
much smaller (by about 10-fold) than those in temperate zone birds (Wikelski
et al. 2000). Some species are, understandably, opportunistic breeders, and
may maintain an activated reproductive system all year round (Hau et al.
2004). Polygynous species, nevertheless, have larger testes than monogamous
species (Wikelski et al. 2000), and, as in temperate zone birds, plasma
testosterone levels are positively correlated with the total number of
spermatozoa produced and proportion of normal sperm morphology
(Wikelski et al. 2000; Garamszegi et al. 2005). Wikelski et al. (2003) consider
that the pace of life is slower in birds in the more sedentary tropical
environment than in the northern temperate migratory individuals of the
same species. That is probably why the tropical species, living in ‘stable’
saturated environments tend to experience less extra-pair copulation and tend
to breed less synchronously throughout the year than do the generally
synchronous temperate or high-altitude species (Stutchbury et al. 1998).
Temperate or high altitude species have a short breeding season, and an
elevated plasma T level throughout the season (Levin and Wingfield 1992;
Moore et al. 2002; Wingfield et al. 1997a).
7.8.1.1 Supplementary environmental signals
7.8.1.1.1 Food availability
Breeding in birds usually coincides with suitable feeding conditions (Lofts
and Murton 1973; Wingfield 1988; Hau et al. 1998; Wikelski et al. 2000). Food
Spermatogenesis and Testicular Cycles !'
availability is regarded as an ultimate factor or as supplemental information

in seasonal breeding activities of tropical birds (Fogden 1972; Snow 1976;
Sinclair 1978; Poulin et al. 1992; Young 1994). Some birds are able to withstand
the deleterious effects of low temperature if they have free access to food
(Wingfield 1984; Wingfield et al. 1982). The availability of nesting sites and
the process of nest-building has been shown, also, at times, to exert proximate
control over the reproductive schedule in the Mountain white-crowned
sparrow (Zonotrichia leucophrys oriantha) (Morton 2002).
7.8.1.1.2 Ambient temperature
The influence of ambient temperature on reproductive activities in birds has
received conflicting appraisal (Farner and Mewaldt 1952; Lewis and Farner
1973; Wingfield et al. 1997b). Low ambient temperature probably stimulates
the hypothalamo-hypophyseal-thyroid axis to cause an increase in blood T3
and/or T4 (Assenmacher 1973; Smith 1982), but T4 levels have been found to
be lower in Mountain white-crowned sparrows (Zonotrichia leucophrys
oriantha) subjected to low temperature (Wingfield et al. 1997b). Earlier studies
showed that low ambient temperature delayed photoperiod-induced gonadal
growth (Marshall 1949; Lofts and Murton 1966; Storey and Nicholls 1982),
but Lewis and Farner (1973) and Wingfield et al. (1997b) have come to the
conclusion that various temperature regimens have marginal or no effect on
testes sizes and growth, as well as cloacal protuberance in birds that are also
exposed to long days. However, low temperature retards gonadal regression,
while high temperature has different effects on gonadal recrudescence, onset
of breeding and termination of breeding, in ways that are not clearly
understood (Wingfield et al. 1997b). The interaction of both proximate and
supplementary environmental cues and stimuli appears to be part of this lack
of understanding. Disturbances during any phase, especially during the
active breeding phase of the reproductive cycle, may trigger off responses that
may compromise gonadal structure and function in birds (Wingfield 1988).
7.8.1.1.3 Overcrowding
Overcrowding has been known to cause a reduction in body weight, testis
dysfunction (as evidenced by a fall in seminiferous tubular diameter and
epithelial height), spermatogenic inhibition, Leydig cell function and therefore
androgen production (De Tapas et al. 1974).
7.9 CYCLICAL MORPHOLOGICAL CHANGES IN THE TESTIS

Most wild birds and a few domestic birds are seasonal breeders. In such birds,
there is a sequence of events, mediated by environmental cues, that occurs in
a cyclical manner in the reproductive organs. During the breeding or sexually
active phase of the reproductive cycle, such birds possess reproductive organs
that are maximally differentiated and hormonally stimulated, and thus have
maximally functioning epithelial cells. During this phase, the reproductive
organ and tract produce and modify the gametes for maximum viability and
fertility. This phase is followed by an involutionary phase, a period of

declining, and ultimately total cessation of, function in the testis and its
excurrent ducts. This phase includes, or is followed by (depending on the
authors), a period of recrudescence during which phase of the reproductive
cycle both the gonad and tract are prepared, morphologically and
functionally, for resumption of full reproductive activity.
Marshall (1961) and Lofts and Murton (1973) have described three main
phases in testicular function, in the annual reproductive cycle of the male
bird, viz. Regeneration phase, Acceleration phase and Culmination phase. On the
other hand, Mehrotra (1962) and Traciuc (1969) have divided the reproductive
cycle of birds into four phases, in their reports on seasonal effects on the
excurrent ducts of birds, as follows:
Mehrotra (birds in India) Traciuc (birds in Central Europe)
(1) Presecretion phase Progressive phase (March)
(October to December)
(2) Reproductive phase Active secretory phase (April)
(January to March)
(3) Regression phase (April to July) Reconstruction phase (May to June)
(4) Refractory phase Resting phase (June to February).
(July to September)
It is clear that the latter authors have split the regeneration phase (Marshall
1961; Lofts and Murton 1973) into their respective regression and refractory
phases of the reproductive cycle. These are minor non-significant differences,
which should, however, be noted.
7.9.1 Basic Distinguishing Structural Features of the
Testis in Juvenile and Reproductively Resting
Sexually Mature Birds
It is essential that certain morpho-physiological features of the testis in the
three main phases of reproductive development or activity (juvenile, sexually
mature and active, and sexually mature but inactive or resting) be established
so that by evaluating the structure of any testis, the reproductive history of the
bird can be appropriately determined. The following table (Table 7.5), sketches
the main differences in the testes of (a) sexually immature/juvenile birds, (b)
sexually mature and active birds and (c) sexually mature but resting birds,
represented histologically in Fig. 7.12.
7.9.2 Structural Changes in the Testes of Sexually Mature Birds

At the end of the active period of reproduction certain profound changes occur
in the structure of the testis during the regeneration phase of the reproductive
cycle. According to Humphreys (1975b), cyclic changes in the testis seem to
commence with the Leydig cells, and subsequently, the tubular and interstitial
tissue in each phase of the reproductive cycle.
Table 7.5 Main morphological features of testes in three different phases of development
and/or activity
Sexually immature Sexually mature and active Sexually mature but resting
a. Small testis, firm to touch. Large testis, soft to touch. Small testis, firm to touch.
b. Cut surface relatively ‘dry’ Cut surface very fluid, and Cut surface relatively ‘dry’
and does not bulge out. bulges out. and does not bulge out.
c. Small seminiferous tubular Large seminiferous tubular Small seminiferous tubular
diameter. diameter. diameter.
d. Intertubular tissue is moderately Intertubular tissue is sparse Intertubular tissue is abundant,
voluminous and uniformly granular. and compact. with foamy islets.
e. Seminiferous epithelium contains Full germ cell complement Seminiferous epithelium is
numerous basal cells, mainly in seminiferous epithelium. distinctly heterogeneous,
supporting (future Sertoli) cells and Sertoli cells contain a few contains abundant, dense
spermatogonia or gonocytes in a dense bodies and residual bodies, lipoidal and lipofuchsin
homogeneous matrix. There are bodies in the apical granules in Sertoli cell
no evident lipid droplets or dense cytoplasm in toluidine blue- cytoplasm; spermatogonia and
bodies in the cytoplasm of the stained plastic sections. only a few, degenerating
supporting cells in plastic sections. Tubular lumen is large and spermatocytes are present.
Tubular lumen is absent or only fluid-filled Tubular lumen is usually
small spaces occur centrally, when obliterated.
fluid secretion commences.
(a) During the regenerative or preparatory phase, in spite of its name,

involutionary or regressive changes occur throughout the testis, from the
testicular capsule to the core of the seminiferous epithelium. This phase is
usually dependent upon environmental cues, such as shortening day-length
and/or declining food availability. The testis is in the refractory phase, a
period of declining, followed by arrest of, reproductive activity during
maximal day-length (Robinson and Follet 1982). This phase is a normal
feature of the reproductive cycle of birds, except for some domestic species,
such as the domestic fowl, that have been bred for continuous reproductive
activity. The testis involutes and its size is greatly reduced. The testicular
capsule sloughs off and is replaced by a new one generated from below by
fibroblast proliferation (Marshall and Serventy 1957). In the early stage, the
interstitial tissue appears foamy, following accumulation of lipid droplets in
Leydig cells and certain peritubular myofibroblasts (Fig. 7.13A). Macrophages
and other mononuclear cells invade the interstitium and remove degenerating
cells. A new set of Leydig cells seems to develop from myofibroblasts
(Nicholls and Graham 1972; Aire 1997). The length of time required for the
rehabilitation of the interstitial cells may vary from species to species (Lofts
and Murton 1973). The Sertoli cells in the seminiferous tubules are filled with
lipid materials including lipofuschin materials, and, along with
spermatogonia, are the main cells left in the seminiferous epithelium. At the
height of this phase, most of the worn and degenerating cells within the
seminiferous tubule and interstitial tissue are removed by macrophage
Fig. 7.12 Struthio camelus (A); Anas platyrhynchos (B, C). A. seminiferous cords
of juvenile testis surrounded by an obvious cellular intertubular tissue. The cords
are devoid of lumina and exhibit mainly supporting cells and gonocytes. B. The
seminiferous epithelium epithelium (E) of a sexually mature and active testis
displays a full complement of germ cells and a wide lumen (L). C. Involuted
seminiferous tubules in a regressed testis. The intertubular tissue is bulky and,
once again, quite cellular, while the seminiferous epithelium contains mainly Sertoli
cells that are laden with lipid droplets, and lipofuschin and dense granules. The
tubular lumen is obliterated. Bars: A = 200 µm, B = 10 µm, C = 200 µm. Original.
Spermatogenesis and Testicular Cycles ! !
activity, and the testicular capsule sloughs off (Lofts and Murton 1973;
Breucker 1978). The testicular framework is ready for regeneration by
gonadotropins and the attendant hormonal action, following stimuli from
appropriate environmental and endogenous cues, such as increasing day-
length, rainfall and abundant food sources.
The seminiferous tubules of sexually mature but gonadally inactive birds
are physiologically atrophic, and contain both Sertoli and germ cells,
comprising spermatogonia and a few primary spermatocytes. Sertoli cells are
relatively preponderant, fill the highly reduced or obliterated tubular lumen,
and are greatly laden with numerous, dense inclusions, as well as numerous,
large, lipid droplets and lipofuschin pigments resulting from phagocytosis of
degenerating germ cells (Fig. 7.13B). Macrophages in the seminiferous tubules
of regressing testes in the Mute swan (Cygnus olor), aid Sertoli cells, with
which they seem to have a preferential contact, in the regressing testes, to
dispose of large amounts of cell debris, by phagocytosis (Breucker 1978). The
Sertoli-Sertoli occluding junctions are intact structurally and functionally in
the seminiferous epithelium in regressed testes of Mallard, although this
junction appears to be apically displaced, due to atrophy of Sertoli cell
cytoplasm (Pellietier 1990). The basal lamina of the seminiferous tubule is
irregular in outline, thickened, relatively electron-dense, and often invaginates
into the germinal epithelium in the form of finger-like plicae or folds (Fig.
7.14A), whose function probably facilitates the flow of raw materials into or
out of the seminiferous tubule (Chakraborty et al. 1976; Aire 1997). These folds
contain numerous electron-lucent globules whose nature and function are
unknown (Aire 1997). The interstitium becomes relatively bulky and foamy in
appearance, as a result of development of lipid droplets in certain
myofibroblasts and particularly in extant Leydig cells (Lofts and Murton 1973;
Aire 1997).
Peritubular tissue remains intact in involuted testes, but the increased
content of rough endoplasmic reticulum (RER), accumulation of lipid droplets
and the change from lamellar to tubular cristae in the mitochondria (Fig. 7.14)
of some of the myofibroblasts lend support to the hypothesis that certain of
these cells undergo morphological and functional cytodifferentiation,
preparatory to transforming into Leydig cells in the recrudescent testis (Aire
1997). Leydig cells in involuted testes contain an unusually large number of
dense granules that are probably lysosomes, an abundance of unextracted
lipid droplets, swollen and vesiculated mitochondria, and highly
heterochromatic nuclei (Fig. 7.15A,B). The SER is profoundly atrophic. Two
types of Leydig cells are present in the interstitium (Fig. 7.15B). The one,
apparently normal, contains electron-dense ground substance, numerous
lipid droplets and well formed polymorphous mitochondria bearing tubular
cristae within an electron-dense matrix, while the other displays a rarified or
reticular, electron-lucent cytoplasm, partially extracted lipid droplets, dense
bodies, which are probably lysosomes, a heterochromatic nucleus, and
vesiculated, disorganized mitochondria. The latter cell appears to undergo
! " Reproductive Biology and Phylogeny of Birds
Fig. 7.13 Anas platyrhynchos. A. Early changes in the intertubular tissue of a

regressing testis. Leydig cells and myofibroblasts display lipid droplets. B. A TEM
micrograph of the seminiferous epithelium of a fully regressed testis. The
epithelium contains Sertoli cells predominantly. Dense bodies (straight arrows)
abound in the Sertoli cell cytoplasm and the basal lamina (curved arrow) is
thickened. S, Sertoli cell nucleus. Bars: A = 10 µm, B = 20 µm. Original.
Spermatogenesis and Testicular Cycles ! #
Fig. 7.14 Anas platyrhynchos. A. The basal lamina (arrows) of the seminiferous
tubule (E) of a regressed testis is highly thickened, electron-dense, irregular in
outline and contains numerous electron-lucent globules. Folds of the basal lamina
(arrows) project into the Sertoli cell cytoplasm (S). Peritubular myofibroblasts (M)
contain large lipid droplets (L) and irregularly-shaped nuclei (N). Electron-lucent
globules also occur in the intercellular boundaries of the myofibroblasts. B. A
myofibroblast contains a large lipid droplet (asterisk) and two, large mitochondria
with tubular cristae (arrowheads). Bars: both figures at 1 µm. From Aire, T. A. 1997
Onderstepoort Journal of Veterinary Research 64: 291-299, Figures 11 and 12
(inset). Reproduced with permission of the Editor.
! $ Reproductive Biology and Phylogeny of Birds
Fig. 7.15 Anas platyrhynchos. A. The interstitial tissue between longitudinal

sections of two seminiferous tubules increases in volume and shows two Leydig
cells (Le) in a state of inactivity. D, an unusual number of dense bodies. B. Two
Leydig cells (A and B) of varying electron-density and mitochondrial structure are
present in the interstitium of a regressed testis. Type A cell is electron-dense and
contains intact mitochondria (M) with tubular cristae, dense bodies (D), and
numerous partially extracted lipid droplets. Type B Leydig cell is electron-lucent and
contains vesiculated mitochondria (arrowheads), numerous lipid droplets and a
heterochromatic nucleus. Bar: A = 20 µm, B = 1 µm. Figure A, original. Figure B is
from Aire, T. A. 1997 Onderstepoort Journal of Veterinary Research 64: 191-199.
Reproduced with permission of the Editor.
Spermatogenesis and Testicular Cycles ! %
degeneration. According to Lofts and Bern (1972), Leydig cells, in the non-
breeding season, disintegrate, are removed by macrophages, and are replaced
by a new generation of Leydig cells.
(b) The accelerative phase: The refractive period prepares the reproductive
organs for receptivity and amenability to internal physiological stimulation.
This process is normally mediated by gonadotropins secreted by the
adenohypophysis, following appropriate environmental cues and
appropriate stimuli from the brain that impinge on this endocrine gland.
Blood levels of FSH and LH undergo increasing elevation (Haase 1983). The
activities engendered in the reproductive organs, as a result of positive
stimulation by gonadotropins are usually quite rapid, and bring about a flurry
of growth activities in the gonads, which phenomenon is known or referred to
as recrudescence. The period of recrudescence is usually short, if inhibiting
factors such as low temperature (Lofts and Murton 1966) or lack of rainfall
(Marshall 1970), are excluded or overridden. The Sertoli cells lose their
lipoidal accumulations, spermatogenesis resumes, and the bird is ready to
commence reproductive activity.
(c) The culmination phase is when the male bird is morphologically and
functionally prepared to be reproductively active, usually before the female
bird is. Blood FSH, LH and T (testosterone) levels are at their peak. The Leydig
cells lose most of their lipid droplets and both the smooth endoplasmic
reticulum and mitochondria are restored to normal structural and functional
states. The appropriate environmental and endogenous cues are present, and
spermatogenesis progresses satisfactorily. Testosterone and semen production
peak, and LH is correlated with testosterone level (Penfold et al. 2000). The
absolute and relative weights of the testis attain maximum levels, and both
the seminiferous tubular diameter and epithelial height attain full dimensions
and functioning. The normal structure of the mature and active gonads and
excurrent ducts are restored.
7.10 CYCLICAL MORPHOLOGICAL CHANGES IN

THE EXCURRENT DUCTS OF THE TESTIS
The seasonal effects on the structure of the excurrent ducts during the
reproductive cycle of animals have received very scant attention. Even in
mammals, there are very few reports of seasonally-induced changes in the
excurrent ducts (Suzuki and Racey 1976; Pudney and Fawcett 1984).
Aire (2002a,b) describes changes in various duct units in the epididymis of
domestic, farm birds in three main phases of the reproductive cycle
(prepubertal, sexually mature and active, sexually mature but inactive or
resting) so that discrepancies may be prevented or minimized in the
interpretation of normal structure in birds, during the sexually active phase,
as distinct from the other two phases as well as their intermediate stages. The
following account of changes in the various excurrent ducts comes, in the
! & Reproductive Biology and Phylogeny of Birds
main, from the reports on fringillid (passerine) birds (Bailey 1953), in the
Knob-billed goose (Sarkidiornis (=Anser) melanotus) (Mehrotra 1962), Jackdaw
(Corvus monedula) (Traciuc 1969), Starling (Sturnus vulgaris) (Barker and
Kendall (1984), Quelea (Quelea quelea) and Wild puffin (Fratercula artica) (Bhat
and Maiti 2000), rooster, Mallard and Guineafowl (Aire 2002a,b). Only salient
features will be emphasized in this account.
7.10.1 The Reproductive (Active Secretory/Culmination) Phase

The normal structure of the various segments of the excurrent duct system of
birds has been described and discussed in considerable detail in Chapter 2.
The rete testis lacunae are lined by a squamous to cuboidal epithelium
comprising non-ciliated cells. Organelles are normal in structure, number and
distribution. Small subapical vacuoles and multivesicular bodies, few small
lipid droplets as well as a large, deeply indented, relatively euchromatic
nucleus, occur in the cell.
The efferent ducts: the lumen of the proximal efferent duct (PED) is large,
irregular in outline and contains a few germ cells, while that of the distal
efferent duct (DED) is regular in outline and contains a concentrated
accumulation of sperm. The non-ciliated (NC) types I and II cells are
maximally stimulated, structurally and functionally, and contain euchromatic
nuclei which are round or oval in shape and situated in the basal half of the
cells. The subapical endocytic or tubulovacuolar system is remarkably
abundant and fully functional in the NC type I, but only scarcely noticeable in
the NC type II, cell. Dense bodies are numerous and mixed with lysosomes in
varying phases of activity in the type I, but are absent in type II, cells. The
width of the mitochondria of both types I and II cells is much greater, by over
70%, than those of ciliated cells (Aire 2002a). The disappearance of lipid
droplets seems to be a little protracted because large lipid droplets occur
basally in the NC type I cells although they appear structurally and
functionally primed, as evidenced by the full restoration of the subapical
endocytic apparatus and dense bodies, that are normally atrophic or absent,
respectively, in the regressed phase of the reproductive cycle. Lofts (1964)
made similar observations in the testes of quelea birds.
In the epididymal duct unit, the ductus epididymidis appears distinct and
full of sperm. It runs distally into an easily recognised, wavy, turgid, and
whitish ductus deferens, also full of spermatozoa. The receptaculum ductus
deferentis is full of sperm. The papilla ductus deferentis is easily seen, projecting
into the dorsolateral aspect of the urodeum. The apical microvilli of the NC
type III cell are numerous, short and evenly distributed. The basally situated
nuclei are oval/oblong in shape and relatively euchromatic. The Golgi
complex is well developed and extensive. Profiles of smooth and sparsely
granulated endoplasmic reticulum (SER and SGER, respectively) are
abundant and moderately distended. The lateral plasmalemma is highly and
intricately folded.
Spermatogenesis and Testicular Cycles ! '
7.10.2 The Regression (Reconstructive/Regeneration) Phase

The rete testis spaces contain accumulations of desquamated testicular germ
cells at varying stages of degeneration, and are phagocytised by a large
number of infiltrated macrophages and other mononuclear cells. Certain ducts
are obliterated in Jackdaw, according to Traciuc (1969), but this has not been
observed in the rooster, Mallard or Guineafowl, even in the most involuted
organs (Aire 2000a,b).
The proximal efferent duct (PED), as in the rete channels, contains an
accumulation of degenerating desquamated testicular germ cells and
mononuclear cell series that are actively spermiophagic. The duct diameters
are, subsequently, greatly reduced, but the epithelium remains cuboidal to
columnar.
The epididymal duct unit contains degenerating spermatozoa and early
germ cell series in an amorphous matrix that is eosinophilic and stretches the
now cuboidal epithelium.
7.10.3 Refractory (Resting) Phase

In the rete testis, a highly irregular nucleus occupies most of the cytoplasm, is
more heterochromatic than in the active phase, and may be indented by
numerous, fairly large, lipid droplets and dense heterogeneous bodies
that abound in the cytoplasm, during this phase (see Fig. 7.16). Several ciliated
(C) cells appear in the epithelium, and are interspersed between the non-
ciliated (NC) cells that normally compose this epithelium (Fig. 7.16B).
Androgen withdrawal seems to provoke ciliogenesis in the excurrent ducts
of the testis in mice (Schleicher et al. 1984) and the middle segment of
the epididymis of mice treated with the estrogen-like compound, 2, 6-
cis-diphenylhexamethylcyclotetrasiloxane (Aire, pers. obs.). Luminal
macrophages and degenerating germ cells are few, having removed the
degenerating luminal germ cell debris.
The efferent duct unit contains granulofilamentous and some membrane-
bound material in the PED lumen (Fig. 7.17), but the DED lumen is greatly
reduced and empty. The columnar epithelial height of the PED is lower, but
increases by over 30% in the DED than in the active phase of the reproductive
cycle (Aire 2002b). In NC type I cells, the microvilli are considerably
shortened, with highly irregular nuclei, especially in Mallard, occupying most
of the shrunken cytoplasm. The subapical tubulovacuolar system atrophies in
both duct segments, leaving only their silhouettes. Dense bodies which are
typical of the NC type I cell are much fewer in number and much larger in size
in Mallard, but are smaller, more numerous and contain lipofuschin-like
granules in Guineafowl, during this phase (Aire 2002b). Mitochondria are
smaller in NC type I cells, and are now similar in size to those of ciliated cells.
Very large lipid droplets, some of which are as large as the nucleus (Fig. 7.18),
occur in the supra- and infra-nuclear regions of the cell (Aire 2002b).
!! Reproductive Biology and Phylogeny of Birds
Fig. 7.16 Numida meleagris (A) and Anas platyrhynchos (B). A. The regressed
rete testis epithelial cells accumulate a large number of lipid droplets (arrowheads)
and dense, heterogeneous bodies, and the nuclei are highly irregular in shape
and heterochromatic. Other organelles are inconspicuous. B. Ciliated cells (c) are
yet to disappear from the rete testis of birds already in the active state, immediately
following recrudescence. Bars: 20 µm for both figures. Figure A adapted from Aire,
T. A. 2002. Anatomy Histology Embryology 31: 113-118, Figure 4. Reproduced with
permission of Blackwell Publishing Ltd. Figure B, original.
The epididymal duct unit has an atrophic epididymal duct that is relatively
straight, and contains caseous, amorphous eosinophilic coagulum. The
epithelium of the duct undergoes profound ultrastructural changes (Aire
2002a). Microvilli are small and inconspicuous, the lateral plasmalemma is
less folded, and highly elongated, heterochromatic nuclei occupy most of the
rarified cytoplasm, SER and SGER are inconspicuous, while short, narrow
profiles of RER appear more numerous (Aire 2002a). The seminal glomus
shrinks, and its epithelial cells appear pyknotic in fringillid birds (Bailey
1953).
Fig. 7.17 Anas platyrhynchos. The PED lumen is empty, save for
granulofilamentous material (asterisk). The nuclei (N) of the non-ciliated type I cells
are elongated, irregular in outline and heterochromatic. Large extracted lipid
droplets (L) occur in the cell. Dense, polymorphic bodies are present in the
supranuclear region. Non-ciliated cells undergo profound structural changes and
tend to be crowded-out, over-shadowed by ciliated cells (C) that undergo minimal
structural changes. Bar: 20 µm. From Aire, T. A. 2002. Journal of Morphology 253:
64-75. Figure 6. Reproduced with permission of Wiley-Liss, Inc.
!! Reproductive Biology and Phylogeny of Birds
Fig. 7.18 Anas platyrhynchos. A. Lipid droplets (L) in the non-ciliated cells of an
involuted PED may be nearly as large as the heterochromatic nucleus (Nu).
Supranuclear dense bodies (D) are few but large. Mitochondria (M) of ciliated cells
(C) are as large as those of non-ciliated cells (N). B. A light microscope section,
and C. A TEM section of the PED, soon after gonadal recrudescence, exhibiting
very large lipid droplets in the infranuclear region of the non-ciliated type I cells.
Bars: A = 2.5 µm, B = 20 µm, C = 2 µm. Figure A is from Aire, T. A. 2002. Journal
of Morphology 253: 64-75, Figure 7a. Reproduced with permission of Wiley-Liss,
Inc. Figures B and C, original.
Spermatogenesis and Testicular Cycles !!!
In general, the PED epithelium undergoes more profound structural

changes than that of all other excurrent ducts of the testis during the
regression and refractory phases of the reproductive cycle. Similarly, the NC
type I cell of the PED is more greatly affected by androgen withdrawal than
the NC types II and III as well as ciliated cells, with the latter showing less
profound effects than any of the three types of non-ciliated cells (Aire 2002a).
7.10.4 Presecretion (Progressive/Acceleration) Phase

Rete testis tubules enlarge, their epithelium flattens into low cuboidal or
squamous cells, with slightly flattened nuclei (Bailey 1953). The numerous,
fairly large lipid droplets in the RT cells during the previous reproductive
phase appear to coalesce and form large ‘globules’, occupying most of the cell
cytoplasm. At the end of this phase, some ciliated cells are still present in the
epithelium (Fig. 7.16).
Diameters of the efferent duct unit increase and the epithelia undergo
hypertrophy. Cellular division of both ciliated and non-ciliated cells occurs in
the ducts (Mehrotra 1962), and organelle content of the cells becomes
conspicuous, again. It has been noted that this phase is extremely short and
not commonly observed in birds. In the drake, large lipid droplets that
accumulate in the PED during the previous reproductive phase are still
observed in the epithelium (Fig. 7.18), well beyond the full resumption of
spermatogenesis and sperm accumulation in the excurrent ducts of the testis.
It appears that some of the lipid droplets are extruded from the cells into the
duct lumen (Aire, T. A., personal observations).
The epithelial cells in the epididymal duct unit divide rapidly as the duct
diameters increase. Some of the daughter cells are pushed inward and obscure
the duct lumen (Bailey 1953). The diameter of the ductus deferens increases,
and the epithelial cells of the seminal glomus proliferate rapidly, changing
into low columnar or cuboidal type with round nuclei. Some of the new
epithelial cells are sloughed into the lumen of the seminal glomus of various
fringillid birds (Bailey 1953).
Based on the review, above, on cyclical changes in the testis and its
excurrent ducts, it is clear that more comprehensive, phase-controlled studies
in both passerine and non-passerine birds are absolutely necessary. Available
reports are few and disjointed, and some of them employ confusing and
erroneous nomenclature for the ducts, in the light of more recent findings.
7.11 ACKNOWLEDGMENTS
I wish to acknowledge University of Pretoria for providing some resources
that aided the writing of this review. The technical assistance of the Electron
Microscope Unit of the Faculty of Veterinary Science of the University of
Pretoria is also highly appreciated. Professor J. T. Soley kindly made several
useful suggestions during the course of the compilation of the review. The
helpful and insightful comments of Professor Tim Birkhead are gratefully
acknowledged. Drs. Peter Ozegbe and Wahab Kimaro provided invaluable
!!" Reproductive Biology and Phylogeny of Birds
technical assistance in the composition of the text and figures. Mrs. Wilma
Olivier made the excellent line diagrams.

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n n
CHAPTER
8
Avian Spermatozoa:
Structure and Phylogeny
Barrie G.M. Jamieson
8.1 INTRODUCTION
It was intended that this chapter would be confined to a review of
ultrastructural works on bird spermatozoa and phylogenetic implications but
it soon became apparent that the work, nearly a century old, of Gustaf Retzius
(1909, 1911, 1912) and to a lesser extent the earlier publications of Emil
Ballowitz (1886, 1888, 1913) still comprised a large proportion of our
knowledge of avian sperm morphology. I have therefore included their light
microscopical observations, and pertinent drawings of Retzius, with the later
ultrastructural works in this chapter. Retzius’ drawings are comparable with
those of Ernst Haekel (1862) for the extraordinary visual acuity, the excellence
of the optical systems, and the dedication of these authors, on which their
production depended. The light microscopical investigations of McFarlane
(1963) are also of great value. The illustrations by Ballowitz are drawn to a
smaller scale than those of Retzius and, though providing significant
information, are not reproduced in the present work. Avian species examined
for sperm morphology by Ballowitz, Retzius and McFarlane are tablulated in
Tables 8.1, 8.2 and 8.3 respectively.
This chapter is largely restricted to sperm morphology and ultrastructure
and a phylogenetic analysis of these. For a consideration of spermatogenesis
see Aire, Chapter 7 of this volume. For sperm biology see such works as
Birkhead and Møller (1992), Briskie et al. (1997), Froman et al. (2002), and
references therein, and, in this volume, Briskie and Montgomerie, Chapter 9,
who give an extensive bibliography, including the important works of
Birkhead and colleagues; and Stepinska and Bakst, Chapter 10.
The School of Integrative Biology, University of Queensland, Brisbane, Queensland 4072,

Australia
!# Reproductive Biology and Phylogeny of Birds
To place the structure of the avian spermatozoon in an evolutionary

perspective it is useful to briefly consider the general characteristics of amniote
spermatozoa and particularly of crocodile sperm as crocodiles are widely held
to be the extant sister group of birds.
8.2 AMNIOTE SPERMATOZOA, BASIC FEATURES

From detailed comparative and cladistic considerations, the following
characteristics of a hypothetical plesiomorphic amniote spermatozoon (Fig.
8.1) may be recognized. This model is virtually identical with that of the
lowest extant amniotes, the Chelonia, Crocodylia and Sphenodontida.
Amniote sperm are seen to have few basal synapomorphies relative to the
tetrapod ground plan which is deduced from common features of the amniote
and lissamphibian sperm (Jamieson 1995, 1999, and references therein) and
reference to sperm of sarcopgterygian fish (Jamieson 1991).
8.2.1 Amniote Sperm Plesiomorphies

Plesiomorphic features of the generalized amniote spermatozoon, retained
from their tetrapod ancestry (see also sarcopterygian fish in Jamieson 1991),
and still seen in Chelonia, Sphenodontida, Crocodylia and to varying degrees
in other amniotes, are as follows. The spermatozoon (Fig. 8.1) is elongate and
filiform, with an anterior hollow conical acrosome vesicle overlying a simple
subacrosomal cone. The base of the acrosome invests the tapered anterior tip
(rostrum) of the nucleus and rests on pronounced nuclear ‘shoulders’. The
subacrosomal space within the acrosome contains two or three axial rods
(putative perforatoria) or, less likely, only one rod. These penetrate the nucleus
deeply, almost to its base, in endonuclear canals. The nucleus is
plesiomorphically elongate and cylindrical in amniotes from Chelonia
through Sphenodon, crocodiles, squamates, birds, monotremes and, in therian
mammals, the pangolin alone (Leung and Cummins 1988), as in
lissamphibians (Scheltinga and Jamieson 2003a, b; Scheltinga et al. 2003). At
the base of the nucleus there is a compact fossa (implantation fossa) with
which are associated two triplet centrioles of which the distal forms the basal
body of the flagellar axoneme. Whether the presence of an annulus in
amniotes is plesiomorphic or an apomorphic reversal is debatable. The
terminal portion of the 9+2 axoneme forms a short endpiece distinguished
from the principal piece by the absence of the fibrous sheath.
8.2.2 Amniote Spermatozoal Synapomorphies

The Chelonia and Sphenodontida are considered the most basal extant
amniotes and have virtually identical spermatozoa (Healy and Jamieson 1992;
Jamieson 1995; Jamieson 1999). The characteristics of these include features
considered synapomorphies of the Amniota which are simultaneously
symplesiomorphies for Chelonia and Sphenodontida and for the remaining
amniotes, including birds. The amniote synapomorphies include:
Avian Spermatozoa: Structure and Phylogeny !#
Fig. 8.1 Hypothetical plesiomorphic amniote sperm. From Jamieson, B. G. M.

1999. Pp. 303-331. In C. Gagnon (ed). Spermatozoal Phylogeny of the Vertebrata.
The Male Gamete. From Basic Science to Clinical Applications, Cache River Press,
Vienna, USA, Fig. 10.
Elongation of the distal centriole. The distal centriole is extremely elongate

and extends the entire length of the long midpiece (the latter defined by its
mitochondria) in turtles, the tuatara, crocodiles (Fig. 8.2), and paleognaths
(Figs. 8.4-8.9), an apparent basal synapomorphy of amniotes. These elongate
centrioles differ from most metazoan basal bodies in being penetrated by two
central singlets from the axoneme. Thus in spermatids of the ratite Rhea, the
distal centriole elongates and, late in spermiogenesis, becomes penetrated by
!# Reproductive Biology and Phylogeny of Birds
a central pair of tubules from the developing axoneme (Phillips and Asa 1989).
The shorter, though still elongate distal centriole in the rooster and the
somewhat shorter centriole in Guineafowl (0.6 µm) and Geopelia striata (0.5
µm) (Jamieson 1995), the short centriole in squamates, and the vestigial
centriole in monotremes possibly represent secondary reduction in length of
the distal centriole (Healy and Jamieson 1992), culminating in almost total
reduction in therian mammals (Jamieson 1999).
Mitochondria, with concentric cristae. In turtles, tuatara (Healy and Jamieson
1992; 1994; Jamieson and Healy 1992; Jamieson 1995, 1999), Caiman crocodylus
and Crocodylus johnstoni (Jamieson 1995; Jamieson et al. 1997; Jamieson 1999)
(Fig. 8.2), the mitochondria have concentric cristae, known elsewhere in
amniotes only in the sperm of some marsupials, notably the Woolly opossum,
Caluromys philander (see Fawcett 1970; Phillips 1970) and the Virginia
opossum, Didelphis virginiana (Temple-Smith and Bedford 1980) and also in
the macropod Lagorchestes hirsutus (Jamieson 1999; Johnston et al. 2004).
The mitochondrial cristae in the three ‘reptilian’ taxa (Chelonia,
Sphenodontida, Crocodylia) usually surround a large central dense body. In
all other amniotes studied, the cristae have a “conventional” appearance,
being linear or curved, as in Lissamphibia, but never concentric, and do not
surround a dense body. In spermatids of Sphenodon (Healy and Jamieson
1992; Jamieson and Healy 1992), the cristae have the linear appearance usual
for metazoan sperm and the concentric arrangement is a late development.
Phylogenetic “reversion” of concentric cristae to the linear condition seen in
other amniotes would need only suppression of this final transformation
(Jamieson and Healy 1992). Concentric cristae also occur in the spermatozoa
of Gymnophiona (Scheltinga et al. 2003) and Urodela (Scheltinga and
Jamieson 2003a). Although noting that multiple, homoplastic origin of
concentric cristae would not be dismissed with certainty, Scheltinga et al.
(2003) proposed that concentric cristae are an autapomorphy of tetrapods and
not of amniotes as had previously been suggested by Jamieson (1999). They
would therefore be symplesiomorphic for amniotes. The concentric
arrangement appears to have been lost in all birds.
The annulus. A dense ring, the annulus, at the posterior end of the midpiece
is a feature of many metazoan sperm. It is clearly plesiomorphic for amniotes,
occurring in all classes (Jamieson and Healy 1992), including paleognaths
and several non-passerine orders, but absence in Dipnoi possibly indicates
apomorphic re-acquisition in tetrapods. Irrespective of such reversal it is
clearly a symplesiomorphy for Aves.
Fibrous sheath. A dense fibrous sheath (Fig. 8.1) must, clearly, have
developed, as an annulated structure, in the earliest amniotes as it is present
in all amniote classes. With the exception of squamates, in which it penetrates
the midpiece, it commences immediately behind the midpiece, as in turtles,
Sphenodon (Healy and Jamieson 1992; Jamieson and Healy 1992) and
crocodiles (Jamieson 1995, 1999; Jamieson et al. 1997); in ratites (Figs. 8.4 - 8.8),
Avian Spermatozoa: Structure and Phylogeny !#!
Fig. 8.2 Diagrammatic longitudinal section of the spermatozoon of Crocodylus

johnstoni. The spermatozoon of the Palaeognathae is closely similar to the
crocodile spermatozoon. From Jamieson, B. G. M., Scheltinga, D. M. and Tucker, A.
D. 1997. Journal of Submicroscopic Cytology and Pathology 29: 265-274, Fig. 1.
galliforms (Fig. 8.11, 8.14, 8.18), anseriforms (Fig. 8.19, 8.20), gruiforms
(reduced, Fig. 8.32), charadriiforms (Fig. 8.34) and in mammals (Jamieson
1995, 1999).
Nine peripheral axonemal fibers. Nine longitudinal dense fibers (coarse fibers)
peripheral to the nine axonemal doublets, or to the distal centriole also where
this is elongated as in chelonians, Sphenodon, crocodiles and paleognath birds,
!#" Reproductive Biology and Phylogeny of Birds
are a fundamental feature of amniote sperm (Fig. 8.1), being found in all classes
(Healy and Jamieson 1992; Jamieson and Scheltinga 1993; Jamieson 1995,
1999). As nine peripheral fibers are seen in lampreys and the fish Pantodon
(references in Jamieson 1991) but also in heterobranch and cephalopod
molluscs (references in Jamieson 1999) it might be considered that nine is the
basic sarcopterygian, rather than merely amniote, number and that amphibians
have lost all but those represented by the fibers at doublets 3 and 8. However,
there is no evidence in extant Lissamphibia for such a reduction and the
presence of only two lateral elements in dipnoans and Latimeria suggests that
nine fibers were an amniote synapomorphy, albeit homoplastic with the other,
non-amniote taxa.
Fibers at 3 and 8. It is possible that a further basal amniote apomorphy is
enlargement and lateral displacement of two fibers, at doublets 3 and 8, and
that all fibers in the centriolar region intruded into the inter-triplet radii, as in
‘lower’ amniotes (Chelonians, Sphenodon and crocodiles) (Jamieson 1995,
1999). This displacement is not retained in birds.
Longitudinal columns. In the principal piece, two longitudinal keel-like
outward projections or thickenings (longitudinal columns) of the fibrous
sheath opposite doublets 3 and 8, may be present, with or without inward
projections to these doublets, as shown for mammalian spermatozoa (Fawcett
1975; Jamieson 1999) and Ostrich sperm (Baccetti et al. 1991; Soley 1993).
Retronuclear body transformation. The striated columns of mammalian
sperm are possibly derivatives of the tetrapod retronuclear body, present in
dipnoans and (as the neck structure) in urodeles (Jamieson 1999). No definite
conclusion can be made as to whether structures in bird sperm are
homologues, viz. the non-segmented columns in Ostrich sperm and the three
projections (stated to be probably equivalent to striated columns) in Crested
pigeon (Ocyphaps lophotes) (Fig. 8.24C).
8.3 SPERMATOZOA OF CROCODYLIA

As the Crocodylia are traditionally considered to be the extant sister group of
the birds a consideration of their sperm ultrastructure provides insights into
the evolution of bird spermatozoa (we presumably will never know the sperm
of theropods). The ground plan for the Crocodylia, as exemplified by
Crocodylus johnstoni (Jamieson 1995, 1999; Jamieson et al. 1997) (Fig. 8.1), is
very similar to that of the Chelonia and Sphenodon. All three have two or three
endonuclear canals and, though requiring further confirmation for crocodiles,
concentric cristae with intramitochondrial bodies. In Crocodylus johnstoni the
mitochondria are subspheroidal to slightly elongate and possess few septate
to (more externally) concentric cristae; a central dense mitochondrial body
reported for Caiman crocodylus (Saita et al. 1987) is questionably present.
Caiman crocodylus, resembles ratites in having only one perforatorium but
this may be a reversion from multiple perforatoria homoplastic with the single
condition in ratites.
Avian Spermatozoa: Structure and Phylogeny !##
Synapomorphic conditions in the Crocodylia have little relevance to

consideration of avian evolution. However, it may be noted that one
condition, a dense sheath investing the two central singlets within the
elongate distal centriole, previously considered an apomorphy of crocodile
sperm (Jamieson 1999) is also seen in Ostrich sperm. It could therefore have
been an apomorphic state of a common (theropod?) + crocodile + avian stock.
Restriction of the endonuclear canal in caiman sperm to the anterior region
of the nucleus indicated by Saita et al. (1987) appears to be apomorphic
relative to the longer condition in Crocodylus johnstoni but requires
confirmation. A similar trend to shortening in ratites and, progressively, in
non-passerines presumably occurred in parallel.
8.4 SPERMATOZOA OF AVESINTRODUCTION

As mentioned in the introduction, the light microscopical works of Ballowitz
(1886, 1888, 1913) and of Retzius (1909, 1911, 1912) retain their significance
in current avian spermatology. Species which they investigated are tabulated
here (Tables 8.1, 8.2).
Table 8.1 Bird species examined by Ballowitz for spermatozoal morphology by light
microscopy
Date Figure Species Valid name if different Common name

Galliformes
1888 135-147 Haushahns Gallus domesticus Rooster
1888 128-134 Truthahns Meleagris gallopavo Turkey
Anseriformes
1888 121-127 Tadorna vulpanser Tadorna tadorna Sheld-drake
Piciformes Great spotted
1888 98-109 Picus major Dendrocopos major woodpecker
Cuculiformes
1888 110 Cuculus canorus Cuckoo
Charadriiformes
1888 111-113 Larus ridibundus Black-headed gull
1888 115 Larus canus Common gull
1888 116-120 Vanellus cristatus Vanellus vanellus Lapwing
Falconiformes
1888 114 Milvus ater Black Kite
Caprimulgiformes
1888 85-90 Caprimulgus europaeus Nightjar
Columbiformes
1888 91-97 Haustaube Columba livia Domestic pigeon
Passeriformes
Corvida
1888 84 Corvus frugilegus Rook
1888 63-75 Oriolus galbula Oriolus oriolus Golden oriole
1888 76-83 Lanius collurio Red-backed shrike
!#$ Reproductive Biology and Phylogeny of Birds

Date Figure Species Valid name if different Common name
Passeriformes
Passerida
1888 48-49 Passer domesticus House sparrow
1888 4, 11-15, Muscicapa grisola Muscicapa striata Spotted flycatcher
25, 38-39
1888 19, 26-28 Hirundo rustica Swallow
1888 5 Chelidon urbica House martin
1888 8, 20-22 Sylvia nisoria Barred warbler
1888 36 Sylvia atricapilla Blackcap
1888 37 Sylvia cinerea Whitethroat
1888 51-52 Sylvia hortensis Garden warbler
1888 16-17 Rubicilla phoenicura Carpodacus rubicilla? Great Rosefinch
1888 18, 50 Motacilla flava Yellow wagtail
1888 6-7 Phyllopneuste hypolais Hippolais icterina? Icterine Warbler
1888 29-34, 40- Phyllopneuste sibilatrix Phylloscopus sibilatrix Wood Warbler
44
1888 35 Sitta europaea Nuthatch
1888 24 Fringilla canabina Carduelis canabina Linnet
1888 1-3, 23, Fringilla caelebs Fringilla coelebs Chaffinch
54-62
1888 9-10 Ligurinus chloris Cardeulis chloris Greenfinch
1888 45-47, 53 Emberiza citrinella Yellowhammer
1913 Uria lomvia Thick-billed murre
(Brünnich’s
guillemot)
Table 8.2 Bird species examined by Retzius for spermatozoal morphology by light
microscopy. (Modified from Afzelius 1995)
Volume Species Valid name if different Common name

(date)
Struthioniformes
16 (1911) Struthio molybdophanes Struthio camelus Somalian ostrich
14 (1909) Galliformes
Gallus gallus Domestic rooster
14 Anseriformes
Anas boschas domestica Anas platyrhynchos Domestic duck
14 Fuligula fuligula Aythya fuligula Tufted duck
14 Gruiformes
Fulica atra Common coot
14 Rallidae
Crex crex Corncrake
14 Charadriiformes
Uria Troile Uria aalge Common guillemot
14 Larus fuscus Lesser black-backed gull
Avian Spermatozoa: Structure and Phylogeny !#%

Volume Species Valid name if different Common name
(date)
14 Vanellus vanellus Lapwing
14 Tringa alpina Calidris alpina Alpine dunlin
14 Totanus ochropus Tringa ochropus Green sandpiper
14 Scolopax rusticola Woodcock
14 Pavoncella pugnax Philomachus pugnax Ruff
14, 16 CoIumbiformes
Columba livia domestica Domestic pigeon
14 Psittaciformes
Psittacus sp. A parrot species
14 Strigiformes
Syrnium aluco Strix aluco Tawny owl
16 Apodiformes
Cypselus apus European swift
14 Piciformes
Dendrocopos major Great spotted woodpecker
14 Passeriformes
Corvida
Corvus cornix Corvus corone Hooded crow
17 (1912) Corvus frugilegus Rook
17 Perisoreus infaustus Siberian jay
16 Coloeus monedula Corvus monedula Jackdaw
14 Pica pica Magpie
16 Lanius collurio Red-backed shrike
14 Passerida
Sturnus vulgaris Starling
14 Turdus musicus Turdus philomelos Song thrush
14 Aedon luscinia Luscinia luscinia Thrush nightingale
14 Muscicapa atricapilla Ficedula hypoleuca Pied flycatcher
14 Phylloscopus sibilator Phylloscopus sibilatrix Wood warbler
14 Alauda arvensis Skylark
14 Anthus obscurus Anthus spinoletta Rock pipit
14 Fringilla coelebs Chaffinch
14 Chrysomitris spinus Carduelis spinus Siskin
14 Chioris chioris Carduelis chloris Greenfinch
14 Passer domesticus House sparrow
14 Emberiza citrinella Yellowhammer
Some of the species of birds for which sperm are described in this chapter
are illustrated in Fig. 8.3.
8.5 NEORNITHES
According to Gauthier and de Queiroz (2001) the name “Aves” refers to the
crown clade stemming from the most recent common of Ratitae (Struthio
camelus Linnaeus 1758), Tinamidae (Tetrao [Tinamus] major Gmelin 1789) and
!#& Reproductive Biology and Phylogeny of Birds
Table 8.3 Orders and families with number of genera and species examined by McFarlane
(1963)
Non-passerines Passeriformes
Procellarilformes Suborder Tyranni
Hydrobatidae (1 gen., 1 sp.) (Suboscines)
Ciconiiformes Dendrocolaptidae (2 gen., 3 spp.)
Ardeidae (2 gen., 2 spp.) Furnariidae (1 gen., 1 sp.)
Anseriformes Formicariidae (2 gen., 2 spp.)
Anatidae (1 gen., 1 sp.) Cotingidae (4 gen., 4 spp.)
Falconiformes Pipridae (2 gen., 3 spp.)
Accipitridae (1 gen., 1 sp.) Tyrannidae (12 gen., 14 spp.)
Galliformes Suborder Passeres
Tetraonidae (1 gen., 1 sp) Corvida
Phasianidae (1 gen., 1 sp.) Corvidae (2 gen., 2 spp.)
Gruiformes Laniidae (1 gen., 1 sp.)
Rallidae (1 gen., 1 sp.) Vireonidae (2 gen., 4 spp.)
Charadriiformes Passerida (Oscines)
Charadriidae (1 gen., 1 sp.) Alaudidae (1 gen., 1 sp.)
Scolopacidae (2 gen., 2 spp.) Hirundinidae (4 gen., 4 spp.)
Recurvirostridae (1 gen., 1 sp.) Paridae (1 gen., 4 spp.)
Sittidae (1 gen., 2 spp.)
Laridae (3 gen., 7 spp.) Troglodytidae (2 gen., 2 spp.)
Rynchopidae (1 gen., 1 sp.) Mimidae (3 gen., 3 spp.)
Alcidae (1 gen., 1 sp.) Muscicapidae (=Turdidae) (3
Columbiformes gen., 7 spp.)
Columbidae (5 gen., 6 spp.) Sylviidae (2 gen., 2 spp.)
Cuculiformes Bombycillidae (1 gen., 1 sp.)
Cuculidae (1 gen., 1 sp.) Sturnidae (1 gen., 1 sp.)
Strigiformes Coerebidae (1 gen., 1 sp.)
Strigidae (1 gen., 1 sp.) Parulidae (13 gen., 27 spp.)
Caprimulgiformes Ploceidae (1 gen., 1 sp.)
Caprimulgidae (1 gen., 1 sp.) Icteridae (8 gen., 8 spp.)
Apodiformes Thraupidae (7 gen., 8 spp.)
Apodidae (1 gen, 1 sp.) Fringillidae (17 gen., 23 spp.)
Trochilidae (5 gen., 5 spp.)
Trogoniformes
Trogonidae (1 gen., 1 sp.)
Piciformes
Picidae (1 gen., 1 sp.)
Ramphastidae (1 gen., 1 sp.)
Neognathae (Vultur gryphus Linnaeus 1758). It seems preferable, however, to

use the term Neornithes for this assemblage.
8.6 PALAEOGNATHAE
This is the Parvclass Ratitae of Sibley and Ahlquist (1990). According to
Gauthier and de Queiroz (2001), “Palaeognathae” refers to the crown clade
CMYK
Avian Spermatozoa: Structure and Phylogeny !#'

CMYK
CMYK
Fig. 8.3 Some bird species examined for spermatozoal structure. A. Mallard (Anas
platyrhynchos) Anseriformes. B. Coot (Fulica atra) Gruiformes. C. Crested pigeon
(Ocyphaps lophotes) Columbiformes. D. Crimson Rosella (Platycercus elegans)
Psittaciformes. E. Magpie lark (Grallina cyanoleuca) Corvida, Passeriformes. F.
Southern ant-eater chat (Myrmecocichla formicivora) Passerida, Passeriformes.
Photos © A, B, C, E, Barrie Jamieson. D, Christopher Tudge. F, Eric Hermann.
CMYK
stemming from the most recent common ancestor of Tinamidae (Tetrao

[Tinamus] major Gmelin 1789) and Ratitae (Struthio camelus Linnaeus 1758).
8.6.1 Order Struthioniformes

The Order Struthioniformes contains the families Apterygidae, Casuariidae,
Rheidae and Struthionidae. The Struthionidae is monotypic for the genus
Struthio; Casuariidae contains the genera Casuarius and Dromaius; Rheidae
contains the genera Pteroicnemia and Rhea.
8.6.1.1 Taxa examined
The following Struthioniformes have been examined for spermatozoal
ultrastructure (see also Table 8.4): Struthionidae, Struthio camelus, Ostrich
(Soley 1989; Baccetti et al. 1991; Soley 1993, 1994; Soley and Roberts 1994;
Soley 1994b, 1996; 1999); Rheidae, Rhea americana albisceus, Rhea (Asa et al.
1986; Phillips and Asa 1989); Casuariidae, Dromaius novaehollandiae, Emu
(Baccetti et al. 1991).
8.6.1.2 Overview of struthioniform spermatozoa
This diagnostic resumé is drawn chiefly from the accounts for Ostrich (Struthio
camelus) sperm (Baccetti et al. 1991; Soley 1993, 1999; Soley and Roberts 1994).
Lesser detail has been obtained from the accounts for Rhea (Rhea americana
albisceus) (Phillips and Asa 1989) and Emu (Dromaius novaehollandiae) (Baccetti
et al. 1991).
The nucleus is an elongate cylinder and extends as a tapering nuclear ros-
trum far apically into the subacrosomal space (amniote symplesiomorphies).
The acrosome vesicle is conical, shorter than the nucleus (amniote
symplesiomorphy), about one tenth (Dromaius) to one fifth (Struthio) its length.
The midpiece is shorter than the head (amniote symplesiomorphy), only about
a quarter to a third of its length. An annulus is present at the junction of
midpiece and principal piece (amniote synapomorphy by reversal?). The prin-
cipal piece, several times the length of the nucleus, is defined by the presence
of an annulated fibrous sheath (amniote synapomorphy) ensheathing the
axoneme. There is a short endpiece consisting of axoneme (and plasma mem-
brane) only. A subacrosomal space contains homogeneous material but
no subacrosomal cone is present (synapomorphy of Aves). The nucleus is
penetrated axially by an endonuclear canal (amniote symplesiomorphy)
which contains the putative perforatorium. The perforatorium does not extend
apically beyond the tip of the nucleus (Struthioniform + Timamiform
synapomorphy), except, at most, for a little filamentous material. The
endonuclear canal and enclosed perforatorium are absent in Dromaius
(autapomorphy of this genus). There is a small basal nuclear fossa behind
which is the proximal centriole orientated at right angles to the long axis of
the spermatozoon (amniote symplesiomorphy). This is followed by the distal
centriole (basal body) in the long axis and continuous with the axoneme. The
distal centriole, also with nine triplets, is extremely elongate (amniote
synapomorphy, reducing in non-paleognaths) and extends through the length
Avian Spermatozoa: Structure and Phylogeny !$
Table 8.4 Ultrastructural studies on bird spermatozoa*
Taxon Reference
Struthioniformes
Rhea americanus albisceus, Rhea Asa et al. 19861
Phillips and Asa 1989 1
Struthio camelus, Ostrich Baccetti et al. 19911 2; Soley 1989 1, 1993 1.
1994a,b1, 19961, 19991; Soley and Roberts 19942
Dromaius novaehollandiae, Emu Baccetti et al. 19911 2
Tinamiformes
Eudromia elegans, Crested tinamou Asa et al. 19861 2; Asa and Phillips 19871
Anseriformes
Anas platyrhynchos, Mallard Humphreys 1972 1; Maretta 1975a, b1
Branta sandvicensis, Hawaiian goose Humphreys 19721
Craciformes None
Galliformes
Gallus gallus/domesticus, Rooster Grigg and Hodge 19491 2 ; Bonadona 19542;
Nagano 19601, 19621; Mclntosh and Porter 19671;
Nicander and Hillstrom 1967 1;
Krustev and Danov 1968 1; Lake et al. 19681;
Nicander 1970b 1; Tingari 1973 1; Bakst and
Howarth 19751; Gunawardana and Scott 19771;
Bakst and Sexton 19791; Bakst 19801; Xia et al.
19851; Xia et al. 19861; Xia et al. 19881; Bae
and Kim 19871; Woolley and Brammall 19871;
Thurston and Hess 1987 1; Sprando and Russell
1988 1; Jamieson 19991
Coturnix japonica, Japanese quail Saita et al.19801; Maretta et al. 19821; Lin
and Jones 19931; Woolley 19951; Tripepi et al.
19911; Vernon and Woolley 19991
Coturnix chinensis, Blue-breasted quail This study1
Meleagris gallopavo, Turkey Marquez and Ogasawara 1975 2; Bakst and
Sexton 19791; Baccetti et al. 19801; Bakst 19801;
Bradley et al. 19861; Thurston and Hess 1987 1 2
Tragopan caboti, Cabot’s tragopan Wen et al. 19971
Numida meleagris, Guineafowl Hess et al. 19861; Thurston et al. 19821 2 ;
Thurston and Hess 1987 2; Aire and Soley
20031
Turniciformes None
Piciformes
Melanerpes carolinus, Red-bellied Henley et al. 19781
woodpecker
Galbuliformes None
Bucerotiformes None
Upupiformes None
Coraciiformes None
Coliiformes None
Cuculiformes

Taxon Reference
Crotophaga ani, Cuckoo Saita et al. 1982; Tripepi et al. 1991 1; this
study1
Psittaciformes
Melopsittacus undulatus, Budgerigar Humphreys 19751; Samour et al. 19861 2;
Jamieson et al. 19951.
Agapornis roseicollis, Peach-faced lovebird Jamieson et al. 19951
Platycercus elegans, Crimson Rosella Jamieson 19991
Nymphicus hollandicus, Cockatiel Jamieson et al. 19951
Apodiformes
Apus (=Cypselus) apus, Common swift Tripepi et al. 1984 1; this study 1; Jamieson
and Tripepi 20051
Apus melba, Alpine swift Tripepi et al. 19911
Trochiliformes None
Musophagiformes None
Caprimulgiformes
Caprimulgus europaeus, European nightjar Tripepi et al. 19911; this study1
Columbiformes
‘Pigeon’ Fawcett et al. 19711
Columba livia, Domestic pigeon Yasuzumi and Yamaguchi 19771
(spermiogenesis only); Vernon and Woolley
19991
Streptopelia roseogrisea, Turtle dove Mattei et al. 19721
Geopelia striata, Peaceful dove Jamieson et al. 19951; 1999 1; this study1
Ocyphaps lophotes, Crested pigeon Jamieson et al. 19951; Jamieson 19991; this
study1
Gruiformes
Grus vipio, White-necked crane Asa and Phillips 19871 2; Phillips et al. 19871 2
The following 7 orders were previously
placed in the order Ciconiiformes sensu
Sibley and Ahlquist (1990)
Charadriiformes
Jacana jacana Saita et al. 19831; Tripepi et al. 19911
Falconiformes
Falco peregrinus, Peregrine falcon Wagley 19802
Pelecaniformes None
Procellariformes None
Podicepediiformes None
Sphenisciformes None
Gaviiformes None
Passeriformes
SUBOSCINES
Tyrannldae
Tyrannus verticalis, Western kingbird McFarlane 19711 3
Tyrannus tyrannus, Eastern kingbird Feduccia 19791
Contopus virens, Eastern wood peewee Feduccia 19791
Avian Spermatozoa: Structure and Phylogeny !$!
Taxon Reference
Myiarchus crinitus/ M. griseisticta, Great McFarlane 19711 3 ; Asa and Phillips 1987 1
crested flycatcher
OSCINES
Corvida
Corvidae
Cyanocitta cristata, Blue jay Henley et al. 19781
Corvus splendens, Crow Bawa et al. 19901 2
Grallinidae
Grallina cyanoleuca, Magpie lark Jamieson 19951,19991
Vireonidae
Vireo olivaceus, Red-eyed vireo McFarlane 19713; Henley et al. 1978 1
Vireo griseus, White-eyed vireo Asa and Phillips 1987 1
Passerida
MUSCICAPOIDEA
Muscicapidae (including Turdidae)
Musclcapidae
Myrmecocichila formicivora, S. Ant-eater chat Jamieson et al., unpublished1; this study1
Turdus greyi, Clay-colored robin McFarlane 19631
Turdus merula, Blackbird Furieri 19611
Turdus migratorius, American robin McFarlane 1971 1 3; Henley et al. 19781
Sturnidae
Sturnus vulgaris , Starling Koehler 1995 1 2 ‘ Vernon and Woolley 1999 12
Sylvioidea
Paridae
Parus bicolor, Tufted titmouse McFarlane 1971 1 3; Henley et al. 19781
Hirundinidae
Tachycineta thalassina, Violet-green swallow McFarlane 19711 3
Certhioidea
Troglodytidae
Thryothorus ludovicanus, Carolina wren Asa and Phillips 1987 1
Troglodytes troglodytes, Wren Tripepi and Perrotta 19911
Certhiidae
Certhia brachydactyla Tripepi and Perrotta 19911
Sittidae
Sitta europaea Tripepi and Perrotta 19911
PASSEROIDEA
Parulidae
Dendroica pinus, Pine warbler Asa and Phillips 1987 1
Dendroica dominica, Yellow-throated Asa and Phillips 1987 1
warbler
Protonataria citrea, Prothonotory warbler Asa and Phillips 1987 1
Fringillidae
Ammodramus maritimus (=Ammospiza
maritima), Seaside sparrow McFarlane 19631
Fringilla coelebs, Chaffinch Furieri 19621; Tripepi and Perrotta 19911
!$" Reproductive Biology and Phylogeny of Birds
Taxon Reference
Pipilo erythrophthalmus, Rufous-sided Henley et al. 19781
towhee
Piranga rubra, Summer tanager Henley et al. 19781
Serinus canaria, Canary Humphreys 19721
Serinus canaria, Canary ¥ Cardeulis Swan 19851
cardeulis Goldfinch, hybrid
Zonotrichia albicollis, White-throated Henley et al. 19781
sparrow
Carduelis (=Chloris) chloris Tripepi and Perrotta 19911
Icteridae
Agelaius phoeniceus, Redwing Asa and Phillips 19871; Koehler 19952
Icterus galbula, Northern oriole Asa and Phillips 19871
Molothrus ater, Brown-headed cowbird Koehler 19951
Quiscalus quiscula, Grackle Koehler 19951 2
Emberizldae
Cardinalis cardinalis, Cardinal Henley et al. 19781; Koehler 1995 1 2
Emberiza cirlus, Cirl bunting Tripepi and Perrotta 19911; Tripepi et al. 19911
Estrildidae
Lonchura striata (=Uroloncha striata), Love Yasuzumi and Sugioka 19661; 19711.
bird Yasuzumi 19741 2; Kondo et al. 19881
Lonchura castaneothorax ¥ L. puntulata Swan and Christidis 19871
Taeniopygia guttata, Zebra finch Nicander 1970a1; Fawcett et al. 19711;
Asa and Phillips 19871
Vernon and Woolley 19991
Passeridae Humphreys 19721; Asa and Phillips 19871 2
Passer diffusus, Grey-headed sparrow This study1
Passer domesticus, House sparrow Koehler 19951 2
Passer italiae, italian sparrow Furieri 19611
Thraupidae
Piranga rubra, Summer tananger McFarlane 19711 3
Pioceidae
Philetairus socius, Social weaver Jamieson et al., unpublished1; this study1
Euplectes orix, Red bishop This study2
Ploceus capensis, Cape weaver This study2
Quelea qualea, Red-billed quelea This study2
Prunellidae
Prunella colIaris, Alpine accentor Chiba and Nakamura 20011 2
1
TEM 2SEM 3fide Koehler (1995)
*For a list of additional species examined by Birkhead et al. (2006) see section 8.10.12.17.
For additional species examined by Tripepi and Perrotta (1991) see section 8.10.10.1.
of the midpiece. It is penetrated by the two central singlets of the axoneme

(amniote synapomorphy) around which is a dense sheath (crocodile-bird
synapomorphy, not described for Rhea and Dromaius and lost in non-
struthioniforms). The midpiece consists of approximately ellipsoid (Struthio,
Avian Spermatozoa: Structure and Phylogeny !$#
Rhea) or spheroidal (Dromaius) mitochondria ensheathing the axoneme (am-

niote synapomorphy), four (Struthio), four to six (Rhea), or five to six (Dromaius)
per transverse section, joined by electron-dense cement, totaling about 20-25
(Struthio), 30 (Rhea) or 40 (Dromaius) in a poorly defined helix, ending posteri-
orly at the annulus; cristae longitudinal plates, not in the basal amniote con-
centric arrangement. The fibrous sheath of the principal piece consists of semi-
circular ribs, enlarging near axonemal doublets 3 and 8 as longitudinal col-
umns (amniote synapomorphies, replaced by amorphous sheath in other non-
passerines). A small dense fiber is attached to each doublet in the principal
piece (Struthio, Rhea, shorter in Dromaius) (the fibers are a crocodile-avian
symplesiomorphy, though reduced in the latter). At the posterior end of the
endpiece, the doublets separate into individual components, and the dense
contents of the A microtubules disappear. The resulting disorganized collec-
tion of 20 lucent microtubules displays a random decrease in number towards
the tip (amniote plesiomorphy?).
8.6.1.3 Struthio camelus
The following account of Ostrich sperm is drawn from (Soley 1984b, 1989,
1993, 1999; Soley and Roberts 1994), with some substitution with the
terminology of the present author, and from the earlier, succinct account of
Baccetti et al. (1991). Spermatogenesis (Soley 1994, 1996) is discussed in
Chapter 7 of this volume.
General morphology. The mature spermatozoon of Ostrich is illustrated in
Figs. 8.4 and 8.5. Examined by scanning electron microscopy (SEM) (Fig. 8.5Q-
U) it is a 60 m long (70-80 mm in Soley 1989, 1999), filiform cell with an
anterior tapering, cylindrical, 16 mm long head (acrosome + nucleus) (13 mm in
Soley 1999) which reaches a diameter of 0.8 mm (Baccetti et al. 1991) near its
base (greatest width of nucleus 0.5 µm, Soley 1999). A slight circular
depression, the posterior ring of Soley (1999), marks the posterior limit of the
2 mm long acrosome. Immediately posterior to the head is the 3 mm long, 0.7
mm wide, midpiece, followed by the 40 mm long, 0.7 mm wide, cylindrical
principal piece of the tail, tapering towards its posterior limit and followed in
turn by 1 mm long, 0.2 mm wide, endpiece (Baccetti et al. 1991). A raised hoop-
like ring at the junction of the midpiece and principal piece was identified as
the site of the annulus (Soley and Roberts 1994). Soley gives detailed tables of
dimensions from a total of 200 sperm from 10 Ostriches, in which mean
lengths, in mm, were: acrosome 1.91; nucleus (excluding the portion covered
by the acrosome) 10.95; total head 12.86; midpiece 3.16; principal piece 51.18;
endpiece 2.39; total tail 56.73; total sperm length 69.59. The mean tail to head
ratio was 4.43. Differences in dimensions relative to those given by Baccetti et
al. (1991) may be real as Soley and Roberts (1994) recognized two populations,
possibly subspecies, in their own data, with one bird intermediate between
the two.
The head. The head of Ostrich sperm is a slightly curved, cylindrical structure
tapering gradually at its most anterior aspect and measures 13 mm in length
!$$ Reproductive Biology and Phylogeny of Birds
Fig. 8.4 Struthio camelus, Ostrich. Schematic representation of a spermatozoon

illustrating the various components of the head (acrosome and nucleus) and tail
(midpiece, principal piece and endpiece). The three figures on the right show finer
details, not drawn to the same scale) of the structures labeled on the whole
spermatozoon (left). A. Acrosome. B. Midpiece. C. Principal piece. Relabeled after
Soley, J. 1999. Pp. 129-158. In D. C. Deeming (ed). The Ostrich: Biology, Production
and Health, CAB International, Fig. 6.5.
Avian Spermatozoa: Structure and Phylogeny !$%
and 0.5 mm in width at its widest point (dimensions from Soley and Roberts
1994, throughout). The tip of the head is invested by the 2 mm long acrosome
vesicle containing fine homogeneous material of moderate electron density
(Fig. 8.5A, B) (Baccetti et al. 1991; Soley 1993). A subacrosomal space, 30 nm
wide, is present between the inner acrosomal and nuclear membranes but this
widens to 150 nm at the tip of the head (Baccetti et al. 1991). Flocculent
material of an electron-density similar to the contents of the acrosome is
observed in this space, often in close association with the nuclear membrane
(Fig. 8.5A, B). Baccetti et al. (1991) state that the apical subacrosomal space
contains a bundle of filamentous material which emerges from the
endonuclear canal, opening at the nuclear tip; it is correctly regarded as a
continuation of the rod (perforatorium) within the endonuclear canal.
However, the prenuclear material is minimal and Soley (1993) did not
recognize its presence. He regarded the avian perforatorium as merely
residual. In contrast, Baccetti et al. (1991). considered that the subacrosomal
filaments were probably actinic, as in other birds (Campanella et al. 1979). An
area of close contact existed between the plasmalemma and nuclear membrane
at the caudal extremity of the acrosome, forming a structure similar to the
posterior ring of mammalian sperm (Fig. 8.5A) (Soley 1993).
Nucleus. The nucleus forms a cylinder except for the part covered by the
acrosome where it tapers sharply, as the nuclear rostrum, to end in a fine point
beneath the tip of the acrosome. From the tip of the nucleus an axial
endonuclear canal, lined by invaginated nuclear membrane, and containing
the rod-like perforatorium, extends through the length of the rostrum for
roughly 1/4 of the length of the main body of the nucleus (Soley 1993) or for
ca 5 mm and about the apical third of the head, being ca 30 nm wide (Baccetti
et al. 1991) (Fig. 8.5A-C). The chromatin is compact and electron-dense (Fig.
8.5C) excepting small light regions indicative of incomplete condensation
throughout the nucleus, particularly in the rostrum. The basal, implantation
fossa consists of a small central concavity surrounded by a shallow circular
moat or series of depressions running around the perimeter of the nuclear base
(Soley 1993). This was interpreted as two parallel implantation fossae, each
with an undulating basal lamina by Baccetti et al. (1991).
The neck and midpiece. Beneath the base of the nucleus, a short (0.3 mm)
proximal centriole displays the characteristic nine sets of triplet microtubules
(Baccetti et al. 1991; Soley, 1993). These are embedded in a ring of dense
amorphous material. The central cavity of the centriole sometimes contains
flocculent or granular material similar to that observed between the
mitochondria of the midpiece (Fig. 8.5E). Dense material associated with the
juxta-nuclear surface of the proximal centriole fills the center of the nuclear
fossa, merging with similar peripheral material provided by dense non-
segmented columns emanating from the walls of the proximal and distal
centrioles (Fig. 8.5D, E). The distal centriole is perpendicular to the proximal
centriole, and occupies the entire length of the midpiece. In transverse section
!$& Reproductive Biology and Phylogeny of Birds
Fig. 8.5 Struthio camelus, Ostrich. A-P. Transmission electron micrographs. A.

Longitudinal section (LS) of acrosome and anterior nucleus. B. Transverse section
(TS) of acrosome vesicle and the enclosed nuclear rostrum with central
Fig. 8.5 Contd. ...
Avian Spermatozoa: Structure and Phylogeny !$'
it consists of a narrow ring of electron-dense material from which nine evenly

spaced dense projections jut into the centriolar cavity. Nine sets of
characteristically arranged triplet microtubules are situated between the
projections. Within the centriolar cavity are two singlets (central tubules)
(Baccetti et al. 1991; Soley 1993, 1999). Around these Soley (1993) describes a
rod, sometimes eccentric, of dense material extending posteriorly as far as the
annulus and containing a pair of microtubules (Fig. 8.5D, E). The rod is
occasionally seen in the form of two closely apposed but separate units, each
containing a single microtubule. Posterior to the annulus a typical central pair
of microtubules extends throughout the rest of the tail.
The 0.3 mm long midpiece is slightly wider (0.65 mm) than the nucleus and
contains about 20 (Soley 199) or 24-25 (Baccetti et al. 1991) mitochondria
arranged in an helical pattern around the proximal and distal centrioles (Fig.
8.5D, F, S) as a single layer. The mitochondria have flattened, rectangular
profiles (Figs. 8.4B, 8.5D), although round or oval forms are sometimes
Fig. 8.5 Contd. ...

endonuclear canal. C. TS nucleus and perforatorium. D. LS midpiece. Note the
termination of the inner dense rod of the distal centriole in the vicinity of the annulus
(white arrow). The dense walls of the distal centriole, which runs the length of the
midpiece are indicated by black arrows. E. LS of the neck region showing the
proximal centriole cut transversely. F. TS midpiece and distal centriole. A dense rod
of material containing two singlet microtubules is eccentrically situated. Small black
arrow heads indicate atypical cristae. G. LS proximal region of principal piece,
showing annulus at posterior limit of midpiece, and fibrous sheath. Arrows indicate
ribs of this sheath. H. Similar section but showing (arrows) longitudinal columns of
the fibrous sheath. I. LS of a more distal region of the principal piece, through
longitudinal columns (arrows) of the fibrous sheath. J. TS of the proximal principal
piece, showing, above and below, the longitudinal columns. Dense fibers 3 and 8
have been incorporated into the columns. K. TS of two sperm tails posterior to the
region with dense fibers but still surrounded by the fibrous sheath. L. LS at junction
of midpiece and principal piece, showing the annulus I tangential section. M. LS
distal region of principal piece. The ribs of the fibrous sheath are shown in cross
section (arrows) and longitudinal profile (squat arrow). N. LS endpiece. Note the
staggered termination of the fibrous sheath (arrows). O. TS intermediate region of
the principal piece. The coarse fibers have disappeared. Doublets 3 and 8 are
connected to the longitudinal columns. The cytoplasmic layer surrounding the
axoneme is thinner than further proximally. P. TS endpiece at disruption of 9+2
pattern, resulting in a collection of doublets (arrows) and dense and translucent
singlet microtubules. Q-U. Scanning electron micrographs. Q. Entire spermatozoon.
R. Junction of principal piece and endpiece. S. Midpiece, with 5 tiers of
mitochondria. T. Tapered tip of the nucleus (rostrum), between arrows, revealed
after loss of the acrosome vesicle. U. Detail of the head and midpiece. A-P modified
after Soley, J. T. 1993. Onderstepoort Journal of Veterinary Research 60: 119-130,
Figs. 1-16. Q-U after Soley, J. T. and Roberts, D. C. 1994. Onderstepoort Journal of
Veterinary Research 61: 239-246, Figs. 1-5.
!% Reproductive Biology and Phylogeny of Birds
observed (Fig. 8.5D, S) and in tangential sections they appear as rectangular

or polygonal structures (Fig. 8.5L) (Soley 1993) but Baccetti et al. (1991) found
them to be elliptic in both longitudinal and transverse section; four in each
cross section, joined by an electron-dense cement. The cristae are longitudinal,
in a dense matrix. Atypical cristae containing paracrystalline inclusions occur
in some mitochondria (Figs. 8.5F, arrow heads). The inclusions display two
forms depending on the plane of section. Those sectioned longitudinally
presented the appearance of tight junctions while oblique sections revealed a
pattern of parallel fibers with a regular spacing and direction. Sandwiched
between the mitochondria are conspicuous accumulations of granular
material (Soley 1993) (Fig. 8.5F).
Annulus. A well developed annulus (Figs. 8.4B, 8.5D, G, H) marks the
boundary between the midpiece and the principal piece (Baccetti et al. 1991;
Soley 1993, 1999), although a retro-annular recess is not apparent. The
annulus is situated beneath the last row of midpiece mitochondria in close
association with the plasmalemma and is composed of homogeneous,
electron-dense material.
Principal piece. The principal piece forms the longest segment of the tail (ca
50 mm) and consists of the 9+2 axoneme surrounded by a ribbed fibrous sheath
(Soley 1993, 1999) (Fig. 8.5D-O). The ribs are semicircular and about 50 nm
thick, enlarging near the axonemal doublets 3 and 8 (Baccetti et al. 1991) as
the ‘longitudinal columns’ described below by Soley (1993). The A
microtubule of each doublet is a circular structure filled with dense material
whereas the B microtubule formed an incomplete lucent cylinder. Dynein arms
project from each A microtubule towards the neighboring doublet and radial
links form a connection with the central microtubules (Figs. 8.5K, O). The
principal piece is a tapered structure and gradual decreases in diameter along
its length, coupled with changes in the composition of the fibrous sheath,
allow three regions to be distinguished:
1. The first region lies immediately posterior to the annulus where the tail
abruptly narrows to a diameter of 0.5 µm. The axoneme is surrounded
by a loosely arranged fibrous sheath consisting of two dense
longitudinal columns connected by circumferential bands of dense
material. The longitudinal columns and the interconnecting ribs
appeared to be composed of alternating layers of electron-dense and
loosely packed material, giving both structures a laminated appearance.
The columns lie in line with the two central microtubules in the position
occupied by coarse fibers 3 and 8 in mammalian sperm. Peculiar to this
region is the presence of nine small (rudimentary, Soley 1999), dense,
coarse fibers (the accessory fibers, reniform in cross section, noted by
Baccetti et al. 1991) lying between the fibrous sheath and the axonemal
doublets, each in close association with an A microtubule. A prominent
cytoplasmic layer, maximally 60-80 nm wide (Baccetti et al. 1991)
containing fine flocculent material is interposed between the fibrous
sheath and the cell membrane (Fig. 8.5G-J, L).
Avian Spermatozoa: Structure and Phylogeny !%
2. In the second region the diameter of the tail narrows to 0.4 µm, the
rudimentary coarse fibers disappear, and the ribs of dense material
appear as solid structures. The longitudinal columns, however, retain
their laminated appearance while the cytoplasmic layer becomes
narrower. Septum-like inward extensions of the longitudinal columns
make contact with the adjacent microtubular doublets (Fig. 8.5J).
3. The third region of the principal piece is characterized by a progressive
decrease in diameter, from 0.3 µm to approximately 0.2 µm. The
longitudinal dense columns became solid, less conspicuous, structures
which eventually disappear leaving a thin dense band of material
surrounding the axoneme. The plasmalemma is closely applied to the
layer of dense material (Fig. 8.5I, K, M) (Soley 1993).
Endpiece. The endpiece forms the short, narrow, 2.4 mm long (Soley and
Roberts 1994) or 2-3 mm (Soley 1999), termination of the tail and consisted of
the axoneme covered only by plasmalemma. Because the transition from
principal piece to endpiece is gradual, remnants of the fibrous sheath are
sometimes seen around the axoneme (Soley 1993) (Fig. 8.5N). The organized
structure of the axoneme is disrupted towards the end of the tail (Baccetti et al.
1991; Soley 1993, 1999). The specific orientation of the axonemal microtubules
is lost, the dynein arms and radial spokes linking the microtubules have
disappeared, the doublets separate into individual components, and the
dense contents of the A microtubules disappear (Fig. 8.5P). The resulting
disorganized collection of 20 lucent microtubules displays a random decrease
in number at the tip of the endpiece (Soley 1993).
The crocodiloid spermatozoon. Ballowitz and Retzius recognized the
‘sauropsid’ features of non-passerine spermatozoa. However, the ratite and
lower non-passerine spermatozoon, especially the former, would more
appropriately be termed crocodiloid. Features of Ostrich sperm which are
similar to those of crocodiles are: the pointed acrosome vesicle; the
perforatorium in a long endonuclear canal; the midpiece with several tiers of
mitochondria surrounding an extremely long distal centriole and terminating
at an annulus; presence of nine dense fibers; and a fibrous sheath consisting
of transverse ribs (Figs. 8.4C, 8.5G). All of these features are also seen in
Chelonia (Healy and Jamieson1992) and are basic (symplesiomorphic) to
amniotes, only the fibrous sheath and the long distal centriole being amniote
synapomorphies. The sole spermatozoal synapomorphy of crocodiles and
birds is the dense sheath investing the two central singlets within the elongate
distal centriole (Jamieson 1999). In birds this sheath is known only in ratites
(Ostrich) and the Galloanserae.
8.6.1.4 Rhea americana albisceus
The following account of the sperm of Rhea, Rhea americana albisceus, is drawn
from Phillips and Asa (1989). Additional data are added from perusal of their
illustrations and current terminology is employed.
!% Reproductive Biology and Phylogeny of Birds
Head. The head is curved and tapered. A substantial acrosome composed of

moderately electron-dense homogeneous material fits over the anterior portion
of the nucleus. A narrow cylindrical structure (endonuclear canal) extends
from the anterior portion of the acrosome to deep within the nucleus (Fig. 8.6C,
D). The center of the cylinder (putative perforatorium) is composed of material
that is about the same electron density as the acrosome, which is
circumscribed by an electron-lucid region, in turn surrounded by a thin region
of moderate electron density (Fig. 8.6C, D). The chromatin is compact but not
as condensed as is observed in spermatozoa of insects or mammals (Fig. 8.6A-
D). A short midpiece lies between the head and principal piece (Fig. 8.6A). The
long principal piece comprises most of the length of the cell.
Neck region and centrioles. As in many other animals, the neck region of
Rhea sperm is characterized by a precisely shaped posterior portion of the
nucleus. Electron-dense material associated with the proximal centriole fits
into the contour of the sperm nucleus and associated nuclear membrane (Fig.
8.6A, D). The proximal centriole is short, only about 0.4 mm long, and is inlaid
with electron-dense material (Fig. 8.6D, E). The distal centriole is much longer.
It apparently extends the entire length of the midpiece. Both centrioles are
embedded in electron-dense material. Although the distal centriole displays
the characteristically disposed triplet subtubules, the central tubules of the
flagellum extend into the center of the centriole (Fig. 8.6E).
Midpiece. The midpiece contains about 30 mitochondria, four to six in
transverse section, and about seven longitudinally, with a very dense matrix
typical of spermatozoal mitochondria. In the interior of the mitochondrion
distal to the centrioles, there is a complex configurations of mitochondrial
membranes (Fig. 8.6A, D, E).
Principal piece. Transverse sections through the principal piece reveal a small
fibrous sheath. The sheath is larger opposite doublet tubules 3 and 8 and is
connected to these two doublets by a thin band of dense material, as in
mammals, but enlargement is only slight, less than in the latter (Fig. 8.6F).
Dense axonemal fibers. There are very tiny dense fibers present only for a
short region of the principal piece (see Fig. 8.6F), given (erroneously?) in the
original account as the very anterior portion of the “midpiece”. They are no
larger than microtubules and are associated with the doublet subtubules A
and B (Fig. 8.6F).
Remarks. The spermatozoon of Rhea is closely similar to that of Ostrich, the
similarities including the deep extension of the endonuclear canal and
perforatorium into the nucleus. Soley (1993) considers Ostrich sperm to differ
from that of Rhea, however, in that the central tubules are embedded in a core
of dense material which only disappears in the vicinity of the annulus. The
account of Phillips and Asa (1989) does not refer to this feature and no sheath
of dense material is visible in the relevant micrograph (Fig. 8.6E).
In both Ostrich and Rhea, modifications of the mitochondrial membranes
have been observed, with those of Rhea (Phillips and Asa 1989) resembling
Avian Spermatozoa: Structure and Phylogeny !%!
Fig. 8.6 Rhea americana albisceus. A. Longitudinal section (LS) midpiece,

penetrated by the long distal centriole, and terminating at the annulus. B. LS basal
region of acrosome vesicle enclosing the nuclear rostrum and, at the center of this,
the perforatorium. C. Transverse section (TS) of a nucleus and (right) the acrosome
vesicle and nuclear rostrum. D. LS neck region showing short proximal centriole
perpendicular to the distal centriole. E. TS midpiece; right, through the proximal
centriole; left, through the distal centriole, showing two central singlets. F. TS
through the principal piece of several spermatozoa. Relabeled after Phillips, D. M.
and Asa, C. S. 1989. Anatomical Record 223: 276-282, Fig. 1. Reprinted with
permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
myelin figures and those of Ostrich adopting the form of atypical cristae
containing paracrystalline material. The significance of these structural
modifications is unknown. Although organized and arranged in a fashion
!%" Reproductive Biology and Phylogeny of Birds
similar to that of mammalian sperm, the ribbed sheath of Ostrich sperm is

more flimsy in structure, resembling that described in Rhea (Phillips and Asa
1989) and tinamou (Asa et al. 1986; Soley 1993). The latter author correctly
observes that the ribbed form of the fibrous sheath seen in ratites does not
occur in other non-passerines. We may add that the transverse ribbing is a
basic amniote (Fig. 8.1) and crocodile (Fig. 8.2) feature. This unique
crocodiloid feature of ratite sperm endorses the basal position of ratites in bird
phylogeny.
The proximal segment of the principal piece of Ostrich sperm displays a
prominent cytoplasmic layer situated between the axoneme and the
plasmalemma. This layer is filled with fine amorphous material and resembles
a similar region seen in Rhea sperm (Phillips and Asa 1989). Tinamou sperm
are structurally similar in this respect although particulate material which
morphologically resembles glycogen is found throughout this region (Asa et
al. 1986). A similar region is absent in other non-passerine birds (Soley 1993).
As Soley (1993) observes, penetration of the long distal centriole by the
central singlets of the axoneme is a feature shared by Rhea and Ostrich but is
absent from Tinamou. As noted by the present author, this penetration is a
crocodilian (and chelonian) feature and is therefore a symplesiomorphy of
Rhea and Ostrich signifying an unchanged basal amniote condition but not
necessarily indicating rheid-struthionid monophyly.
8.6.1.5 Dromaius novaehollandiae
General morphology. The mature sperm cell of Emu seen by SEM (8.7A-C) is
ca 65 mm long. The cylindrical, tapered head measures ca 12 mm in length and
0.8 mm in maximum diameter. The acrosomal region is 1.5 mm long. The
midpiece is 3 mm long and not as deeply marked as in Ostrich. The principal
piece is ca 47 mm and the endpiece 3 mm long. Compared to Ostrich, the
spermatozoon of Emu has a longer tail and a shorter head, while the midpiece
(i.e. the distal centriole) is the same length (Baccetti et al. 1991). TEM sections
of the spermatozoon are shown in Fig. 8.7D-I.
Fig. 8.7 Dromaius novaehollandiae, Emu, spermatozoa. A-C. Scanning electron

micrographs. D-I. Transmission electron micrographs. A Whole spermatozoon. ¥
2200. B. Enlarged view excepting posterior flagellum. ¥ 4400. The head is shorter,
the midpiece equal in length, and the tail longer than in Ostrich. C. Posterior end,
showing the endpiece. ¥ 19000. D. Acrosome vesicle (A) on the anterior tapering
rostrum of the nucleus (N). ¥ 49600. E. Longitudinal section (LS) of the posterior
nucleus and the midpiece. ¥ 63600. F. LS midpiece and anterior principal piece. ¥
33400. G. Transverse section (TS) of the acrosome vesicle and nuclear rostrum. ¥
47700. As in A, note the absence of an endonuclear canal and perforatorium. H. TS
of the principal piece showing the fibrous sheath. ¥ 44200. I. TS endpiece; note
absence of fibrous sheath. ¥ 45000. A, acrosome vesicle; AF, accessory fibers; AX,
axoneme; dc, distal centriole; FS, fibrous sheath; M, mitochondria; N, nucleus;
Modified after Baccetti, B., Burrini, A. G. and Falchetti, E. 1991. Biology of the Cell
(Paris) 71(1-2): 209-216. Figs. 3-5, 7, 12, 13, 9, 21, 20.
Avian Spermatozoa: Structure and Phylogeny !%#
Fig. 8.7
!%$ Reproductive Biology and Phylogeny of Birds
Acrosome. Examined in sections (Fig. 8.7D, G) by transmission electron

microscopy (TEM), the acrosomal complex is made up of a truncate-conical,
100 nm thick acrosome vesicle, which holds the apical tapered portion of the
nucleus, and of an extremely thin bundle of microfilaments evident in the
apical subacrosomal space (Fig. 8.7D). This space is extremely reduced if
compared to that of Ostrich, and no endonuclear canal and no rod-like
subacrosomal structure are evident. Therefore, the perforatorium, represented
only by the sparse extranuclear microfilaments, is almost absent.
Nucleus. The nucleus, which is strongly condensed, is cylindrical and similar
to that of Ostrich.
Centrioles and midpiece. The same is true for the centrioles, while the
mitochondria are smaller, spheroidal, 5-6 in a cross-section of the midpiece,
numbering 8-10 in longitudinal section (Fig. 8.7E) and reach a total number of
40 or more. They are closely juxtaposed, and the intermitochondrial cement is
sparse. This organization explains the reduced demarcation of the midpiece,
seen from the exterior by SEM. No penetration of the distal centriole by the
two axonemal singlets is apparent in the micrograph (Fig. 8.7E) but absence
perhaps requires confirmation.
Tail. The axoneme has the same characteristics as in Ostrich, but the region
containing the small accessory dense fibers (Fig. 8.7H) is even shorter, and the
fibrous sheath (Fig. 8.7H) is progressively thinner toward the end part of the
tail and is, by definition, lacking from the endpiece (Fig. 8.7I). The tail is longer
than in Ostrich, and contains the basic 9 + 2 axoneme (Baccetti et al. 1991).
Remarks. The spermatozoon of Emu resembles that of Ostrich and Rhea in
most respects but shows three remarkable departures. Two of these, the
absence of an endonuclear canal and contained perforatorium, are clearly
correlated. The third is the rounded form of the apex of the acrosome vesicle.
Loss of the perforatorium is enigmatic if, as the author hypothesizes, it
contributes to the acrosome reaction at fertilization. Soley (1993) remarks that
no definite function has yet been ascribed to the avian perforatorium and
states that it would appear merely to represent a residual structure. However,
Campanella et al. (1979) found that the turkey perforatorium consisted of actin
and Baccetti et al. (1980) believed that it supported the conical shape of the
acrosome.
A role in the acrosome reaction and fertilization has been shown for the
perforatorium which lies in an endonuclear canal penetrating most of the
nucleus in the lamprey. In Lampetra fluviatilis, the central fiber (putative
perforatorium) is capable of extrusion as a 50 µm long ‘head filament’
(Afzelius and Murray 1957; Kille 1960). In the acrosome reaction, the plasma
membrane is drawn out into a slender sheath containing the central fiber
(Stanley 1967). The putative perforatorium undergoes no observable change
on extrusion (Follenius 1965). In L. planeri sperm in the egg coatings show a
true acrosome reaction in which the central fiber is extended in an acrosomal
tubule which penetrates the egg envelopes to reach the egg surface (Nicander
Avian Spermatozoa: Structure and Phylogeny !%%
and Sjödén 1968, 1971). In the hagfish Eptatretus an acrosome reaction occurs
with formation of an acrosomal process with a filamentous core and is
deduced to involve polymerization of actin (Morisawa and Cherr 2002).
Analysis of proteins in the rat perforatorium, which does not form an
acrosome process, failed to detect actin (Oko et al. 1990).
It is thus difficult to accept a merely residual status for the avian
perforatorium. On the other hand, even if it is involved in an acrosome
reaction in Ostrich and Rhea, which has yet to be demonstrated, it may also
have a supportive function for the conical acrosome vesicle as Baccetti et al.
(1980) suggested and its loss in Emu could conceivably be related to the
rounded form of the vesicle. It is clear that in Emu, as in many other birds, e.g.
pigeons and passerines, similarly lacking a perforatorium, an acrosome
reaction must nevertheless occur.
8.6.2 Order Tinamiformes

The Tinamiformes contain only the family Tinamidae, the Tinamous, with 9
genera and 48 species.
8.6.2.1 Eudromia elegans
The spermatozoon of Crested tinamou has been described by Asa et al. (1986)
and Asa and Phillips (1987). Their accounts are paraphrased and augmented
here.
The sperm head. The head of Tinamou spermatozoon is cylindrical and
slightly curved. The smooth, tapered anterior-most end is characterized by a
small bump at the tip (Fig. 8.8A).
Acrosome. Although the form of acrosome vesicle is undescribed, it is seen in
the illustration to be a hollow cone, the inner and outer membranes of which
are almost in contact at the tip and are more widely separate at about mid-
length than elsewhere. The vesicle contents appear homogeneous and
moderately electron-dense. In thin sections, a small space is observed between
the cell membrane and the outer acrosomal membrane (Fig. 8.8B).
Nucleus. The nucleus (Fig. 8.8A) is a moderately elongate cylinder. In contrast
with the acrosomal region, the cell membrane appears to be closely associated
with the nuclear membrane in the region immediately posterior to the
acrosome. The chromatin rarely appears completely condensed. In oblique or
longitudinal sections, at the edge of the nucleus thick strands of chromatin
are seen disposed obliquely to the long axis of the nucleus. This arrangement
suggests that the chromatin spirals around the endonuclear canal.
Endonuclear canal and perforatorium. Transverse and longitudinal sections
of the nucleus reveal a tube-like structure (endonuclear canal) which extends
from one end of the nucleus to the other and abuts the tip of the acrosome
(Fig. 8.8B).
Neck Region and Midpiece. The posterior edge of the sperm nucleus is
indented as a concave disk. A short proximal centriole is situated near the
!%& Reproductive Biology and Phylogeny of Birds
Fig. 8.8 Eudromia elegans elegans, Crested tinamou. A. Scanning electron

micrograph of the anterior portion of a spermatozoon. About 20 mitochondria are
Fig. 8.8 Contd. ...

Avian Spermatozoa: Structure and Phylogeny !%'
base of the nucleus (Figs. 8.8C, D, 8.9A). Dense columns of the neck piece
surround this centriole and extend into the distal centriole for, apparently,
about half of its length. The distal centriole is 3 mm in length, the entire length
of the midpiece, and is embedded in dense material (Figs. 8.8C, 8.9A). The
mitochondria are roughly spherical. The midpiece contains about 20
mitochondria arranged in seven tiers with about five around the centriole
(Figs. 8.8C, D, 8.9A). Flocculent material is observed between the mitochondria
(Figs. 8.8C, D, 8.9A) (Asa et al. 1986).
Annulus and principal piece. A distinct annulus is situated posterior to the
short midpiece (Figs. 8.8C, 8.9), as in mammalian spermatozoa (Fawcett,
1975). Asa et al. (1986) state that no dense fibers were observed in tinamou
spermatozoa, either in the midpiece or the principal-piece. However, they later
state (Asa and Phillips 1987) that dense fibers are present in the proximal
principal piece to which they are restricted. Periodic structures are observed
in longitudinal sections of the fibrous sheath (Figs. 8.8C, 8.9C). In transverse
section, the fibrous sheath shows the typical ribs opposite dense fibers 3 and
8. Material which appears morphologically similar to glycogen surrounds the
fibrous sheath in the anterior region (Fig. 8.9C) (Asa et al. 1986).
Tinamou sperm and ratite phylogeny. Spermatozoal characters have been
considered equivocal as to whether the Tinamiformes or Struthioniformes are
the most primitive (Soley 1993) or, in other words, if they are sister-groups,
which is the apomorph sister-group. Asa et al. (1986) observe that Tinamou is
exceptional among avian species in the great length of what is here termed the
endonuclear canal, extending to the base of the nucleus. They considered it
possible that the contents of the canal possess actin which can polymerize to
cause the acrosome to be propelled through egg investments during the
acrosome reaction as shown for other groups.
Fig. 8.8 Contd. ...

observed in the midpiece. ¥ 6800. B. Longitudinal section of the anterior portion of
the sperm head. The contents of the acrosome vesicle and moderately electron
dense. A perforatorium lying in an endonuclear canal runs through the center of the
nucleus where it abuts the tip of the acrosome vesicle. ¥ 32500. C. Midsagittal
section through the midpiece. The short proximal centriole and the long distal
centriole are each seen in longitudinal section. The distal centriole, surrounded by
spheroidal mitochondria, extends the entire length of the midpiece as is crocodile
and other ratite sperm. The posterior end of the nucleus has a concave circular
fossa. ¥ 22000. D. Transverse sections through the midpiece. The distal centriole,
embedded in electron dense material, is observed throughout the length of the
midpiece. Flocculent material is interspersed between some mitochondria. ¥
53000. Av, acrosome vesicle; ax, axoneme; dc, distal centriole; m, mitochondrion;
nr, nuclear rostrum; pc, proximal centriole; Adapted and relabeled after Asa, C.,
Phillips, D. M. and Stover, J. 1986. Journal of Ultrastructure and Molecular Structure
Research. 1986; 94(2): 170-175, Figs. 1, 2, 5-7. With permission from Elsevier.
Fig. 8.9 A. Longitudinal section (LS) showing the midpiece of a tinamou

spermatozoon. A, annulus; M, mitochondrion; F, fibrous sheath; G, glycogen; p,
proximal centriole. ¥ 17400. B. LS showing portions of three spermatozoa of
Thryothorus ludovicanus, the Carolina wren. The mitochondria (M) which spiral
around the flagellum (FLA) have been referred to as the undulating membrane.
Fig. 8.9 Contd. ...
Avian Spermatozoa: Structure and Phylogeny !&
They also considered the Tinamou spermatozoon to be unusual in that

there is a single distal centriole which extends the entire length of the
midpiece. However, this has also been described for Struthioniformes (see
Section 8.6.1), and for chelonians, and Sphenodon (Healy and Jamieson 1992;
Jamieson and Healy 1992) and therefore appears to be a symplesiomorphy of
Palaeognathae. Asa et al. (1986) observed that other birds such as fowl and
ducks also have long distal centrioles, but that the midpiece of spermatozoa of
these species is longer and the centriole only occupies the anterior portion of
the midpiece. However, it is here noted that in addition to being shorter than
the midpiece, the absolute length of the distal centriole in these is less than
that in Struthioniformes and Tinamiformes. The presence of glycogen, around
the fibrous sheath, appears to be unique to the Tinamou spermatozoon and
therefore an autapomorphy insofar as tinamiforms have been studied.
Soley (1993) reasonably considered that there is a trend to enlargement of
axonemal dense fibers in birds. He therefore considered Tinamou to be the
most primitive ratite in this respect in the erroneous belief that it lacked dense
fibers in contrast with the rudimentary dense fibers observed in the proximal
segment of the principal piece of Ostrich and Rhea (Phillips and Asa 1989)
sperm. Therefore consideration of dense fibers does not contribute to
phylogenetic analysis within the ratites.
Furthermore, although (Soley 1993) rightly recognizes a trend to reduction
in the length of the endonuclear canal and contained perforatorium in the
non-passerines, with their loss in passerines, it is by no means certain that the
fact that these structures are longer in tinamou than in struthioniforms
indicates that Tinamou is more primitive. Doubt is cast on the latter inference
by the fact that the canals and perforatoria are shorter in Crocodylia than in
struthioniforms.
As Soley (1993) argues, Ostrich and Rhea sperm appear to be more
primitive than those of Tinamou in respect of the structure of the distal
centriole. In these birds a central pair of microtubules occupies the centriolar
cavity, a feature which is typical of chelonian (Furieri 1970; Hess et al. 1991;
Healy and Jamieson 1992) and, we may add, crocodilian sperm (Jamieson et
al. 1997). Tinamou sperm (and, apparently, see Section 8.6.1.5, Emu sperm) are
said to lack this microtubular arrangement and to display an empty distal
centriole (Asa et al. 1986; Asa and Phillips 1987) as in other non-passerine
birds. However, central singlets appear to reach almost halfway into the
Fig. 8.9 Contd. ...

A, acrosome; AP, lateral projection of acrosome; D, dense fibers; ED, electron-
dense material of the neck; N, nucleus. ¥ 17400. C. Transverse section (TS)
through the principal piece of tinamou spermatozoa. E, endpiece; F, fibrous sheath;
G, glycogen. ¥ 48700. D. TS of spermatozoa from the vas deferens of the Carolina
wren. Viewed in cross section the dense fibers are approximately circular and are
similar to one another. M, mitochondria. ¥ 45900. After Asa, C. S. and Phillips, D. M.
1987. Pp. 365-373. In H. Mohri (ed). New horizons in sperm cell research, Japan
Science Society Press, Gordon and Breach Scientific Publications, Tokyo/New York,
Figs. 1-4.
Fig. 8.10 Drawings by light microscopy of spermatozoa of non-passerine birds.

Rooster (Gallus gallus); Tufted duck (Aythya fuligula); Domestic duck (Anas
platyrhynchos); Common guillemot (Uria aalge); Corncrake (Crex crex); Lesser
black-backed gull (Larus fuscus); Alpine dunlin (Calidris alpine); Green sandpiper
(Tringa ochropus); Lapwing (Vanellus vanellus); Woodcock (Scolopax rusticola);
Common coot (Fulica atra). After Retzius, G. 1909. Biologische Untersuchungen,
Neue Folge 14(10): 89-122 Taf XIX-XXXVII.
Avian Spermatozoa: Structure and Phylogeny !&!
centriole in Crested tinamou sperm (see Fig. 8.9A, above, from Asa and
Phillips 1987).
The phylogeny of ratites is discussed in Section 8.11.
8.7 NEOGNATHAE
According to Gauthier and de Queiroz (2001) “Neognathae” refers to the
crown clade stemming from the last common ancestor of Charadrius pluvialis
(Pluvialis apricaria) Linnaeus 1758 and all extant birds sharing a more recent
common ancestor with that species than with Struthio camelus Linnaeus 1758
and Tetrao (Tinamus) major Gmelin 1789. Neognathae consists of two primary
crown clades, Galloanserae and Neoaves.
8.8 GALLOANSERAE
This is the Parvclass Galloanserae of Sibley and Ahlquist (1990). Its
monophyly versus paraphyly are discussed by Harshman in Chapter 1.
Spermatozoal ultrastructure is consistent with monophyly.
The ancestor of the Galliformes and Anseriformes was presumably a
generalized form lacking the highly derived filter feeding apparatus of
Anseriformes.
8.8.1 Order Galliformes
8.8.1.1 Gallus gallus (=domesticus)

The spermatozoon of the rooster, Gallus gallus (= domesticus), Phasianidae, has
been described ultrastructurally (Grigg and Hodge 1949; Bonadona 1954;
Nagano 1960, 1962; McIntosh and Porter 1967; Nicander and Hillstrom 1967;
Krustev and Danov 1968; Lake et al. 1968; Nicander 1970b; Tingari 1973; Bakst
and Howarth 1975; Gunawardana and Scott 1977; Bakst and Sexton 1979;
Bakst 1980; Xia et al. 1985; Xia et al. 1986; Bae and Kim 1987; Thurston and
Hess 1987; Woolley and Brammall 1987; Sprando and Russell 1988; Xia et al.
1988; Jamieson 1999). The following account is based chiefly on a re-
examination. Reference is made to other accounts where they are not in
agreement or give additional or important supporting information.
General morphology. The Gallus spermatozoon (Figs. 8.11, 8.12, 8.16J, P, R)
has the usual non-passerine components in anterior-posterior sequence: an
acrosome vesicle; perforatorium (apical spine of Grigg and Hodge 1949);
acrosome spine of Lake et al. 1968; acrosomal spine of Tingari 1973) lying in
the subacrosomal space and extending posteriorly into an endonuclear canal,
here in the form of a deep anterior nuclear fossa; elongate nucleus; proximal
and distal centrioles; midpiece, consisting of mitochondria encircling the 9+2
axoneme and ending posteriorly at the annulus; principal piece, consisting of
the axoneme surrounded by a fibrous, here amorphous, sheath; and the
endpiece, consisting of the axoneme surrounded by the plasma membrane but
lacking a fibrous sheath. The endpiece contains a central dense ‘tip granule’
!&" Reproductive Biology and Phylogeny of Birds
Fig. 8.11 Gallus gallus. Transmission electron micrographs. A. Longitudinal section

(LS) of the acrosome and adjacent nucleus, showing perforatorium in the
subacrosomal space and extending into the nuclear fossa (endonuclear canal). B.
LS basal portion of nucleus, midpiece and anterior portion of principal piece. C. LS
Avian Spermatozoa: Structure and Phylogeny !&#
(terminology of Woolley 1995 for Coturnix), also observed in Gallus by Woolley

and Brammall (1987). Grigg and Hodge (1949) give a total length for the sperm
“head” (nucleus but contrary to usual definitions apparently excluding the
acrosome) of 14 µm and a diameter not exceeding 0.5 µm, and a length for the
sperm of more than 100 µm, c.f. 90 µm or more (Thurston and Hess 1987).
Acrosome. The acrosome vesicle is approximately 2.2 µm long (present study),
agreeing with 2 µm or greater (Thurston and Hess 1987). It has the form of an
elongate cone, asymmetry of which correlates with a slightly curved shape.
The base of the vesicle overlaps a short, abruptly narrowed anterior region of
the nucleus (Figs. 8.11A, C, 8.12A). In the large, conical subacrosomal space
which occupies a little more than the basal third of the acrosome, there lies a
dense rod, the perforatorium, ca 1.2 µm long (1.0 µm, Thurston and Hess
1987), the tip of which closely abuts the apex of the subacrosomal space.
Approximately the posterior fourth of the perforatorium is contained in, and
closely fits, the anterior nuclear fossa which is here considered the homologue
of the ratite endonuclear canal.
Amorphous material in the subacrosomal space is said to be continuous
with the perforatorium and to be part of it by Xia et al. (1988). This condition
is seen in the present study in Fig. 8.11C but may represent a dissolution of
the perforatorium as the latter is a distinct rod in the absence of amorphous
material, except that closely ensheathing the rod, in Fig. 8.11A.
Micrographs of cross sections at the base of the perforatorium of tannic
acid-fixed rooster sperm demonstrate the plasmalemma, inner and outer
acrosomal, and double nuclear membranes (Thurston and Hess 1987) (Fig.
8.16J).
Nucleus. The nucleus is an elongate cylinder, slightly tapering anteriad, with
a slight shoulder supporting the base of the acrosome vesicle (Fig. 8.11A, B,
C). It is slightly curved, at least in fixation. The chromatin is strongly
condensed and electron-dense. At its base there is a very shallow, slightly
asymmetrical basal (implantation) fossa.
Centrioles. The short proximal centriole lies at right angles to the long axis of
the spermatozoon, with its anterior region in the shallow implantation fossa
(Fig. 8.11B). It consists of 9 triplets embedded in a dense ring. It is linked by an
unstriated connecting piece to the base of the nucleus (Bakst and Howarth
1975), termed the non-striated connecting piece and considered to include the
proximal centriole by Thurston and Hess (1987). This is termed the capitulum

base of acrosome vesicle, perforatorium and anterior nuclear fossa. D. Transverse
section (TS) through anterior nuclear fossa, showing enclosed perforatorium. E.
TS nucleus, showing circular profile. F. TS midpiece, showing four mitochondria
and the axoneme with nine outer dense fibers. G. TS principal piece, distinguished
by presence of a fibrous (amorphous) sheath around the axoneme. H. TS
endpiece, distinguished by the axoneme lacking a fibrous sheath. I. TS terminal
region of endpiece, showing doublets undergoing disruption and central singlets
replaced by a dense ‘tip granule’. Original.
!&$ Reproductive Biology and Phylogeny of Birds
Fig. 8.12 Gallus domesticus. A. Longitudinal section of the acrosomal region. The
acrosomal vesicle overlaps a perforatorium which inserts into a nuclear concavity.
The perforatorium is not membrane bound and is surrounded by amorphous,
granular material. B. Longitudinal section of the centrioles and midpiece, the
proximal centriole (PC) is orientated at right angles to the distal centriole (DC)
which is surrounded by mitochondria (M). Bars: 0.1 µm. From Thurston, R. J. and
Hess, R. A. 1987. Scanning Microscopy 1: 1829-1839, Figs. 2b, 4b.
by Tingari (1973) who states that it forms in the epididymis by fusion of

previously existing dense masses and that in the excurrent ducts the
reduction in size of the central cavity of the proximal centriole may be due to
deposition of dense matrix.
The distal centriole, contiguous with the proximal centriole, lies in the
longitudinal axis of the sperm and is continuous with the axoneme. It is
surrounded by the most anterior mitochondria of the midpiece. Unlike the
condition in struthioniforms, the central singlets do not extend to the
proximal end of the centriole. It is considered that these commence at its
posterior end (Nagano 1962; Bakst and Howarth 1975; Gunawardana and
Scott 1977) but some micrographs suggest a considerable penetration of the
singlets into the distal end of the centriole (Fig. 8.12B). From a micrograph of
Bakst and Howarth (1975) the length of the distal centriole is ca 1.8 µm and it
extends for ca 0.4 of the length of the midpiece. It has nine triplets embedded
in a ring of dense material (Lake et al. 1968; Bakst and Howarth 1975; present
study). The distance from the proximal end of the distal centriole to the
commencement of the inner paired microtubules is 0.9 µm (Thurston and
Hess 1987).
Avian Spermatozoa: Structure and Phylogeny !&%
Tannic acid fixation reveals for the proximal (Fig. 8.16J) and distal
centrioles 13 protofilaments for subtubule A and 10 for each of B and C
(Thurston and Hess 1987).
In the round spermatid the two centrioles are said to be of the same size
and to lie end to end and almost in a straight line (Nagano 1962), the
condition seen in the mature Coturnix sperm. However, Xia et al. (1986) clearly
indicate a small proximal centriole at right angles to a long distal centriole in
the round spermatid, as confirmed here for the spermatozoon in Fig. 8.11B.
Midpiece. The midpiece is ca. 3.7 µm long (agreeing with 4 µm, Grigg and
Hodge 1949). It shows, in transverse section, four mitochondria encircling the
axoneme (Fig. 8.11F) and 7 or 8 along its length (Fig. 8.11B), totaling ca 28-32,
agreeing with approximately 30 according to Bakst and Howarth (1975). Their
arrangement is helical (Lake et al. 1968; Bakst and Howarth 1975; Thurston
and Hess 1987). The cristae form stacks of plates orientated longitudinally
(Figs. 8.11B, 8.12B). The mitochondria in tangential section appear as closely
fitting slightly elongate polygons (usually hexagons) (Bakst and Howarth
1975). The axoneme in the midpiece, posterior to the distal centriole, has nine
conspicuous dense fibers (osmiophilic masses of Bakst and Howarth 1975);
each fiber being in the radius of its doublet and enveloping the latter in its
inner extremity. The dense fibers do not extend into the principal piece (Lake
et al. 1968; Bakst and Howarth 1975; present study).
Annulus. A small, compact annulus marks the posterior limit of the midpiece
(Fig. 8.11B).
Principal piece. This commences at the annulus and is defined by the
presence, encircling the axoneme, of a fibrous sheath (Figs. 8.11B, G, 8.16P).
This is amorphous in that it does not show the annulation or ribbing seen in
ratites.
Endpiece. The endpiece (Figs. 8.11H, 8.16R) consists of the axoneme and
plasma membrane and lacks the fibrous sheath. As in the principal piece, the
A subtubule of each of the 9 doublets has dense contents, as first noted by
Nagano (1962), and the outer dynein arms are more conspicuous than the
inner arms. Posteriorly the two central singlets are replaced by a large dense
structure, the so-called ‘tip granule’ (Fig. 8.11I). At this level and posteriorly
the doublets are progressively disrupted. Location of the tip granule at the end
of the two central singlets and not terminally is deduced from the
observations of Woolley (1995) for Coturnix coturnix.
8.8.1.2Coturnix japonica
In the present account the valid name Coturnix japonica is used in place of
Coturnix coturnix, Coturnix coturnix japonica and Coturnix coturnix var. japonica
employed in the various accounts summarized. The spermatozoon of C.
japonica (Phasianidae) has been described by Marquez and Ogasawara (1975);
Saita et al. (1980); Maretta et al. (1982); Lin and Jones (1993) and Woolley
(1995). The dynamics of spermatozoal motility are described and illustrated
!&& Reproductive Biology and Phylogeny of Birds
Fig. 8.13 Coturnix japonica. Light microscopy of spermatozoon. A. Immotile

spermatozoon, wet preparation. Upper arrow indicates the midpiece-principal
piece boundary. Lower arrow, the distal limit of the principal piece and its sheath.
The long unsheathed more posterior region is here termed the endpiece. B.
Flagellum after trypsin digestion, showing helical midpiece. C. The acrosome,
nucleus, and proximal flagellum contrasted supravitally with eosin-yellow. After
Woolley, D. M. 1995. Acta Zoologica (Copenhagen) 76: 45-50, Figs. 1-3.
Avian Spermatozoa: Structure and Phylogeny !&'
by Vernon and Woolley (1999) who repeat the ultrastructural data of Woolley
(1995). The following account is drawn chiefly from Woolley (1995).
General morphology. Eosin-yellow in vivo staining of the spermatozoon
reveals a pointed conical acrosome many times shorter than the cylindrical
nucleus (Fig. 8.13C). Light micrographs of the flagellum revealed three zones
of decreasing thickness, later confirmed to be the midpiece, the proximal
(sheathed) principal piece and the “distal principal piece” (endpiece) (Fig.
8.13A). The mean overall length of the flagellum was 207.6 mm. The mean
lengths of the three zones (± one standard deviation) were: midpiece 161.4
(± 2.8) mm, proximal (sheathed) principal piece 5.4 (± 0.7) mm and endpiece
40.8 (± 1.7) mm (n = 10, a single bird).
Acrosome. The acrosome is conical, about 2.6 mm (Fig. 8.13C). The mode of
attachment of the acrosome (Fig. 8.14A) involves an overlapping joint, as is
usual in galliforms, with a perforatorium (length 1.5 mm) engaged in conical
depressions in both the acrosome and the nucleus, and connected to each
through a granular matrix.
Nucleus. The sperm nucleus is a curved cylinder, 20.6 mm long. The nuclear
envelope is thickened where it lies against the inner acrosomal membrane.
Centriolar region. The neck of the spermatozoon contains two separate
centrioles lying almost on the same axis. The proximal centriole is thickened
abaxially to support an implantation plate; distally it ends in an annular
thickening into which the base of the longer distal centriole is anchored (Fig.
8.14F-H). The triplet microtubules are not continuous between the two
centrioles (Fig. 8.14F). Beyond the distal centriole there is a transition region
characterized by the appearance of the central pair, by electron dense material
peripheral to the nine doublets and by additional densities associated with
the radial spokes, showing their 96 nm periodicity (Fig. 8.14F, I). The
transition region is within the mitochondrial sheath.
Midpiece. Each mitochondrion is disc-shaped, diameter about 0.25 mm, and
curved against the axoneme, with the packing of adjacent ones often making
the profiles slightly hexagonal (Fig. 8.14J). Transverse sections typically show
four mitochondria at any level (Fig. 8.14K). The arrangement can be modeled
as four parallel ‘out-of-register’ chains of mitochondria surrounding the
axoneme. The total number of mitochondria per sperm is estimated as
ca 2,500. The midpiece terminates at a thin annulus (Fig. 8.14M).
Principal piece. The short “proximal principal piece” consists of the axoneme
encased in a cylindrical fibrous sheath that tapers distally (Fig. 8.14N).
Beyond this sheath, the axoneme is simple (Fig. 8.14O). Although the fibrous
sheath is amorphous in the mature sperm, it develops as a series of
circumferentially orientated hoops in the stage 2 spermatid (Lin and Jones
1993).
Endpiece. The endpiece exceeds 1.5 mm in length. In it the central pair of
microtubules terminates first. Posterior to this, for about 0.4 mm, the center of
!' Reproductive Biology and Phylogeny of Birds
Fig. 8.14 Coturnix japonica. TEM of sperm. A. Longitudinal section (LS) through
the junction between the acrosome vesicle (av) and the sperm nucleus (n). The
perforatorium (p) inserts into each structure. B. Transverse section (TS) of nucleus,

Avian Spermatozoa: Structure and Phylogeny !'
the axoneme is occupied, as in Gallus, by an electron dense body tip granule

(Fig. 8.14L, P). Thereafter the nine doublets become progressively simplified
and end as singlets attached to the plasmalemma (Fig. 8.14Q, R) (Woolley
1995).
Remarks. Woolley (1995) gives an interesting discussion of the sperm of
Coturnix coturnix, some points of which will be discussed here. The
morphology of the acrosome and sperm nucleus is typical of galliform birds.
Furthermore a conical acrosome, with a perforatorium occurs in most of the
other non-passerine orders that have been examined ultrastructurally
(reviewed by Asa and Phillips 1987).
As Woolley (1995) notes, the ultrastructure of the sperm neck in Coturnix
has an unusual feature. In spermatozoa generally, there are two centrioles (at
least, during development): a distal centriole that gives rise to the flagellum
and a proximal one at right angles to it. This arrangement is also general in
birds. In Coturnix japonica, however, the two centrioles lie almost in line. This
condition is also seen in C. chinensis (8.8.1.3) and Numida meleagris (8.8.1.6).
As also noted by Woolley (1995), the general features of the flagellum in
Coturnix japonica spermatozoa are qualitatively typical of non-passerine birds.
Its dimensions, however, are unusual. First, at 208 mm, the flagellum is more
than twice as long as in the other galliforms noted. In passerine bird sperm
the flagella range up to 263 mm in length, in Dendroica petechia, the yellow
warbler (Briskie and Montgomerie 1992). Flagellar length is useful
taxonomically within avian genera, as shown for Dendroica by McFarlane
(1963). The significance, taxonomic or physiological, of the flagellar
which has a circular profile at all levels. C. TS of the region where acrosome and
nucleus are interlocked. D. TS of caudal acrosome, showing the perforatorium
centrally. E. TS of rostral acrosome. F. LS through the neck region, which consists
of a proximal centriole (pc), a distal centriole (dc) and a transitional region with
periodic densities suggestive of mechanical re-enforcement. G. TS through one of
the centrioles, probably the distal one. H. A further LS of the neck to show the
separate identity of the centrioles, indicated by their lack of continuity and slightly
different axes. I. TS of the transition region. The microtubular triplets have been
reduced to doublets, a central singlet has appeared and there are extra densities
both peripherally and centrally. J. Tangential section of the midpiece to show the
shape and arrangement of the mitochondria. K. TS of midpiece showing the most
common arrangement of the mitochondria. L. LS of the flagellar tip showing
particularly the tip granule. M. LS showing annulus (arrows) at the distal limit of the
midpiece. N. TS proximal principal piece, where fibrous sheath occurs between
the axoneme and the cell membrane. O. TS distal principal piece. P. TS flagellum
at the level just beyond the termination of the central pair, showing the central tip
granule (tg). Q, R. TS showing progressive reduction of the axoneme in the
flagellar tip. After Woolley, D. M. 1995. Acta Zoologica (Copenhagen) 76: 45-50,
Figs. 4-21.
!' Reproductive Biology and Phylogeny of Birds
elongation in Coturnix is unknown. However, the physiological explanation

may lie in the positive correlation shown by Briskie and Montgomerie (1992)
between sperm flagellar length in birds and the length of the sperm storage
tubules in the female of the species.
The length of the midpiece in Coturnix japonica is noteworthy (Woolley
1995) and requires confirmation. Thurston and Hess (1987) estimated that the
turkey, the domestic fowl and the Guineafowl all have 25-30 mitochondria in
each spermatozoon (confirmed here for Gallus). For Coturnix, Saita et al. (1980),
using electron micrographs of testicular tissue, estimated ca 350 mitochondria
per sperm. The estimate of midpiece length (161 mm) in Coturnix japonica
(=Coturnix coturnix var. japonica) by Woolley (1995) was made from light
micrographs, supported by the very high frequency of midpiece profiles seen
in the thin sections. This length measurement led to an estimate of
mitochondrial number of ca 2500 per sperm. This would suggest a surprising
616 tiers of mitochondria. The only report of a midpiece approaching this
length in non-passerine sperm is for the Order Columbiformes. In contrast, in
the Passerida the mitochondria make a single spiral thread along much of the
axoneme (reviewed by Asa and Phillips 1987, and this chapter).
8.8.1.3 Coturnix chinensis
General morphology. The ultrastructure of the spermatozoon of Coturnix
chinensis, the Blue-breasted quail (Fig. 8.15A-L) is closely similar, excepting
some dimensions, to that of Gallus and, particularly, of Coturnix japonica. It has
the usual non-passerine components described above for Gallus. By light
microscopy, approximate dimensions are: total length of the spermatozoon 91
µm; head (acrosome + nucleus) 9.7 µm; midpiece 24.4 µm (n=1).
Acrosome. The acrosome vesicle, by TEM, is approximately 1.9 µm long (n=2).
It has the form of an elongate, straight cone. The base of the vesicle overlaps a
short, narrower anterior region of the nucleus (Figs. 8.15C, E). The nuclear
shoulders are rounded and not as distinct as in Gallus. In the large, conical
subacrosomal space which occupies more than half the length of the acrosome
vesicle, there lies a dense rod, the perforatorium, ca 1.3 µm long. This agrees
with C. japonica and Gallus in abutting the apex of the subacrosomal space but
differs from that of Gallus in more closely fitting the sides of the space, there
being a relatively thin later of granular material between it and the vesicle
laterally. Approximately the posterior third of the perforatorium is contained
in, and closely fits, the anterior nuclear fossa (reduced endonuclear canal).
Nucleus. The nucleus is an elongate cylinder, ca 6.4 µm long (cf 20.6 µm for C.
japonica), circular in cross section (Fig. 8.15B), slightly narrowing anteriad, but
conspicuously clubbed basally on one side (Fig. 8.15C). Seen in the plane of
the clubbing (Fig. 8.15C, F) the nucleus is tilted at an angle to the long axis of
the midpiece and flagellum. In a plane at right angles to this (Fig. 8.15G)
clubbing is absent and the nucleus appears to be in the long axis. The
chromatin is homogeneous and electron-dense (Figs. 8.15C, F, G, H). Where it
Avian Spermatozoa: Structure and Phylogeny !'!
appears granular (Fig. 8.15E) this may be due to incomplete maturity, as a

transition from granular to condensed is noted by Maretta et al. (1982) for C.
japonica. At its base there is a very considerable, slightly asymmetrical basal
(implantation) fossa (Figs. 8.15F, G, H). As in C. japonica, the extreme tip of the
nucleus is attenuated around the anterior half of the anterior fossa (Fig.
8.15E), being thinner on each side than in Gallus, and is asymmetrical so that
in transverse section it may appear interrupted on one side (Fig. 8.15A).
Centriolar region. The neck of the spermatozoon contains two separate
centrioles lying almost on the same axis (Fig. 8.15G, D), as in C. japonica. The
triplet microtubules are not continuous between the two centrioles. The
posterior limit of the distal centriole is difficult to define but appears to be
three or four times the length of the proximal centriole. It is penetrated for
about half of its length by a central axonemal element. This consists of a dense
sheath around and capping the two central singlets (Fig. 8.15G). At the level
of the tip of this sheath, the nine triplets of the centriole lie in a dense ring and
their triplet structure is almost obscured though persistent (Fig. 8.15I). The
ring is surrounded by the mitochondria of the midpiece.
Midpiece. Transverse sections of the midpiece typically show four
mitochondria at any level (Fig. 8.15I, J). The length of the midpiece has not
been determined by TEM, nor has its posterior limit been sectioned. Small
outer dense fibers, one in the radius of and contiguous with, each doublet are
present in the midpiece (Fig. 8.15J); in some sections they are considerably
larger than those shown in this figure.
Principal piece. This is defined by the presence, encircling the axoneme, of a
fibrous sheath (Fig. 8.15K). This is amorphous in that it does not show the
annulation or ribbing seen in ratites.
Endpiece. The endpiece (Fig. 8.15L) consists of the axoneme and plasma
membrane and lacks the fibrous sheath. As in the principal piece, the A
subtubule of each of the 9 doublets has dense contents. A tip granule has not
been demonstrated in the few sections obtained.
Remarks. The difference in length of the nucleus between Coturnix coturnix
and C. chinensis, 20.6 µm versus 6.4 µm is remarkable. Even more disparate is
the midpiece length of 161 µm contrasting with approximately 24 µm
determined by light microscopy for C. chinensis. The two species agree, and
differ from Gallus, in the much greater length of the midpiece, and in the
almost co-linear arrangement of the proximal and distal centrioles, the
proximal centriole being at right angles to the distal in Gallus as in other birds
studied, with the exception of Numida meleagris.
The clubbing and angular deflection of the nucleus in C. chinensis is echoed
in a light micrograph by Woolley (1995) of the sperm of C. japonica (Fig. 8.13A)
but may reflect a dynamic situation as it is not evident in a second micrograph
(Fig. 8.13C).
!'" Reproductive Biology and Phylogeny of Birds
Fig. 8.15 Coturnix chinensis. TEM of sperm. A. Transverse section (TS) acrosome
through the tip of the nucleus, showing asymmetry of the anterior nuclear fossa
containing the base of the perforatorium. B. TS nucleus showing circular profile.

Avian Spermatozoa: Structure and Phylogeny !'#
Fig. 8.15

C. LS of the entire acrosome and nucleus, with centriolar and anterior midpiece. D.
Detail of centriolar region, showing proximal centriole almost in line with the distal
!'$ Reproductive Biology and Phylogeny of Birds
8.8.1.4 Meleagris gallopavo

The spermatozoon of the turkey, Meleagris gallapavo (Phasianidae), has been
described ultrastructurally (Marquez and Ogasawara 1975; Bakst and Sexton
1979; Baccetti et al. 1980; Bakst 1980; Bradley et al. 1986; Thurston and Hess
1987). The account of Thurston and Hess (1987) gives a valuable comparison
of the sperm of Turkey, Guineafowl and rooster and is summarized here for
the turkey, with some reference to the other species and other accounts.
General morphology. As shown by SEM, the general shape of turkey,
Guineafowl (‘guinea’) and rooster spermatozoa is remarkably similar. As in
Gallus, the spermatozoa are long and narrow with a vermiform appearance,
and an acrosome, nucleus, midpiece (Fig. 8.16A). The nucleus is usually
curved. The anterior end of the sperm consists of a conical acrosome which is
most prominent in Guineafowl sperm (Fig. 8.16B). The acrosome of the turkey
is 1.0-2.6 µm (mean 1.8) µm long (Marquez and Ogasawara 1975), compared
to 2 µm or greater (Thurston and Hess 1987), reaching 2.5 µm (Marquez and
Ogasawara 1975) for the rooster acrosome. The nucleus gradually increases
in diameter from its junction with the acrosome to its distal end at the
beginning of the midpiece. The turkey sperm nucleus is said to be shorter (7 to
9 µm) than that of Guineafowl or rooster (10 to 14 µm in length) (Thurston
and Hess 1987). However, there is considerable overlap between species as
Marquez and Ogasawara (1975) give a length of 7.2-11.0 µm, with a mean
width of 0.8, for the turkey. Junction of the nucleus with the midpiece at the
neck region is not as conspicuous as in Guineafowl sperm (Thurston and
Hess 1987).
The flagellum comprises most of the length of the spermatozoon, although
the junction between the principal and end piece could not be discerned with
SEM. Turkey and Guineafowl sperm flagella are usually 60-65 µm long
(Thurston and Hess 1987) (61 µm, Marquez and Ogasawara 1975), cf often
more than 70 µm in rooster spermatozoa. The overall sperm length of 75-80
µm in turkey and Guineafowl sperm is also less than that of rooster sperm (90
µm). For all three species, the spermatozoa increased in width from the
acrosome to a maximum of 0.5-0.7 µm at the junction of the nucleus with the
midpiece. The width then decreased to 0.1-0.2 µm at the end of the flagellum
(Thurston and Hess 1987).

centriole. E. LS acrosome and adjacent nucleus. F. LS base of nucleus, centriolar
region and anterior midpiece. G. Same, in a plane approximately at right angles.
H. Same but showing some mitochondria around the nucleus. I. TS distal centriole,
within the midpiece, and through dense sheath at commencement of central
singlets. J. TS midpiece and contained axoneme showing minute outer dense
fibers. K. TS principal piece, showing amorphous sheath surrounding axoneme.
L. TS endpiece. Original.
Avian Spermatozoa: Structure and Phylogeny !'%
Acrosome. As seen by TEM, the membrane-bound cap-like acrosomal vesicle

(Fig. 8.16C, D) contains a granular, amorphous material which surrounds the
perforatorium, and adjacent to the perforatorium is fine, granular material of
moderate density (more abundant in rooster spermatozoa. At its distal end,
the acrosomal cap encircles projections of chromatin from the apical portion
of the nucleus. At its posterior end the perforatorium inserts into a concavity
of the nucleus and extends obliquely forward approximately half the length of
the acrosomal cap in turkey as in rooster, contrasting with nearly the entire
length of the cap in Guineafowl sperm. Thus, the perforatorium of the turkey
and rooster, at 1.0 µm, is appreciably shorter than that of the Guineafowl (1.9
µm). The base of the perforatorium of turkey is narrower than that of rooster
and Guineafowl sperm (Fig. 8.16A).
The substance of the perforatorium is dense and amorphous, and often
interrupted by lucent channels which contain granular material similar to
that adjacent to the perforatorium (Fig. 8.16D, E).
Nucleus. A longitudinal section of the base of the nucleus and anterior portion
of the midpiece is shown in Fig. 8.16F. The nuclear chromatin is dense and
granular with occasional small lucent areas giving it a mottled appearance
(Fig. 8.16G). The distal end of the nucleus terminated in a concavity, the
implantation fossa (Fig. 8.6F, H).
Centrioles and neck. For turkey (Figs. 8.16F, H) and rooster sperm, dense
processes extended radially from the proximal centriole wall to abut against
the nuclear membrane in the implantation fossa. The centriole complex plus
the projections constitute the non-striated connecting piece of the neck of the
spermatozoon (Bakst and Howarth 1975). Turkey and rooster have a proximal
centriole orientated perpendicular to the distal centrioles (contrast
Guineafowl below).
In Numida, as for all three species, cross sections of the centrioles have the
typical ‘pinwheel’ arrangement of nine triplet microtubules embedded in a
cylindrical, dense wall (Fig. 8.16H). Each projection of the non-striated
connecting piece is associated with one set of the triplet microtubules (Fig.
8.16H).
Midpiece. From SEM micrographs of turkey sperm (Fig. 8.16D) and those of
midpiece cross sections (Fig. 8.16K) where the mitochondrial length varied
progressively from long to short, it is ascertained (Thurston and Hess 1987)
that the midpiece had 25-30 mitochondria arranged in an helical pattern.
Marquez and Ogasawara (1975) observed lengths for the midpiece of 4.0-6.0
µm; plate-like mitochondria numbering four per turn surround the axoneme;
this arrangement is repeated seven times to give approximately 28
mitochondria per midpiece. In surface view (Thurston and Hess 1987), the
mitochondria are polygonal with the dimensions of approximately 0.8 ¥ 0.11
¥ 0.3 µm. In contrast to Guineafowl, cristae of turkey and rooster sperm
mitochondria are parallel to the outer membrane (Fig. 8.16F).
!'& Reproductive Biology and Phylogeny of Birds
Fig. 8.16 A-H, K-M, O, Q-T. Meleagris gallopavo. Turkey spermatozoa. I, N. Numida
meleagris, Guineafowl. J, P, R. Gallus gallus, Rooster spermatozoa. A, B. SEM of
turkey sperm. A. The narrow, vermiform shape of the turkey spermatozoon is typical
Avian Spermatozoa: Structure and Phylogeny !''
Stages in transition from the distal centriole caudally are as follows

(Thurston and Hess 1987) (Fig. 8.16K-M). The distal centriole microtubules
come to lie in a circle and the dense wall material disintegrates (Fig. 8.16K).
Central, singlet microtubules then become visible (Fig. 8.16L). Further
caudally, the 9 + 2 microtubular pattern of the axoneme is visible and then
(Fig. 8.16M) vestiges of dense material remain with the A doublet microtubule
and (not mentioned) dense fibers are present. The centriole has a lucent center
mottled with sparse granular material which extends from the apical end of
the centriole caudally to the origin of the inner paired microtubules (Fig.
8.16F) of the axoneme. This distance varied, being 2.2, 0.9 and 0.65 µm for
turkey, rooster and Guineafowl sperm, respectively.

for sperm of non-passerine birds. Apical tip = acrosome, N, nucleus, M, midpiece,
F, flagellum. B. A damaged turkey sperm with a denuded midpiece demonstrates
helical arrangement of mitochondria (M). N, nucleus. Bars: A 5 µm; Fig. B 0.5 µm.
C. Acrosome vesicle (AV) ensheathing the perforatorium which projects into the
short endonuclear canal. Bar: 0.1 µm. D, E. The perforatorium has lucent areas (L)
containing granular material. Transverse section of a turkey sperm nucleus. Dense
chromatin (NC) granules are surrounded by lucent areas. Bars: D 0.1 µm, E 0.05
µm. F. Anterior portion of the midpiece; the proximal centriole is at right angles to
the long distal centriole. M: mitochondria. G. TS nucleus. Dense chromatin (NC)
granules are surrounded by lucent areas. H. The non-striated connecting piece
consists of dense projections from the proximal centriole (PC) in turkey sperm and
I. what appears to be the distal centriole (DC) in Guineafowl sperm. One projection
is associated with one set of the nine triplicate centriolar microtubules. Bars: F-H,
I 0.1 µm. J. Triplet microtubules of the proximal centriole of rooster sperm fixed
with tannic acid. The tubulin protofilaments are visible: 13 for microtubule A, 10 for
B and C. Bar: 0.05 µm. K-M. Turkey sperm midpiece at progressively distal levels.
K. The distal centriole microtubules become circular and the dense wall material
disintegrates (DC lumen of the distal centriole). L. Central, singlet microtubules
then become visible (arrow). M. Further caudally, appearance of the 9 + 2
microtubular pattern of the axoneme. Vestiges of dense material remain with the A
doublet microtubule (arrow). M – mitochondria. N. Annulus (arrows) at the
termination of the Guineafowl sperm midpiece and beginning of the flagellum.
Bars in K-N 0.1 µm. O-R. Figs. O and Q represent turkey sperm flagella fixed while
motile. The axonemal doublets have a complete, dense A (A) microtubule
connected to dynein arms (D) and radial links (R), and an incomplete, lucent B
microtubule (B). The outer amorphous sheath (Fig. O) disappears in the distal end
of the flagellum (Fig. Q). The inner matrix is coalesced in rooster flagella fixed after
being immobilized by hypertonicity (Fig. P), obliterating the radial links, but the
double microtubules, with 73 protofilaments in A and 10 in B, are intact (Fig. R, A
and B; see also inset above). Bars: O-R 0.05 µm. S, T. LS of turkey sperm
flagellum. S. The central tubules are bridged by material spaced 12 nm (arrow). T.
Axonemal microtubules extend to the end of the flagellum. Bars 0.1 µm. Adapted
from Thurston, R. J. and Hess, R. A. 1987. Scanning Microscopy 1: 1829-1839,
Figs. 1a, d; 2a; 3a, b; 4a; 5a-c; 7a-d; 8a-d; 9a, b.
Cross sections of the flagella showed typical 9 + 2 microtubular axonemes

(Fig. 8.16O). As in other galliforms, the A microtubule of the outer doublets is
completely circular and filled with dense material. Surrounding the outer
doublets is an amorphous sheath (Lake et al. 1968), defining the principal
piece.
Endpiece. The portion of a flagellum in which the cell membrane is in
juxtaposition to the doublet microtubules, in the absence of the amorphous
sheath, is the end piece (Fig. 8.16Q). The dense A microtubule of the doublet
extends uninterrupted along the length of the flagellum (Fig. 8.16S, T), but
doublet microtubules probably become single near the end of the flagellum as
shown for rooster.
Remarks. The structure of the sperm of the turkey is typical of the
Galloanserae.
8.8.1.5 Tragopan caboti
The spermatozoon of Cabot’s tragopan, Tragopan caboti (Phasianidae) has
been described ultrastructurally by Wen et al. (1997). The following account is
augmented by examination of their micrographs.
General morphology. The ultrastructure of the spermatozoon of Cabot’s
tragopan is similar to that of the rooster.
Acrosome. The conical acrosome is located in front of the nucleus, its caudal
part slightly overlaps the anterior end of the nucleus. The perforatorium
(‘acrosomal spine’) lies in the subacrosomal space with its base in the anterior
nuclear fossa (‘nuclear pocket’). It is not homogeneous in electron density.
Nucleus. The nucleus narrows abruptly for a short length anteriorly and
forms shoulders supporting the base of the acrosome vesicle. It is a slender
cylinder widened basally and has a length of ca 12 µm. The implantation
fossa is occupied by the centriolar complex and non-striated connecting piece,
which makes up the neck of the spermatozoon.
Centrioles. The short proximal centriole is at right angles to the long axis of
the sperm. The distal centriole, contiguous with it, is in the long axis and is
elongate, being ca 1.3 µm long, approximately half the length of the midpiece.
It continues as the axoneme. It is not penetrated by the central axonemal
singlets.
Midpiece. In transverse section of the midpiece, there are a few mitochondria
encircling the axonemal complex. The axonemal complex has the typical 9+2
structure. There is said to be no annulus between the midpiece and principal
piece but this requires confirmation; in micrographs a typical annulus is not
distinguishable, though presence as a rudiment cannot entirely be rejected.
Within the midpiece the doublets have each an outer dense fiber.
Principal piece. The principal piece is demarcated by the presence of an
amorphous fibrous sheath surrounding the axoneme. The A subtubule of each
doublet has dense contents as in the endpiece.
Avian Spermatozoa: Structure and Phylogeny "
Endpiece. The endpiece is demarcated by absence of the fibrous sheath.

Terminally, the typical axonemal sequence gradually becomes disorderly and
unsystematic and the A subtubules have lost their dense contents. The B
subtubules become open crescents in transverse section. There is no evidence
for the tip granule seen in Gallus sperm.
8.8.1.6 Numida meleagris
The spermatozoon of the Guineafowl (Numida meleagris, Numididae), has been
described ultrastructurally (Thurston et al. 1982; Hess et al. 1986; Thurston
and Hess 1987; Aire and Soley 2003).
General morphology. The general structure of the Numida spermatozoon
(Fig. 8.17A, B) is similar to that described for turkey and rooster spermatozoa.
The apex of the spermatozoon consists of a conical acrosome ca 1.8 mm long
and 0.47 mm wide at the point of articulation with the nucleus (Fig. 8.18A, B).
The acrosome is a homogenous, membrane-bound, “cap-like” structure
superimposed on a lanceolate spine (here termed the perforatorium), and
Fig. 8.17 Numida meleagris, Guineafowl. A. Scanning electron micrograph of the

entire spermatozoon. B. Transmission electron micrograph of a longitudinal
section through the posterior nucleus to anterior tail. Approximately six
mitochondria span the length of the midpiece. A, acrosome; AX, axoneme; N,
nucleus; M, midpiece; T, flagellum. Thurston, R. J., Hess, R., Hughes, B. L. and
Froman, D. P. 1982. Poultry Science 61: 1738-1743.
Fig. 8.18 Numida meleagris, Guineafowl. A. SEM of sperm apex. ¥ 22000. B. TEM
of a longitudinal section (LS) of the acrosome and anterior nucleus. ¥ 42000. C.
SEM midpiece; arrow marks articulation between nucleus and midpiece. ¥ 19000.
Avian Spermatozoa: Structure and Phylogeny "!
distally it extends over the nuclear chromatin (Fig. 8.18B). The nucleus and
acrosomal cap are concentrically contiguous where they overlap (Fig. 8.18G)
(Thurston et al. 1982). The total length of the spermatozoon is 75-80 µm,
compared with 90 µm or more for the rooster (Thurston and Hess 1987).
Perforatorium. The perforatorium (Fig. 8.18B) is similar to, but longer than,
that of rooster sperm but it was not discerned whether the perforatorium was
hollow as in turkey spermatozoa. It is 1.9 µm long, versus 1.0 µm for turkey
and rooster. In most spermatozoa, the perforatorium is inserted into a deep
anterior nuclear concavity or fossa (here considered a reduced endonuclear
canal) and projects angularly, terminating near the end of the cap (Fig. 8.18B).
Granular material separates the perforatorium from acrosomal material and
nuclear chromatin (Fig. 8.18F, H) (Thurston et al. 1982; Thurston and Hess
1987).
Nucleus. Distal to the acrosome is the nucleus, approximately 12.8 mm long
and 0.49 mm wide (Figs. 8.17A, 8.18A). The plasmalemma and nuclear
membranes are not distinguishable as they are intermeshed to form a wavy
network (Fig. 8.18E, I). However, a double nuclear membrane is discernible in
the subacrosomal space and where the perforatorium inserts into nuclear
fossa (Fig. 8.8B, G). At the distal end of the nucleus there is an implantation
fossa (Fig. 8.18E).
Midpiece. The midpiece, approximately 3.9 mm long, and 0.59 mm wide,
consists of a distal centriole and anterior axonemal complex circumferentially
encased by helically arranged mitochondria and the plasmalemma (Figs.
8.17B, 8.18D). The 25-30 mitochondria project from distal extensions of the
nucleus caudally to the annulus (Fig. 8.18D, E), which marks the terminus of
the midpiece. They are said to be arranged in a helix. This is consistent with
the arrangement of mitochondria in rooster and turkey sperm (Thurston et al.
1982; Thurston and Hess 1987).
Outlines of mitochondria are visible on the surface of the midpiece as a
result of possibly artefactual depressions (Fig. 8.18C). In most species,

D. TEM distal midpiece. E. TEM of LS of distal midpiece and anterior tail; arrow
indicates concave implantation fossa. ¥ 34700. F. Transverse section (TS) of
acrosome tip ¥ 42000. G. TS region where distal extension of acrosome vesicle
overlaps the nucleus ¥ 33600. H. TS. Where perforatorium projects into the
nucleus. ¥ 42000. I. TS of the nucleus. J. TS distal centriole. ¥ 42000. K. TS of
midpiece immediately distal to point of origin of the central microtubules of the
axoneme; arrow indicates dense fiber. ¥ 42000. L. TS flagella at their proximal (P)
and distal (D) ends. ¥ 42000. Abbreviations: A, acrosome; An, annulus; Ar, arms
extending from the dense material of the centriole and terminating in juxtaposition
to the nuclear membrane; B, double bridge across central microtubules; C,
acrosome vesicle; CM, central singlet microtubules of axoneme; DC, distal
centriole; M, midpiece and mitochondria; N, nucleus; S, perforatorium; Sp, spaces
between mitochondria. Partly relabeled after Thurston, R. J., Hess, R., Hughes, B.
L. and Froman, D. P. 1982. Poultry Science 61: 1738-1743, Figs. 1-16.
including roosters and turkeys, the cristae of germinal cell mitochondria are
arranged parallel to the outer limiting mitochondrial membrane, but in
Guineafowl sperm, they are both parallel and obliquely aligned (Fig. 8.18D, E,
K). A fine, granular amorphous material occupies the cisternae created by the
cristae, often making it difficult to resolve the inner membranes (Fig. 8.18K).
Usually six mitochondria are visible in longitudinal section (Fig. 8.17B)
and four in cross section (Fig. 8.18K), totaling 24 for the spermatozoon. Their
three-dimensional structure is probably that of a polygonal plate as for rooster
spermatozoa (Bakst and Howarth 1975), but dimensions measured from cross
and longitudinal sections are approximately 0.6 mm ¥ 0.4 mm ¥ 0.16 mm. The
distal termination of the midpiece is marked by an annulus which appeared
dense and triangular in longitudinal sections (Fig. 8.18D). Guineafowl sperm
differ from those of turkey and rooster in having mitochondrial cristae that are
often oblique and the inner matrix is more dense.
Centriole. Although rooster and turkey spermatozoa contain a proximal
centriole orientated transversely to the distal centriole, (Thurston et al. 1982)
states that in the Guineafowl spermatozoon, the midpiece contains only a
distal centriole (Fig. 8.18E). However, it was later considered that in-line
orientation of a proximal centriole with the distal centriole could not be
discounted (Thurston and Hess 1987) and presence of both centrioles in line
has been confirmed by Aire and Soley (2003). The non-striated connecting
piece of the Guineafowl spermatozoon consisted of projections originating
from the wall of the distal centriole. To accommodate this arrangement, the
implantation fossa is deeper and more curved, the caudal end of the nucleus
forming a semicircular concavity (Fig. 8.18E).
Dense material similar to the nonstriated connecting piece of rooster and
turkey sperm projects laterally as “arms” from the wall of the distal centriole
toward the nuclear membrane (Fig. 8.18J). Although not clearly discernible,
there appears to be one “arm’’ per group of three centriolar microtubules.
When the region of the implantation fossa is sectioned transversely, the nine
groups of three microtubules of the distal centriole, arranged in a “pinwheel”
fashion, are observed embedded in dense material contiguous with the “arms”
of the nonstriated connecting piece (Fig. 8.18J). For rooster and turkey
spermatozoa, where the centrioles are mutually perpendicular, such a section
would longitudinally bisect the proximal centriole and connecting piece. The
distance from the apical end of the centriole to the commencement of the
central singlets is 0.65 mm (Thurston and Hess 1987).
Flagellum. The flagellum averages 59 mm (Thurston et al. 1982) but reaches 65
mm (Thurston and Hess 1987) in length and 0.52 mm wide (at the junction with
the midpiece), and its ultrastructure is similar to that described for rooster and
turkey spermatozoa. As in those species, dense outer fibers are absent from
the axoneme. The central region of the A doublet microtubule retains dense
material throughout the flagellum similar to that of the centriolar wall (Fig.
8.18L). A wide amorphous fibrous sheath (Fig. 8.18L) surrounds the axoneme
Avian Spermatozoa: Structure and Phylogeny "#
in the principal piece (Thurston and Hess 1987). An endpiece is illustrated

(Fig. 8.18L) demarcated by absence of the fibrous sheath.
The central singlet microtubules originated from the basal granule (distal
centriole) approximately 1.0 mm from the implantation fossa (Fig. 8.18E). Thus,
the distal centriole in guinea spermatozoa is stated by Thurston et al. (1982) to
be shorter than of rooster or turkey sperm. From the illustration this appears
to be the case but it must be remembered that in ratites the central singlets
deeply penetrate the distal centriole.
At the terminus of the endpiece the doublets “probably become singlets”.
Remarks. The spermatozoon of Guineafowl is closely similar to, but shorter
than, that of the rooster and Turkey. The distal centriole, or at least the length
from the apical end of the centriole to the commencement of the central
singlets, is shorter, at 0.65 mm, compared with 2.2 mm and 0.9 mm for Turkey
and rooster respectively (Thurston and Hess 1987). As the terminal region of
the endpiece is not described it is not known whether the central “tip granule”
seen in Gallus and Coturnix is present.
8.8.2 Order Anseriformes

Ultrastructural accounts of anseriform sperm are those for Anas platyrhynchos,
Mallard (Humphreys 1972; Maretta 1975a, b) and Branta sandvicensis,
Hawaiian goose (Humphreys 1972).
Light microscope accounts exist for the spermatozoa of Tadorna tadorna,
Common Shelduck (Ballowitz 1888), Anas platyrhynchos, Mallard, Aythya
fuligula, Tufted duck (Retzius 1909) (Fig. 8.10), and Somateria mollissima, Eider
(McFarlane 1963) (Fig. 8.27C).
General morphology. Like the Galliformes, Trogoniformes and
Charadriiformes, inter alia, the anseriform sperm, as seen by light microscopy,
is not helical and the acrosome and midpiece are shorter than the nucleus. As
in galliforms, the acrosome is conical and the midpiece only a small fraction
of the length of the elongate nucleus (Ballowitz 1888; Retzius 1909; McFarlane
1963).
8.8.2.1 Anas platyrhynchos
The following ultrastructural account is taken chiefly from Maretta (1975a, b)
but employs current terminology.
The drake spermatozoon (Fig. 8.19A-M) has the form of a cylinder, apically
terminated by the pointed acrosome. The length and diameter [of the head]
range within 10-11 mm and 0.6-0.7 mm, respectively.
Acrosome. The acrosome vesicle (Fig. 8.19A-D) consists of homogeneous
material of moderate density surrounded by a single membrane. It is inverted
V-shaped and reaches 2.2 mm in length. The thickness of its wall is 0.1-0.2 mm,
being thinnest at the site where the nucleus is attached. The perforatorium
(termed the acrosome spine), composed of electron-dense material, is needle-
shaped, reaching an average length of 2.6 mm and a diameter of 0.2 mm. Of this
length, 1 mm lies in an invagination (anterior nuclear fossa or endonuclear
Fig. 8.19 Anas platyrhynchos, Mallard. A. Longitudinal section (LS) of an acrosome.

The acrosome vesicle (AC) covers the perforatorium (AS). Along the inner part of
the perforatorium vacuoles are seen. The space between the perforatorium and
the vesicle is filled with granular material (GM). B-E. Transverse sections (TS) of an
acrosome at different levels. The vacuoles (V) inside the perforatorium are clearly
visible. F. LS of the caudal region of the head. The chromatin is coarsely granular
with a small vacuole present in the middle. Cytoplasmic membrane closely applied
Avian Spermatozoa: Structure and Phylogeny "%
canal in current terminology) of the proximal portion of the nucleus. The

remaining, more anterior portion of the rod projects into the subacrosomal
space and is covered by the acrosome vesicle. Longitudinally running
vacuoles are present within the rod along its entire length, their number
varying from two to five and sometimes more, and varying in size (Fig. 8.19B-
D). The vacuoles communicate with the space between the acrosome vesicle
and the rod. They are empty or filled with a moderately dense substance.
The acrosome vesicle and the perforatorium are separated by a 10-40 nm
wide space, widest around the processes of the nucleus (nuclear rostrum). The
space is filled with a granular substance of low electron-density. Anteriorly
the perforatorium touches the posterior membrane of the acrosome vesicle
whereas basally it may or may not reach the base of the short endonuclear
canal. Within the endonuclear canal the perforatorium is closely applied to
the inner nuclear membrane lining the canal (Fig. 8.19A).
Nucleus. The chromatin of the nucleus is of uniform density and composed of
large granules of about 60 nm which are closely attached to each other. The
nucleus is surrounded by a double membrane which in mature spermatozoa
is so closely applied to the chromatin that it can hardly be differentiated.
Sometimes the nuclear membrane becomes obvious only in the anterior region
of the head at the site where the acrosome vesicle is attached, at the
endonuclear canal, and at the base of the nucleus where it articulates with the
tail (Maretta 1975a). Posteriorly there is a broad, shallow basal nuclear fossa
(implantation fossa) (Fig. 8.19F).
Neck, midpiece and centrioles. The tail of the drake spermatozoon is
composed of four parts: a neck, a midpiece, a principal piece and an endpiece.
The neck is the place of articulation between the head and the tail. In
contrast to the distal centriole, the proximal centriole (Fig. 8.19F) maintains its
original size. At one end of its lumen an electron-dense material can be seen.
Its wall consists of nine triplet tubules and forms a right angle to the distal

to the nuclear surface (N). G. TS sperm head. At bottom is a TS of the basal fossa
with remnants of the proximal centriole. H. LS of the neck and part of the
mitochondrial sheath. Mitochondria (M) are of irregular subspheric shape. I. TS of
the caudal part of the head and anterior region of the midpiece. The proximal
centriole is surrounded by electron-dense material. J. TS neck in the region of the
proximal centriole (PC). K. LS midpiece. Central singlets (CF) arise from the caudal
end of the distal centriole (DC). The cristae are arranged parallel to the flattened
mitochondrial wall (M). The midpiece terminates at an annulus. L. TS midpiece
through proximal part of distal centriole. M. TS midpiece at different levels. Outer
dense fibers are present (F). A. ¥ 47700. B. ¥ 26000. C. and D. ¥ 27300. E. ¥
33500. F. ¥ 27000. G. and H. ¥ 27000. I. ¥ 36500. J. ¥ 37700. K. ¥ 28200. L. ¥
26100. M. ¥ 22800. A-G. Relabeled after Maretta, M. 1975. Acta Veterinaria
Academiae Scientiarum Hungaricae 25(1): 47-52, Figs. 1-7. H-M. Relabeled after
Maretta, M. 1975. Acta Veterinaria Academiae Scientiarum Hungaricae 25(1): 53-
60, Figs. 1-6.
centriole. In cross sections it appears as a thick-walled ring in which some

tubules of the triplets can be partly identified (Fig. 8.19H, I, J).
The midpiece (Fig. 8.19H-M) is defined by the length of the mitochondrial
sheath measuring in the drake sperm 3-4 mm, and having a diameter of 0.6-
0.7 mm. The main portion of the midpiece consists of’ the centrally placed
distal centriole and part of the axial filament complex. In mature spermatozoa
the distal centriole reaches a length of up to 2 mm and a diameter of 0.2 mm. It
appears to be 3-4 times longer than the proximal centriole, the latter being
shown in Fig. 8.19F, J, K). The lumen of the proximal portion of the centriole is
usually filled with electron-dense material (Fig. 8.19L). The proximal centriole
is orientated with its long axis transverse relative to the longitudinal axis of
the sperm (Fig. 8.19F, H, K). The distal centriole is connected with the proximal
centriole by dense material, which also fills the space under the projecting
parts of the proximal centriole. Within the caudal end of the centriole a small
amount of electron-lucent material is placed where the central fibrils arise.
Annulus. The annulus is a ring-shaped formation (Fig. 8.20B) located at the
junction of the mitochondrial sheath of the midpiece and the amorphous
sheath of the principle piece (Figs. 8.19K, 8.20A). In cross section it is almost
triangular with a wall length of 100-150 nm. It is composed of homogeneous
material suggesting sometimes a fibrous structure. The cytoplasmic membrane
covering the entire length of the tail is closely applied to the underlying
structures. It is fixed only to the annulus. In the posterior portion of the
principal piece the membrane is separated from the amorphous sheath by a
narrow lighter space.
Axoneme. The axoneme has the usual 9+2 pattern. The denser subtubule of
the each doublet (subtubule A) is smaller and lies nearer to the axis of the tail
than does the larger, less dense subtubule B. Subtubule A bears two dynein
arms. Outer dense fibers are present (Fig. 8.19M), they reach a diameter of
about 40-50 nm and are closely attached to the doublets. They are visible only
in the posterior region of the midpiece reaching as far as the beginning of the
principal piece. Further externally, mitochondria are attached to the distal
centriole and part of the axoneme. In the anterior part of the midpiece
mitochondria closely approach the head and are caudally defined by the
annulus. They are of irregular spherical shape (Fig. 8.19H) and their total
number ranges between 24-30. They are flattened at the sides and about 0.2 mm
thick. Mitochondrial cristae run parallel to the flattened mitochondrial wall
(Fig. 8.19 J, K, L).
Principal piece. The principal piece of the tail (Fig. 8.20A, C, D) is defined by
the length of the amorphous sheath. Cranially, it starts at the annulus and
passes caudally into the endpiece. Remnants of the outermost dense fibers are
visible only in its anterior region.
Amorphous sheath. The amorphous sheath consists of moderately electron-
dense material and its wall has a diameter of 0.1 mm in its proximal portion. It
is separated from the axoneme by a 20 nm space. It is composed of an inner
Avian Spermatozoa: Structure and Phylogeny "'
Fig. 8.20 Anas platyrhynchos, Mallard. A. Longitudinal section of principal piece in

the anterior and posterior region. The midpiece and principal piece are separated
by an annulus (An). An amorphous sheath (AS) surrounds the axoneme (AFC).
B. Transverse section (TS) of the tail at the annulus (An). Outer dense fibers (F) are
present. C. TS of the tail in the proximal region of the principal piece. The
amorphous sheath (AS) is composed of an outer less dense and an inner dense
layer. D. TS posterior region of principal piece. The amorphous sheath (AS) here
consists only of the inner dense layer. E. TS endpiece. F. TS termination of
endpiece, showing disruption of 9+2 pattern. A. ¥ 3200. B-D. ¥ 39700. E. ¥ 38000,
F. ¥ 37900. After Maretta, M. 1975. Acta Veterinaria Academiae Scientiarum
Hungaricae 25(1): 53-60, Figs. 7-12.
more dense layer and an outer less dense layer. Candally, the sheath
gradually becomes narrower; the outer layer disappears first, then, gradually,
the inner layer. The site where the amorphous sheath disappears is the
junction with the endpiece.
Endpiece. The endpiece (8.20E) is composed of the axial filament complex
(axoneme). Near the tip of the endpiece the arms of the doublets disappear
and subtubule A loses it dense contents. The doublets are reduced to single
tubules and gradually decrease in number (Maretta 1975b) (Fig. 8.20F).
8.8.2.2 Branta sandvicensis
The sperm of Branta sandvicensis, the Hawaiian goose, compared with those of
the Mallard by Humphreys (1972) had an identical appearance which
conformed closely to that of Gallus sperm. Each spermatozoon was about 100 mm
long, with a nuclear diameter of about 0.5 mm. Humphreys (1972) gave a
graphical comparison of a mallard and canary sperm.
8.8.2.3 Conclusion for Galloanserae
The spermatozoa of the Anseriformes are closely similar to those of the
Galliformes but a notable distinction appears to be that in anseriform sperm,
as exemplified by Anas branchyrhynchos, the perforatorium extends almost to
the tip of the spermatozoon, as the acrosome vesicle is apically very narrow,
whereas in galliforms a large amount of material is present in the acrosome
vesicle anterior to the tip of the perforatorium, correlated with a much shorter
subacrosomal space. Demonstration that this difference is constant would
require examination of a larger sample of species.
The galloanseran spermatozoon resembles that of crocodile and
paleognaths in the conical acrosome vesicle, much shorter than the nucleus;
perforatorium penetrating the nucleus in an endonuclear canal; mitochondria
in tiers surrounding the distal centriole; and presence of an annulus and of a
fibrous sheath. It differs in the short stout form of the perforatorium; short
endonuclear canal; and the amorphous, not ribbed, fibrous sheath.
Anseriforms possess what appears to be the most primitive avian spermato-
zoon above the paleognaths.
8.9 METAVES
As noted by Harshman (Chapter 1 of this volume), Fain and Houde (2004)
divided Neoaves into two basal clades: Metaves, consisting of
Caprimulgiformes, Apodiformes, Podicipedidae, Phaethontidae,
Phoenicopteridae, Opisthocomidae, Mesitornithidae, Rhynochetidae,
Eurypygidae, Pteroclidae, and Columbidae; and Coronaves, consisting of the
remaining Neoaves. This subdivision of the Neoaves requires confirmation
from other analyses as it was based solely on an analysis of intron 7 of the
b-fibrinogen gene. It cannot be said to be supported by spermatozoal ultra-
structure (see section 8.11).
In the Metaves, only the Apodiformes, Caprimulgiformes and

Columbiformes have been examined for spermatozoal ultrastructure.
8.9.1 Apodiformes
Spermiogenesis in Apus (=Cypselus) apus, the Common swift was briefly treated
by Baccetti et al. (1980), who reported it as a passerine, in a valuable work on
the vertebrate perforatorium and by Tripepi et al. (1984) in an abstract.
Jamieson and Tripepi (unpublished; see also 2005) made a more detailed
investigation of the late spermatid. The only other ultrastructural account for
the Apodiformes is a brief account of microtubules in the spermatid of Apus
melba, the Alpine swift (Tripepi et al. 1991).
8.9.1.1 Apus (=Cypselus) apus
Acrosome. The acrosome vesicle (Fig. 8.22A, E-H) forms a slender, smooth,
pointed cone approximately 3 µm in length and 0.75 mm at its greatest, basal
width. Its base is rounded and closely fits a depression of the anterior end of
the nucleus. A perforatorium is absent. From a light microscope drawing by
Retzius (1911) (Fig. 8.21) the acrosome: nucleus ratio is very approximately
0.03.
Nucleus. The nucleus is an elongate cylinder (Fig. 8.22A, B, E, I, K, P) tapering
only slightly towards its tip. Its full length has not been determined but it
exceeds 8 µm, with a basal width of 0.6 µm. In young spermatids the
chromatin is finely granular and the nuclear membrane is surrounded by
microtubules, in single layer, which lie under the plasma membrane (Fig.
8.22J). In the more mature nucleus (Fig. 8.22K) the chromatin forms dark
clumps interspersed sporadically throughout its length with pale areas, some
of which impinge on its surface, and few microtubules remain beneath its
investing membrane (Fig. 8.22B). In the mature nucleus the chromatin is
electron dense and almost homogenous and microtubules are absent (Fig.
8.22I). The nuclear surface is almost smooth. The anterior nuclear fossa is
matched by a concave posterior fossa, the implantation fossa (Fig. 8.22B, P).
Midpiece and centrioles. The elongate cylindrical midpiece in which the
mitochondria are located is wider, at 1.1 µm, than the nucleus. Its length is
approximately 3.5 µm (Fig. 8.22M). Its central axis is occupied by the proximal
centriole, which lies partly within the implantation fossa, and by the distal
centriole. The proximal centriole is short, with its longitudinal axis
perpendicular to the sperm axis. It shows the usual nine triplets of
microtubules (Fig. 8.22B, N-P) but its central space is occupied by a structure
which is annular in transverse section (Fig. 8.22B, N-P). The distal centriole,
perpendicular to the proximal centriole and in the long axis of the cell, also
shows a triplet configuration (Figs 8.22L, Q). It extends for the whole length of
the midpiece. Its axis is empty except for intrusion of the central singlets of the
axoneme a very short distance into its base (Figs 8.22C, D, M).
Fig. 8.21 Drawings of some non-passerine sperm by light microscopy.

Philomachus pugnax, Ruff; Psittacus sp.; Strix aluco, Tawny owl; Dendrocopos
(=Picoides) major, Great spotted woodpecker; Columba livia, Domestic pigeon.

The mitochondria form a circle around the distal centriole, numbering five
or six in a transverse section of the cell (Fig. 8.22L, Q). There are six or seven
in longitudinal sequence (Fig. 8.22D, M) but some of these may be partly
conjoined (Fig. 8.22M). Posteriorly the midpiece narrows slightly but is not
demarcated by a recognizable annulus (Fig. 8.22 D, M). Each mitochondrion
has several cristae which appear transverse in cross- and oblique in
longitudinal section of the midpiece. They are initially subspherical (Fig.
8.22D) but become more elongate nearer maturity (Fig. 8.22B, C, D, M).
Axoneme. Immediately behind the midpiece, the axoneme commences as
indicated by the presence of central singlets. A moderately electron-dense
mass at the anterior end of these protrudes a little into the midpiece (Fig.
8.22C, D, M). An amorphous sheath (Fig. 8.22C, D, M) surrounds the axoneme
behind the midpiece and the long ensheathed region constitutes the principal
piece. Dense fibers have not been observed. A presumed endpiece, with
axoneme lacking the amorphous sheath is surrounded by a transient
cytoplasmic canal and sheath during development (Fig. 8.22R).
Remarks. The long distal centriole is a remarkably plesiomorphic feature of
the swift sperm, being seen only in paleognaths, as it is somewhat shortened
even in the Galloanserae. In the Palaeognathae, struthioniforms differ,
however, in penetration of the distal centriole by the two central axonemal
singlets (Baccetti et al. 1991; Phillips and Asa 1989; Soley 1993, 1999), though
these reach only about halfway into the centriole in Crested tinamou (see Fig.
1 of Asa and Phillips 1987). On the other hand, loss of the perforatorium in
Apus is a notable apomorphic departure from paleognaths and Galloanserae.
The phylogenetic implications of the long centriole and other features of the
sperm are further discussed in section 8.11.
In Apus apus, microtubules in the spermatid are restricted to a transient
layer encircling the cell, though longitudinal microtubules are also present in
the Sertoli cell which invests the spermatid, as also seen in A. melba (Tripepi et
al. 1991). This condition contrasts with passerines in which, in the spermatid,
an ‘helical membrane’ consists of multiple microtubules forming a thick
strand helically coiled around at least the flagellum (e.g. Asa and Phillips
1987; Jamieson 2006).
8.9.1.2 Apus melba
Tripepi et al. (1991) comment on, and illustrate, the microtubular arrangement
in the spermatid of the Alpine swift (Apus melba). Many microtubules,
arranged parallel to the longitudinal axis of the elongating spermatid appear
in the cytoplasm of the Sertoli cell and surround the head of the spermatid.

After Retzius, G. 1909. Biologische Untersuchungen, Neue Folge 14(10): 89-122
Taf XXX, Fig. 14, XXXI, 13, 23, 27. Apus (=Cypselus) apus, Common swift. After
Retzius, G. 1911. Biologische Untersuchungen, Neue Folge 16: 89-92 Taf XXVII,
Fig. 24.
Fig. 8.22 Apus apus, Common swift. Transmission electron micrographs of

spermatids. A. Oblique longitudinal section (LS) of acrosome on tip of nucleus.
B. LS late spermatid showing elongate nucleus, with scattered uncondensed
areas, and anterior portion of midpiece with enclosed proximal and distal
centrioles. C. Same cell, showing posterior end of midpiece and anterior portion of
principal piece. D. LS of the midpiece of a younger spermatid in which
mitochondria are subspherical. Arrow indicates absence of an annulus.
E. LS of two acrosomes. F-H. Progressively posterior transverse sections (TS) of
acrosome vesicle, H, at the level of the anterior nuclear fossa. I. TS of an advanced
nucleus with strongly condensed chromatin and lacking peripheral microtubules.
J. TS nucleus of younger spermatid with uncondensed granular chromatin and
peripheral single layer of microtubules. K. TS nucleus at intermediate stage still
showing pale uncondensed areas. L. TS immature midpiece, showing six
mitochondria surrounding the distal centriole. M. Longitudinal section (LS) of an
advanced spermatid through the entire length of the midpiece and through the

There is therefore a simultaneous occurrence of the longitudinal manchette in

the spermatid and the microtubules of the Sertoli cell, as in Crested tinamou.
8.9.2 Order Caprimulgiformes

The Caprimulgiformes were subsumed in the Strigiformes by Sibley and
Ahlquist (1990), as endorsed by ITIS (http://www.itis.usda.gov/). However,
as relationship of the aegolothelids (owlet-nightjars) is closer to apodiforms
than to strigeids, inclusion of caprimulgiforms in the Strigiformes is
contraindicated. The order Caprimulgiformes is therefore recognized here
while recognizing that paraphyly or even polyphyly of the order (see Chapter
1) may preclude association of caprimulgids with the aegoleothelids. Strigidae
are now placed in the Coronaves (see Harshman, Chapter 1) and this is
supported by sperm ultrastructure (Section 8.10.2)
We owe to Ballowitz (1888) a drawing of a spermatozoon of Caprimulgus
europaeus, the European Nightjar. The spermatid of C. europaeus has been the
subject of a paper on microtubules by Tripepi et al. (1991) and has been
described in some detail by Tripepi, Jamieson and Brunelli (unpublished)
from whose account the following description is largely drawn.
8.9.2.1 Caprimulgus europaeus
The structure of the spermatozoon of Caprimulgus europaeus has been deduced
from the description by TEM (Fig. 8.23) of the late spermatid (Tripepi
unpublished) considered in conjunction with the light microscope description
of the mature spermatozoon by Ballowitz (1888).
Acrosome. The acrosome consists of the acrosome vesicle and associated
material of a presumed perforatorial nature. Condensation of dense material
at the nuclear surface at the base of the subacrosomal space results in
compaction to form an electron dense, stout, spine-like structure, the putative
perforatorium. This penetrates a short distance into the tip of the nucleus
within an endonuclear canal and protrudes into the subacrosomal space (Fig.
8.23A). In transverse section, the acrosome vesicle forms a thick ring, with
moderately electron-dense contents, around the tip of the nucleus with its
central endonuclear canal and perforatorium (Fig. 8.23B). The maturing
acrosome vesicle (Fig. 8.23A) assumes a conical form; the perforatorium
becomes less electron-dense than formerly and the portion within the
endonuclear canal is considerably longer than the ill-defined portion in the
now conical subacrosomal space.

principal piece. N,O. TS proximal centriole and adjacent midpiece, N being detail of
O, showing the central structure of unknown significance. P. TS proximal centriole
and adjacent nucleus and midpiece. Q. Transverse sections (TS) through two
midpieces. R. TS endpiece still surrounded by a transient cytoplasmic canal and
cytoplasmic sheath. as = amorphous sheath; av, acrosome vesicle; ax, axoneme;
dc, distal centriole; m, mitochondrion; mt, microtubules; n, nucleus; pc, proximal
centriole. From Jamieson and Tripepi, unpublished, see also Acta Zoologica
(Stockholm) 86: 239-244, Figs. 1 and 2.
Fig. 8.23 Caprimulgus europaeus. A. LS more mature acrosome, showing

perforatorium lying in the endonuclear canal and extending somewhat amorphously
into the subacrosomal space. The acrosome vesicle is assuming a conical form.
B. Transverse section of an advanced acrosome showing the base of the acrosome
By light microscopy, the mature spermatozoon of Caprimulgus europaeus

appears in a drawing (Fig. 85 in Ballowitz 1888) to have a conical acrosome
tapering evenly from the much longer but fairly stout, curved nucleus.
However, this species is not specifically mentioned in this regard in the
description of the two types of sperm head recognized for birds by Ballowitz,
though in the legend for Fig. 86 the head is described as “an der Spitze
deutlich das kleine blasse Spitzen-stueck zeigend”. This pale point is
consistent with the conical form of the maturing acrosome observed here. It is
in marked contrast to the button-like acrosome of the only described member
of the Strigiformes, the Tawny owl (Retzius 1909), the order in which
caprimulgids were subsumed by Sibley and Ahlquist (1990).
Nucleus. In the elongating spermatid the chromatin comes to form coarse,
electron-dense granules (Fig. 8.23A, C). At maturity, as seen in the advanced
spermatid, the nucleus is cylindrical with strongly condensed, homogeneous,
electron-dense chromatin (Fig. 8.23D). Nuclear elongation is accompanied by
growth of a longitudinal manchette of microtubules in the surrounding
cytoplasm. No circular manchette is present.
Midpiece. The Caprimulgus midpiece, about half the length of the nucleus, has
some 10 tiers of mitochondria; the free axoneme is about the same length as
the head plus midpiece (see Ballowitz 1888). In the advanced spermatid,
mitochondria group around the proximal axoneme. A maximum number of
six mitochondria has been observed, in transverse section, at one level
(Fig. 8.23C, top left).
Centrioles. Each spermatid has a proximal and distal centriole, mutually
perpendicular, and the distal centriole is continuous with the flagellum. The
centrioles have the normal configuration of nine triplets of microtubules. The
lumen of the distal centriole is not, initially, penetrated by the central singlets
of the axoneme but in the young elongating spermatid the distal centriole is
penetrated by these singlets.
Axoneme. The axoneme forms an extension of the distal centriole. In
transverse section, the axoneme has the typical ‘9+2’ arrangement of

vesicle enclosing the tip of the nucleus with its endonuclear canal and perforatorium.
C. Transverse sections (TS) of midpieces and nucleus of advanced elongating
spermatids. Four to six mitochondria surround the proximal axoneme as the
midpiece. Dense fibers associated with the axonemal doublets are small in these
sections. The chromatin of the nuclei has become clumped as condensation
proceeds. Longitudinal but no circularly running cytoplasmic microtubules are
present. D. LS nucleus fully condensed but still accompanied by the manchette. E. TS
elongating spermatids. Left through the transient cytoplasmic canal and contained
axoneme. Center right, TS through a developing midpiece which has assembled
only four mitochondria at this level. F. TS axoneme with weakly developed amorphous
sheath. av, acrosome vesicle; ax, axoneme; cytoplasmic canal; ec, endonuclear
canal; m, mitochondrion; mp, midpiece; mt, microtubules of manchette; n, nucleus;
p, putative perforatorium. Tripepi, Jamieson and Brunelli, unpublished.
microtubules: Within the midpiece, a dense fiber is attached to the outer aspect
of each of the nine doublets (Fig. 8.23C). The free axoneme is surrounded by a
weakly developed amorphous sheath, defining the principal piece (Fig. 8.23F).
Phylogenetic considerations. The structure of the spermatozoon of
Caprimulgus europaeus can be deduced from the description by TEM of the late
spermatid given above when considered in conjunction with the light
microscope description of the mature spermatozoon by Ballowitz (1888). It is
typical of other non-passerines in many respects. Features shared with
paleognaths (ratites and tinamous) and the Galloanserae (e.g. rooster and
duck) are the conical acrosome, shorter than the nucleus; presence of a
perforatorium and endonuclear canal; presence of a proximal as well as distal
centriole; the elongate midpiece with mitochondria grouped around a central
axis (here maximally six mitochondria in approximately 10 tiers); and
presence of a fibrous or amorphous sheath around the axoneme. Most of these
features characterize non-passerines in general. A major (apomorphic)
difference from paleognaths and galloanserans is the short distal centriole, the
midpiece being penetrated by the axoneme and not by the centriole.
In lacking an appreciable annulus, the sperm of Caprimulgus, like those of
Psittaciformes (Jamieson 1999; Jamieson et al. 1995), Gruiformes (Grus vipio,
Phillips et al. 1987), Apodiformes (Jamieson and Tripepi 2006), and passerines
(e.g. Asa and Phillips 1987; Jamieson 1999), differ from those of paleognaths
(e.g., Baccetti et al. 1991), galloanserans, and charadriiforms as represented by
Jacana (Saita et al. 1983). An annulus is basal to paleognaths and these non-
passerines. Absence of the annulus is therefore an apomorphic feature of
caprimulgid sperm.
In Caprimulgus europaeus, the circular manchette has been lost and only a
longitudinal manchette is present in the developing spermatid (Tripepi et al.
1991). A similar arrangement is seen in Jacana jacana and (Jamieson and
Tripepi 2005) in the apodiform Apus apus. In contrast, passerines differ from
non-passerines in possessing, in the spermatid, an ‘helical membrane’,
consisting of multiple transverse and longitudinal microtubules forming a
thick strand helically coiled around at least the flagellum (e.g. Asa and
Phillips 1987; this study). Tripepi et al. (1991) consider the arrangement of
microtubules in C. europaeus to be the second above a “reptilian” level.
For further phylogenetic considerations see section 8.11.
8.9.3 Order Columbiformes

Columbiforms for which sperm ultrastructure has been described are: ‘Pigeon’
(presumably Columba livia) (Fawcett et al. 1971), restricted to a consideration
of nuclear shaping in the spermatid; Columba livia, Domestic pigeon
(Yasuzumi and Yamaguchi 1977), spermiogenesis, mainly concerning
development of the acrosome and nuclear microtubules; Turtle dove
(Streptopelia roseogrisea), chiefly spermiogenesis (Mattei et al. 1972); and brief
descriptions of mature spermatozoa of Peaceful dove (Geopelia striata, Jamieson
et al. 1995, 1999); and of Crested pigeon (Ocyphaps lophotes, Jamieson et al.
1995, 1999). Sperm of G. striata and O. lophotes are illustrated (Figs. 8.24, 8.25)
and described more fully below.
Valuable accounts by light microscopy are those of Ballowitz (1888),
Retzius (1911, 1912) and Vernon and Woolley (1999; also giving
ultrastructure) for Columba livia. Five genera, with six species, were examined
by McFarlane (1963); all except Blue Ground-Dove (Claravis pretiosa) were
unnamed. These results are, however, summarized in his drawing showing a
pointed acrosome much shorter than the very elongate cylindrical nucleus,
and a long cylindrical midpiece, stated to extend far down the tail, in a non-
helical sperm (Fig. 8.27D).
Acrosome. The acrosome vesicle forms an elongate blunt-tipped cone which
is shorter, at ~1.0-1.2 mm (n=2), in Ocyphaps lophotes (Fig. 8.24A, B) than the
~2.6-2.8 mm (n=2) in Geopelia striata (Fig. 8.25A, B). The subacrosomal space
forms a convex sided cone less than half the length of the vesicle. Transverse
sections of the vesicle reveal a circular profile (Figs. 8.24E, F, 8.25G-J). A
striking contrast with Galloanserae, psittaciforms and most other non-
passerines is the absence of a perforatorial rod. Instead of the subacrosomal
space being occupied by a perforatorium, it is filled by a narrow anterior
extension of the nucleus, the nuclear rostrum. However, the electron-dense
chromatin does not fully occupy the investing conical nuclear membrane but
leaves a varying length of the tip of the rostrum free of chromatin (O. lophotes,
Fig. 8.24A, B; G. striata, Fig. 8.25A, B). In some cases (Fig. 8.25B) the pale tip of
the rostrum has an appearance reminiscent of a perforatorium but this seems
to be an artifact of eccentric sectioning of the rostrum as the electron dense
rostrum extends far into the subacrosomal space in other sections (Fig. 8.25A).
Nevertheless, it is possible that some of the finely granular material at the tip
of and surrounding the rostrum represents a reduced perforatorium. In
support of this it is noteworthy that some transverse sections of the rostrum
reveal a central pale core (O. lophotes, Fig. 8.24E; G. striata, Fig. 8.25I) which
cannot merely be dismissed as uncondensed chromatin as in some sections
(Fig. 8.25J) it is in continuity with the subacrosomal matrix. This central
lacuna in the rostrum, also seen in longitudinal section (Figs. 8.24, 8.12A),
may, therefore, be a vestigial endonuclear canal.
Nucleus. The nucleus forms a stout cylinder, circular in cross section (Figs.
8.24C, 8.25E) with shoulders, supporting the base of the acrosome vesicle,
where it narrows to form the rostrum. The chromatin is electron-dense and, at
maturity, almost homogeneous. The base of the nucleus widens slightly and
is indented by a shallow but distinct basal fossa which forms an arc in
longitudinal sections of the nucleus and houses the anterior part of the
proximal centriole (O. lophotes, Fig. 8.24C, D; G. striata, Fig. 8.25K, L, M). The
nuclei illustrated by light microscopy for Columba livia by Retzius (1909) (Fig.
8.21) and Claravis pretiosa by McFarlane (1963) are moderately elongate,
though that for C. livia by Ballowitz (1888) appears shorter than that
illustrated by Retzius.
Fig. 8.24 Ocyphaps lophotes, Crested pigeon. Transmission electron micrographs

of spermatozoa. A and B. Longitudinal section (LS) acrosome and anterior portion
of nucleus. C. LS base of nucleus, centrioles and anterior midpiece. D. LS almost
mature, testicular showing same regions. E. Transverse section (TS) of base of
acrosome vesicle surrounding the nuclear rostrum, the latter showing a central
vacuole or canal. F. TS showing nuclear rostrum nearer its base. G. TS nucleus.
H. TS distal centriole at the transition from 9 triplets to doublets, surrounded by
mitochondria of the anterior midpiece. I. TS midpiece through 9+2 axoneme. J. LS
midpiece, showing mitochondria aligned in single file and occasional dense
bodies. K. TS axoneme posterior to midpiece. Original.
Columbiforms appear plesiomorphic in retaining the nuclear rostrum,

though it is possible that presence is a reversal (Jamieson 1999).
Midpiece. In transverse section the midpiece has four to six mitochondria
surrounding the distal centriole (Fig. 8.24H) and axoneme (Figs. 8.24I, 8.25C,
N, O). In both species compact electron-dense bodies are interspersed between
or lie medially to the mitochondria (Figs. 8.24J, 8.25M, N), an unusual
condition for Aves but common in squamates. This co-occurrence, although
appearing homoplastic, may well indicate persistence of a genetic basis laid
down in early amniotes (Jamieson 1999). In longitudinal section (Figs. 8.24D,
K, 8.25M) the mitochondria are seen to lie end to end in single file. Four or five
parallel chains of mitochondria were described, and illustrated in transverse
section, for Columba livia by Vernon and Woolley (1999).
The length of the midpiece in Ocyphaps lophotes and Geopelia striata was not
ascertained. Woolley (1995) estimated that in Columba livia “var. domestica”,
the flagellar length is ca 150 µm, but that the midpiece occupies the proximal
114 mm (dimensions estimated from Ballowitz 1888, proportions confirmed
from drawings by Retzius 1909) (see Fig. 8.21 of this chapter). He notes that
this is the only record of a midpiece of an avian spermatozoon approaching
the great length (161 mm) of that of Coturnix japonica, though here considered
to require confirmation in the latter. Slightly different dimensions were given
for the domestic pigeon by Vernon and Woolley (1999). The sperm head was
about 16 µm long. The mean flagellar length with SD was 132.0 ± 11.1 µm;
length midpiece 98.1 ± 11.2 µm (n = 13 sperm from 1 individual). Midpiece
length and flagellar length were closely correlated: r = 0.89, P < 0.001.
An annulus terminating the midpiece, basic to bird sperm, and seen in
paleognaths, rooster and Guineafowl, has been reported for columbiforms
(Asa and Phillips 1987) and specifically for Columba livia (Vernon and
Woolley 1999).
Centrioles. The proximal centriole is orientated with its long axis at right
angles to the sperm axis (Figs. 8.24C, D, 8.25K, L, M), as stated and illustrated
by Vernon and Woolley (1999) for Columba livia. Three projections (probably
equivalent to striated columns) are seen to link it to the thickened basal
membrane of the nucleus in Ocyphaps lophotes (Fig. 8.24C). The distal centriole
forms the basal body orientated in the long axis of the axoneme with which it
is continuous (Figs. 8.12C, D, 8.25C, D, L, M). Its posterior limit is difficult to
determine but it is only a little longer than the proximal centriole, its length
being estimated as 0.5 mm in Geopelia striata (Jamieson 1995). Both centrioles
have the usual 9 triplets but where the distal centriole enters the midpiece the
triplets progressively reduce to doublets (Figs. 8.24H, 8.25C). Each centriole is
embedded in a ring of dense material.
Axoneme. The axoneme shows the usual 9+2 pattern of microtubules. Within
the proximal region of the midpiece of apparently mature sperm of Geopelia
striata (Fig. 8.25N) and Ocyphaps lophotes (not illustrated) the axoneme has
outer dense fibers (Jamieson 1995, 1999). These fibers are said by Mattei et al.
Fig. 8.25 Geopelia striata, Peaceful dove. Transmission electron micrographs of

spermatozoa. A and B. Longitudinal section (LS) of acrosome and anterior portion
of nucleus. B cut off center through the nuclear rostrum. C. TS distal centriole at the
Avian Spermatozoa: Structure and Phylogeny " !
(1972) to be present in the spermatid but to be lost by maturation of the sperm

in Streptopelia roseogrisea and they have not been observed in the sperm of O.
lophotes. In the more distal region of the midpiece dense fibers are absent (Fig.
8.2O) and the axoneme posterior to the midpiece is simple (Figs. 8.24K, 8.25F).
Vernon and Woolley (1999) state, for Columba livia, that accessory fibers occur
proximally on the flagellum, in what were clearly mature sperm, but are
extremely short.
Fibrous sheath. A fibrous or amorphous sheath which surrounds the
principal piece in lower non-passerines is absent from mature sperm of those
columbiforms examined for this feature ultrastructurally (Streptopelia
roseogrisea, Mattei et al. 1972; Geopelia striata and Ocyphaps lophotes, this
chapter, and Columba livia, Vernon and Woolley 1999) as from psittaciforms.
It was, however, demonstrated by Mattei et al. (1972) for the spermatid of S.
roseogrisea.
Remarks. The spermatozoa of columbiforms have many features of those of
Galloanserae but differ notably in loss of the perforatorium and the fibrous
sheath by maturity and the exceptionally great length of the midpiece, though
the latter is possibly also greatly elongate in Coturnix japonica (section 8.8.1.2).
8.10 CORONAVES
The Coronaves includes the majority of taxa in the division of the Neoaves
into Metaves and Coronaves by Fain and Houde (2004).
8.10.1 Order Piciformes

These constituted the Parvclass Picae in the system of Sibley and Ahlquist.
8.10.1.1 Melanerpes carolinus
The spermatozoa of the Piciformes are known only from the light microscope
accounts of Ballowitz (1888) and Retzius (1909) for the Great spotted
woodpecker (Dendrocopos (=Picus) major) (Fig. 8.21) and the brief
ultrastructural account of Henley et al. (1978) for the Red-bellied woodpecker
(Melanerpes carolinus) (Fig. 8.26A). Picoid sperm are markedly different from
those of oscines and are considered by Henley et al. (1978) to be referable to

transition from 9 triplets to doublets, surrounded by mitochondria of the anterior
midpiece. D. Detail of same. E. TS nucleus. F. TS axoneme posterior to midpiece.
G. TS anterior region of acrosome vesicle. H. TS basal region of acrosome through
the nuclear rostrum. I and J. Same showing central vacuole in nuclear rostrum
which in J communicates with the material in the subacrosomal space. K. LS
showing base of nucleus, proximal centriole and anterior midpiece. L. Detail of
proximal centriole. M. LS cut at right angles to K showing base of nucleus, proximal
centriole and anterior midpiece. N. TS Midpiece showing a dense body between
mitochondria and axoneme with nine uniform dense fibers. O. TS midpiece
putatively further posterior, with the axoneme lacking dense fibers. Original.
" " Reproductive Biology and Phylogeny of Birds
Fig. 8.26 Melanerpes and Crotophaga A. M. carolinus, Red-bellied woodpecker.

Transverse section through a group of maturing spermatozoa. The section passes

Avian Spermatozoa: Structure and Phylogeny " #
the type seen in ‘various fowls’ by Retzius (1909), McFarlane (1963), and in a
duck by Henley et al. (1978).
The acrosome in such sperm is said by Henley et al. (1978) to be “button-
like”, rather than tapering and long as in oscines. However, the galliform and
anseriform acrosome is here considered elongate conical, though less elongate
than in oscines. The acrosome of Dendrocopos illustrated by Retzius (1909)
(Fig. 8.21) is also a pointed cone though shorter than that of the latter orders.
It does appear to be a small knob in the less clearly defined drawing by
Ballowitz (1888) but is said (p. 449), like Cuculus, Columba, Gallus, Tadorna and
Anas, to be “nach vorne hin verschmälert …so dass sie ein mehr nadelförmiges
oder pfriemenartiges Aussehen darbietet”, thus needle- or bodkin-shaped.
The nucleus of the Dendrocopos sperm is longer and more slender than in
oscines, and the midpiece differs from that of the Passerida in not extending
as a helix along the flagellum; the flagellum therefore differs in having a long
free portion. There is no trace of an helical (“undulating”) membrane.
Although spermatogenesis in Melanerpes, as in oscines, occurs from a
syncytial mass, the components are irregularly arranged and it does not result
in precisely aligned bundles of sperm which is diagnostic of oscines (a corvid
and six passeridans were examined). The testicular sperm are motile in saline
whereas those of oscines are not (Henley et al. 1978).
By TEM the sperm are seen to consist of an acrosome, a densely compact
nucleus and a midpiece composed of mitochondria of conventional
morphology and varying number [about 6 or 7 in transverse sections in the
micrograph] surrounding the axoneme (Fig. 8.26A). Although microtubules
are present in the syncytium in which the spermatids develop, there is no
microtubular sheath, correlating with the absence of an helical membrane.
The sperm are also said to differ from those of “sauropsid” sperm, which they
generally resemble, and those of oscines in lacking dense fibers peripheral to
the axonemal microtubules (Henley et al. 1978) though this absence requires
confirmation for all regions of the axoneme. An amorphous sheath appears to
be present around the axoneme in a single micrograph (Fig. 8.26A).

transversely through different levels of the components, in the region of the nucleus
(N), the mitochondria (M) of the midpiece surrounding the axoneme (A), and in (S)
through the tail (principal piece) posterior to the mitochondrial region and showing
the amorphous sheath. Note the absence of dense fibers. PM, plasma membrane.
From Henley, C., Feduccia, A. and Costello, D. P. 1978. The Condor 80: 41-48, Fig.
7. B-D. Crotophaga ani, Smooth-billed ani. Late spermatids. B. Longitudinal
sections of two nuclei surmounted by acrosomes lacking perforatoria. C.
Transverse sections of an acrosome vesicle, midpiece and nuclei. D. Detail of
midpiece from C. AV, acrosome vesicle; M, mitochondria of midpiece; N, nucleus.
Scales are approximate. From micrographs provided by Dr. Sandro Tripepi. E.
Longitudinal section of midpiece of elongated spermatid. After Saita et al. (1982).
Bolletino de Zoologia 49: 115-123, Fig. 12. A, axoneme; AN, annulus; DC, distal
centriole; M, mitochondria.
" $ Reproductive Biology and Phylogeny of Birds
Remarks. Henley et al. (1978) appear correct in recognizing the similarity of

the woodpecker sperm to those of galliforms and anseriforms but it is not
known whether a perforatorium, present in the latter two orders, is present.
For a phylogenetic discussion see section 8.11.
8.10.2 Order Strigiformes

It is remarkable that the sperm of the Strigiformes have almost entirely been
neglected as objects of study. Retzius (1909) gave a drawing of that of Strix
aluco, the Tawny owl (Fig. 8.21).
8.10.2.1 Strix aluco
The spermatozoon of Strix aluco, illustrated by Retzius (1909) (Fig. 8.21) has,
in contrast to Caprimulgus with which is was associated in an enlarged
Strigiformes by Sibley and Ahlquist (1990), a morphology typical of some
other Coronaves, notably charadriiforms, trogoniforms, and falconiforms,
with their button-like acrosomes. The spermatozoon is particularly similar to
that illustrated by SEM for Falco (Fig. 8.35), having besides the button-like
acrosome, a stout moderately long, cylindrical nucleus, a short midpiece, here
with about six tiers of mitochondria, and a moderately long free flagellum.
8.10.3 Order Trogoniformes

8.10.3.1 Trogon collaris
Trogon sperm are known only from the light microscope account for Trogon
collaris, the Collared trogon, of McFarlane (1963) (Fig. 8.27A).
Trogon sperm (Fig. 8.27A) resemble those of the Charadriiformes,
exemplified by the gull Larus marinus (Fig. 8.27B) in having a button-like
acrosome and straight, cylindrical nucleus and midpiece. The length of the
midpiece is less than half that of the nucleus (McFarlane 1963).
8.10.4 Order Cuculiformes

The spermatozoon of Cuculus canorus, the European cuckoo, illustrated by
Ballowitz (1888) (his Fig. 110) is straight, non-helical, with pointed acrosome
much shorter than the stout cylindrical nucleus, midpiece shorter than the
nucleus, and a long free flagellum.
Spermiogenesis of another cuckoo Crotophaga ani, the smooth-billed ani,
has been described ultrastructurally by Saita et al. (1982a, 1982b). Tripepi et al.
(1991) have commented on, and illustrated the microtubules of the spermatid.
Additional micrographs have been provided by Dr. Tripepi (Fig. 8.26B-D). The
acrosome vesicle of the late spermatid forms a cone, with rounded tip and is
more than twice as long, at 1.5 mm, as wide (Fig. 15 in Saita et al. 1982b). Less
developed acrosomes are shown in Fig. 8.26B. Its rounded base rests in a
shallow concavity of the tip of the long cylindrical nucleus (Fig. 8.26B). A
perforatorium is absent. In a transverse section the midpiece has five
mitochondria encircling the axoneme (Fig. 8.26C, D) and in longitudinal
section three or four somewhat elongate mitochondria which appear to be
Avian Spermatozoa: Structure and Phylogeny " %
Fig. 8.27 Drawings by light microscopy of bird spermatozoa. A-F. Some basic
designs. A. Trogon collaris, Trogoniformes. B. Larus marinus, Charadriiformes.
C. Somateria mollissima, Anseriformes. D. Claravis pretiosa, Columbiformes.
E. Gallus gallus, Galliformes. F. Dendroica coronata, Passeriformes. G-K. Some
variations in passerine sperm. G. Deconychura typica, Dendrocalaptidae.
H. Tyrannus tyrannus, Tyrannidae. I. Corvus brachyrhynchos, Corvidae. J. Vireo
olivaceus, Vireonidae. K. Cardeulis (=Spinus) pinus. Fringillidae. Relabeled after
McFarlane, R. W. 1963. Proceedings of the XIII International Ornithological
Congress: 91-102, Figs. 1 and 2.
" & Reproductive Biology and Phylogeny of Birds
longitudinally conjoined are observed; the midpiece is terminated by a small

annulus (Fig. 8.26E). The axoneme in the sections illustrated (Fig. 8.26C, D)
appears to be transitional from the distal centriole. The distal centriole
penetrates for a short distance into the midpiece (Fig. 8.26E). An helical
membrane is absent. The principal piece has an amorphous sheath (Fig. 14,
Saita et al. 1982b). Dense fibers are absent from the principal piece but there
appears to be a very small density, interpretable as a minute dense fiber, on
the outer aspect of each doublet in the anterior region of the midpiece (Fig.
8.26C, D).
Tripepi et al. (1991) consider the arrangement of microtubules in the
spermatid to be at a third level above the “reptilian” arrangement. There has
been a total loss of the longitudinal manchette and only the longitudinal
microtubules of the Sertoli cells are present.
Phylogenetic remarks. The loss of the perforatorium is a notable development,
distinguishing the cuculiform spermatozoon from that of the Galloanserae.
The acrosome is also considerably shorter and the distal centriole penetrates
for a much shorter distance into the midpiece.
8.10.5 Order Psittaciformes

Accounts of spermatozoal ultrastructure exist for Melopsittacus undulatus,
Budgerigar, Psittacinae (Humphreys 1975; Samour et al. 1986; Jamieson et al.
1995); Agapornis roseicollis, Peach-faced lovebird, Psittacinae (Jamieson et al.
1995); Platycercus elegans, Crimson Rosella (Jamieson 1999; adult in Fig. 8.3D)
and Nymphicus hollandicus, Cockatiel, Cacatuinae (Jamieson et al. 1995).
Additional micrographs are included here for N. hollandicus. Retzius (1909)
illustrated the sperm of a Psittacus sp. by light microscopy.
As shown in the accounts below, psittaciform sperm have the following
characteristics: (1) a conical acrosome vesicle; rod-like perforatorium;
cylindrical, highly condensed nucleus; proximal and distal centriole
embedded in dense material; elongate periaxonemal mitochondrial midpiece,
(2) nine dense peripheral axonemal fibers (coarse fibers), (3) they have lost the
fibrous sheath around the axoneme which occurs in ratites and lower non-
passerines, (4) mitochondria with linear cristae, lacking intra- (or inter-)
mitochondrial dense bodies, (5) restriction of the endonuclear perforatorial
canal to the anterior region of the nucleus, (6) a short proximal and distal
centriole, and (7) nuclear tip not or only very slightly penetrating the acrosome
vesicle.
Psittaciforms differ fundamentally from passerines and resemble galliforms,
in the elongate nucleus and the much shorter acrosome relative to this, the
ratio of length acrosome:length nucleus being 0.2 in Nymphicus hollandicus.
The drawing of a Psittacus sperm by Retzius (1909) supports ultrastructural
observations for psittaciforms, there being a short pointed acrosome, a shorter
rod-like penetration of the anterior end of the nucleus, an elongate though
stout nucleus many (ca 13) times the length of the acrosome, together with a
Avian Spermatozoa: Structure and Phylogeny " '
short straight midpiece about 0.4 the length of the nucleus, and a long free
flagellum (Fig. 8.21).
8.10.5.1 Melopsittacus undulatus
Differentiation of the acrosome in the Budgerigar spermatid has been
described ultrastructurally by Humphreys (1975) and some features of the
mature spermatozoon by Samour et al. (1986). The following account is taken
chiefly from the account of Jamieson et al. (1995).
General. The spermatozoon of Melopsittacus undulatus is a filiform cell
consisting of a head region containing the nucleus and acrosomal structures,
a midpiece, and tail region. Its ultrastructure is summarized,
semidiagrammatically, in Fig. 8.28. The anteriorly tapering cylindrical head
was estimated from both light microscopy and transmission electron
microscopy to be 13 mm long. The nuclear and acrosomal lengths reported for
the Budgerigar by Jamieson et al. (1995) and from the work of Samour et al.
(1986) are close to those ascertained for three galliform species (Thurston and
Hess 1987). The length of the entire head (acrosome + nucleus) is 8 ± 1.97 mm
and its width is 0.5 ± 0.05 mm (n = 100) (Samour et al. 1986).
Acrosome complex. The anteriormost region of the head consists of the
acrosomal complex, which is composed of a smooth, elongated, conical
acrosomal vesicle and a rod-like perforatorium lying free in the subacrosomal
space (Figs. 8.28, 8.29A, C, D, G, H). The acrosomal vesicle is 1.9 mm long (n=2)
(Jamieson et al. 1995), (1.42 ± 0.16 mm (n = 100) Samour et al. 1986), and
terminates anteriorly in a blunt tip. Its width is greatest at its distal end. Here
its diameter is 0.5 mm and its walls are ~0.15 mm thick (Fig. 8.29C, D). The
perforatorium extends proximally very nearly to the tip of the spermatozoon
(as in anseriforms), and distally extends within a cylindrical end nuclear
canal another 0.6 mm into the anterior region of the nucleus. At its widest
point, the perforatorium measures 0.2 mm (Fig. 8.29C). The contents of the
acrosomal vesicle are homogeneous and of moderate electron density. The
perforatorium is slightly more electron-dense, but of a less uniform
composition, with irregularly spaced, electron-lucent channels penetrating it.
Granular material surrounds the perforatorium within the subacrosomal
space (Fig. 8.29C, D). The space between the outer membrane of the acrosomal
vesicle and the plasma membrane is also occupied by granular material. This
gap is ~20 nm wide at the distal end of the acrosomal vesicle. The acrosomal
vesicle terminates adjacent to the proximal end of the nucleus. These two
structures do not overlap or join directly. It appears that only the p1asma
membrane and the shared perforatorium hold them in position. This fragile
connection is considered to form a point of mechanical weakness and
“decapitated” spermatids and isolated acrosomes are often found in testicular
sections (Humphreys 1975). This condition contrasts with other investigated
avian orders, thus giving a psittaciform synapomorphy and autapomorphy.
"! Reproductive Biology and Phylogeny of Birds
Fig. 8.28 Melopsittacus undulatus, Budgerigar. Semidiagrammatic representation

of the ultrastructure of a spermatozoon from the ductus deferens (drawn by C.
Tudge). From Jamieson, B. G. M., Koehler, L. and Todd, B. J. 1995. Anatomical
Record 241(4): 461-468, Fig. 3. Reprinted with permission from Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.
Parrot sperm, like those of ratites and other birds, differ from reptiles in
reduction of the subacrosomal material (subacrosomal cone) to a negligible
amount. The psittaciform restriction of the endonuclear canal, housing the
perforatorium, to the anterior region of the nucleus is an apomorphic
condition (Jamieson et al. 1995) shared with other non-passerines (e.g.
Galloanserae and the White-naped crane, Grus vipio).
Nucleus. The nucleus, which is a gently curved cylinder (Fig. 8.29A), has an
approximate length of 10 mm, a width of about 0.5 mm at its proximal end, and
0.6 mm at the nuclear-midpiece junction (Fig. 8.29C-F). The endonuclear canal
(and perforatorium) occupies only about the proximal 5% of the nuclear
length and is 0.2 mm wide (Fig. 8.29C, D). The chromatin is compact and
electron-dense, although sporadic, small, electron-lucent areas are
interspersed throughout the nucleolus. The surface is rough and the nuclear
membrane is in close association with the plasma membrane (Fig. 8.29C, D, H,
I). The distal end of the nucleus forms a broad, shallow, concave fossa (Fig.
8.29E, F).
Midpiece, centrioles, and axoneme. The cylindrical midpiece region is 5.1 mm
long (n =1) and ~ 0.6 mm wide along much of its length. At its anterior end, a
basal lamina lines the concave surface of the nuclear base. Associated with
this lamina is a band of granular, electron-dense material (Fig. 8.29E).
Immediately posterior to the lamina is a 0.3-mm-long proximal centriole,
positioned centrally, parallel to the base of the nucleus. Lying perpendicular
to the proximal centriole is a 0.4-mm-long distal centriole, occupying only a
very small fraction of the midpiece length. Both centrioles display the typical
pattern of nine triplets of microtubules arranged in a cylinder, and both are
embedded in electron-dense pericentriolar material (Fig. 8.29E, F, J, K).
Whereas the center of the proximal centriole appears completely electron-
lucent, mottled granular material occupies the lumen of the distal centriole
(Fig. 8.29J).
The axoneme begins at a point ~ 1 mm along the length of the midpiece (Fig.
8.29E, F), continuous with the distal centriole, and extending the remainder of
the length of the spermatozoon. It is organized according to the usual “9+2”
pattern (Fig. 8.29K-M). The A microtubule of each doublet is completely
circular and electron-dense and bears two dynein arms, whereas the lumen of
the incomplete subunit B is electron-lucent. In the axoneme of the midpiece,
associated peripherally with each doublet and in the same radius is a dense
peripheral fiber (coarse fiber). In cross section, these fibers, which are
approximately equal in size, are circular with a diameter of approximately
30 nm (Fig. 8.29K, L).
A single layer of mitochondria surrounds the centrioles and the axoneme
along the length of the midpiece. In longitudinal section, the mitochondria
appear elongate elliptical and rather loosely aggregated, particularly in the
centriolar region where they tend to a spherical form, with flocculent material
present between them. They are apparently arranged longitudinally in a long
period spiral (Fig. 8.29B, E, F). In transverse section of the spermatozoon, they
"! Reproductive Biology and Phylogeny of Birds
Fig. 8.29 Melopsittacus undulatus, Budgerigar. A. Longitudinal section (LS) of the

full length of the nucleus. B. LS of the full length of the midpiece. C and D. LS of the
acrosomal complex and the anterior region of the nucleus. E and F. LS of the
posterior end of the nucleus and part of the midpiece. Note the nine triplets of the
transversely sectioned proximal centriole (E), which lies at right angles to the distal
centriole, the latter forming the basal body of the axoneme. G. Transverse section
(TS) of the acrosomal region through the perforatorium. H. TS nucleus through the
endonuclear canal. I. TS nucleus posterior to the endonuclear canal. J. TS through
the distal centriole showing granular central element and surrounding
mitochondria. K. TS midpiece at the point of transition between distal centriole and
axoneme. L. TS midpiece posterior to the distal centriole showing the 9 + 2

Avian Spermatozoa: Structure and Phylogeny "!!
appear more circular, and approximately nine are contained, tightly

juxtaposed, around the axoneme (Fig. 8.29J-L). Mitochondrial dimensions are
roughly 0.3 ¥ 0.15 ¥ 0.15 mm. The mitochondrial matrix is moderately electron-
dense and the cristae, although difficult to discern, appear to be linear or
slightly curved.
The estimate of 28-30 mitochondria in budgerigar sperm made by Samour
et al. (1986) may be a severe underestimate as the midpiece has as many as
nine mitochondria in transverse section and eight or more in longitudinal
section.
No annulus is discernible at the distal end of the midpiece, but the
beginning of the tailpiece can be identified by a reduction in the diameter of
the spermatozoon, to ~0.2 mm (Fig. 8.29B). By seemingly lacking an annulus,
parrot sperm differ from those of paleognaths (e.g., Baccetti et al. 1991) and
lower non-passerines including the mallard duck (Humphreys 1972), and the
turkey, chicken, and Guineafowl (Thurston and Hess 1987; this study). An
annulus is basal to amniotes and these non-passerines. Absence is therefore
an apomorphic feature of budgerigar sperm but appears to be homoplastic
relative to absence in passerines.
Despite lacking an annulus, the midpiece-tail junction in parrot sperm is
clearly demarcated, because at this point there is a rather abrupt narrowing of
the spermatozoon. As a fibrous sheath is absent, only a narrow rim of
cytoplasm separates the plasma membrane from the axonemal complex
throughout the length of the axoneme behind the midpiece. (Samour et al.
1986) states the principal piece is composed of a sheath surrounding the axial
filament complex but in the micrograph to which he refers only the plasma
membrane closely unsheathes the axoneme. However, a narrow, 10-nm-wide
ring of cytoplasm with granular inclusions separates the doublets from the
surrounding plasma membrane (Jamieson et al. 1995) but has not been
considered to constitute an amorphous sheath.
The length of the tail (excluding the midpiece) has been estimated from light
micrographs to be 53-55 mm (n = 5), for an entire sperm length of ~70-71 mm
(n = 7) (Jamieson et al. 1995). The total length of the tail, including the
midpiece, is given by Samour et al. (1986) as 54.31 ± 5.97 mm (n = 100).
Transverse sections of the flagellum reveal the typical “9 + 2” microtubular
axoneme, but the peripheral accessory fibers seen in the midpiece region do
not exist (Fig. 8.29M).

axoneme with peripheral dense fibers. M. TS of the axoneme behind the midpiece.
Scale bars = 1 µm (C-M same scale). av, acrosome vesicle; dc, distal centriole; ec,
endonuclear canal; m, mitochondrion; n, nucleus; p, perforatorium; pc, proximal
centriole; pd, pericentriolar density; pf, dense peripheral fiber (coarse fiber). From
Jamieson, B. G. M., Koehler, L. and Todd, B. J. 1995. Anatomical Record 241(4):
461-468, Fig. 1. Reprinted with permission from Wiley-Liss, Inc., a subsidiary of
John Wiley & Sons, Inc.
"!" Reproductive Biology and Phylogeny of Birds
Seminal glomera. The features and parameters of the ductal spermatozoa of

Melopsittacus undulatus correspond very closely with those previously reported
for semen samples by Samour et al. (1986) who conclusively confirmed the
presence of seminal glomera in the cloaca of this species. Conventionally
considered an exclusively passerine feature (Birkhead et al. 1994), the seminal
glomera are situated on either side of the cloaca of male birds and are formed
by convolutions of the terminal end of the ductus deferens. These sacs have
been likened to the eutherian epididymis in that they serve as sites for sperm
storage and appear to play a role in sperm maturation (Middleton 1972). The
studies of Samour et al. (1986) and Jamieson et al. (1995) indicate that in M.
undulatus no significant changes in sperm morphology occur within the
seminal glomera as compared with sperm in the ductus deferens.
8.10.5.2 Nymphicus hollandicus, Platycercus elegans,
Agapornis roseicollis
The spermatozoa of the Cockatiel (Nymphicus hollandicus) (Figs. 8.30A,E, 8.31A-
N), Crimson rosella (Platycercus elegans) (Fig. 8.30B), and Peach-faced lovebird
(Agapornis roseicollis) (Fig. 8.30C, D) essentially agree with the above account
for Melopsittacus undulatus, with some differences in acrosome vesicle size and
midpiece length. Thus a hollow conical acrosome vesicle encloses a stoutly
rod-like perforatorium around which is a small or negligible amount of
subacrosomal granular material. The perforatorium, which is penetrated by
pale canals, extends almost to the tip of the acrosome vesicle and posteriorly
for a short distance into the anterior end of the nucleus within an endonuclear
canal (Fig. 8.31A, B, C). The cylindrical nucleus has a shallow concave basal
fossa housing only the anterior region of the proximal centriole. This centriole
lies parallel to the base of the nucleus and approximately at right angles to the
distal centriole. Both centrioles are embedded in dense material. The axoneme,
arising from the distal centriole, again lacks a fibrous sheath or an amorphous
sheath. In the midpiece, nine coarse fibers are attached externally to the
doublets in the same radii. The axoneme, with its coarse fibers is surrounded
by a single layer of mitochondria, few and ovoid in cross section of the sperm
but seen in longitudinal section to be slightly elongated and apparently
arranged in a long period spiral. No annulus has been demonstrated. The
coarse fibers do not extend behind the midpiece. The midpiece is very short in
N. hollandicus (~2 mm; n=4) (Fig. 8.31D, G, K), but in A. roseicollis (Fig. 8.30D)
Fig. 8.30 Longitudinal section (LS) of the acrosome vesicle, perforatorium and
endonuclear canal of the spermatozoon of A. Cockatiel (Nymphicus hollandicus).
B. Crimson Rosella (Platycercus elegans). C Peach-faced lovebird (Agapornis
roseicollis). D. LS of the distal end of the elongate midpiece of A. roseicollis. E. N.
hollandicus. Adult female. Both sexes have the crest; the only crested parrot. Photo
Barrie Jamieson. av, acrosome vesicle; ec, endonuclear canal; m, mitochondrion,
nucleus; p, perforatorium. A, C and D. From Jamieson, B. G. M., Koehler, L. and
Todd, B. J. 1995. Anatomical Record 241(4): 461-468, Fig. 2A, C, D. Reprinted with

Avian Spermatozoa: Structure and Phylogeny "!#
permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. B. From
Jamieson, B. G. M. 1999. Pp. 303-331. In C. Gagnon (ed). Spermatozoal Phylogeny
of the Vertebrata. The Male Gamete. From Basic Science to Clinical Applications,
Cache River Press, Vienna, USA, Fig. 13J. A, C, and D are to the same scale.
"!$ Reproductive Biology and Phylogeny of Birds
the midpiece has similar dimensions to that of the budgerigar, ~7 mm (n=1).

Nuclear and acrosomal dimensions are in the same order as those of M.
undulatus. They are: for N. hollandicus, length of acrosome 1.4 mm (n = 3);
length of nucleus 6.5 mm (n = 2, width of nucleus proximally 0.5 mm, distally
0.6 mm; for A. roseicollis, length acrosome 1.5 mm (n=2); length nucleus 9.3 mm
(n=1); width of nucleus proximally 0.5 mm, distally 0.6 mm (Jamieson et al.
1995; present study).
Phylogenetic considerations. The conical acrosome vesicle; rod-like
perforatorium; cylindrical, highly condensed nucleus; proximal and distal
centriole embedded in dense material; and the elongate periaxonemal
mitochondrial midpiece of psittaciform sperm are tetrapod
symplesiomorphies. The nine dense peripheral axonemal fibers (coarse fibers)
are an amniote synapomorphy; the fibers differ from those of reptiles, and
particularly of mammals, in being uniform in size. Loss of the fibrous sheath
around the axoneme which is present in paleognaths and lower non-
passerines is an apomorphy known elsewhere in birds only in columbiforms
and passerines. Mitochondria with linear cristae, lacking intra- (or inter-)
mitochondrial dense bodies are apomorphic relative to basal amniotes
(Chelonia, Sphenodon, and Crocodylia). Restriction of the endonuclear
perforatorial canal to the anterior region of the nucleus is an apomorphic
condition shared with other non-passerines (galliforms and the white-naped
crane) and crocodilians. The short distal centriole appears to be a reversal
from the elongate distal centriole of crocodiles, ratites, galliforms and
anseriforms. The acrosomal-nuclear junction, in which the nucleus abuts on
but does not intrude into the acrosome is a synapomorphy of parrot sperm
relative to other non-passerines and crocodile. It is also, homoplastically, a
Fig. 8.31 Nymphicus hollandicus, Cockatiel. TEM micrographs of sperm. A-H, K.

Longitudinal sections. A. Showing the entire length of the nucleus, surmounted by
but not intruding into the acrosome vesicle. B. Acrosome vesicle with perforatorium.
C. Acrosome vesicle and perforatorium which penetrates a considerable
endonuclear canal. D. Centriolar region, showing mitochondria here grouped
around the proximal centriole and the base of the nucleus. Note absence of a
fibrous sheath around the axoneme. E. Developing acrosome vesicle and
perforatorium in a spermatid. F. Proximal and distal centrioles and short midpiece.
G. The same, with longer midpiece. H. Proximal and distal centrioles and
mitochondria of midpiece also extending along base of nucleus. I. Transverse
section (TS) through apex of nucleus, showing perforatorium in endonuclear canal.
J. TS nucleus, showing circular profile. K. LS centriolar region and midpiece. L. TS
showing small number of mitochondria grouped around axoneme in midpiece;
nine uniform dense fibers are attached one to each axonemal doublet. M. TS
axoneme with typical 9+2 arrangement. N. TS of an abnormal axoneme with 18
doublets and four singlets. av, acrosome vesicle; dc, distal centriole; df, dense
fiber; ec, endonuclear canal; m, mitochondrion, nucleus; p, perforatorium; pc,
proximal centriole. F. After Jamieson, B. G. M., Koehler, L. and Todd, B. J. (1995),
Anatomical Record 241(4): 461-468, Fig. 2B. Reprinted with permission from Wiley-
Liss, Inc., a subsidiary of John Wiley & Sons, Inc. A-E, G-N. Original.
Avian Spermatozoa: Structure and Phylogeny "!%
Fig. 8.31
"!& Reproductive Biology and Phylogeny of Birds
synapomorphy of passerine sperm but there the acrosome and nucleus are
more closely contiguous. The short midpiece of N. hollandicus distinguishes
this cacatuine from the four psittacines. The sperm of other cockatoos have
not been investigated but this difference may prove to answer in the positive
the query as to whether cockatiels are true cockatoos. For further phylogenetic
considerations, see section 8.11.
8.10.6 Order Gruiformes
8.10.6.1 Fulica atra, Crex crex

For the gruiform Common coot (Fulica atra) (Retzius 1909) (Fig. 8.10) depicts a
spermatozoon with a needle-like acrosome much shorter than the elongate
nucleus and with a short apparently spiral midpiece with approximately five
gyres. In contrast, the spermatozoon of another rallid, Corncrake (Crex crex)
(Fig. 8.10), appears to have a button-like acrosome and the midpiece consists
of seven tiers of mitochondria grouped around the proximal axoneme but not
notably spiral. Both species have a long flagellum. A single genus and species
of Rallidae was examined by McFarlane (1963) but he gave no account.
8.10.6.2 Grus vipio
The sperm of White-necked crane (Grus vipio) have been examined by light
microscopy (LM), SEM and TEM (Asa and Phillips 1987; Phillips et al. 1987).
By LM sperm are pleomorphic, with nuclei which are straight or curved or S–
shaped or have droplet, spherical or irregular shapes. The acrosome appears
as a spherical structure and these motile cells appear to have a simple
flagellum. There is no obvious midpiece except for a small phase-dense region,
of varying size and density, at the base of the flagellum.
Acrosome. By TEM the acrosome (Fig. 8.32A) is described as a spherical
moderately electron-dense structure. It is perhaps more accurately described
as having the form of a very thick inverted U. The slightly more electron-dense
structure extending from the center of the acrosome into the nucleus is clearly
a well developed but fairly short perforatorium, the portion of which lying in
the short endonuclear canal (anterior nuclear fossa) is approximately equal in
length to the portion filling the subacrosomal space; the length of the entire
perforatorium is here estimated at about 0.56 µm and the length of the
acrosome vesicle 0.57 µm for a width of 0.48 µm.
Nucleus. Crane spermatozoa are unique for Aves, so far as is known, in that
the degree of condensation varies among spermatozoa, but in no
spermatozoon does chromatin become highly compacted. Incomplete
condensation of chromatin may be responsible in part for the variety of sperm
head shapes (Phillips et al. 1987). From an SEM micrograph, the nucleus
appears stout and moderately elongate.
Midpiece. The midpiece (Fig. 8.32B) appears by TEM to consist of a cluster of
small mitochondria grouped at the base of the nucleus with a length of only
about 0.8 µm. It is here considered that a small density seen on each side at its
Avian Spermatozoa: Structure and Phylogeny "!'
Fig. 8.32 Grus vipio, White-naped crane. A. Longitudinal section (LS) of the
acrosome vesicle, perforatorium and short endonuclear canal. B. LS midpiece
showing that the irregular mitochondria are located in an enlarged region at the
base of the flagellum. C. Sperm chromatin is never highly compacted and the
degree of compaction varies between spermatozoa. A distal centriole is seen in
TS. D. The variability in size of the nucleus is not always a function of chromatin
condensation. A. ¥ 42000. B. ¥ 49300. C. ¥ 16000. D. ¥ 16000. Relabeled after
Phillips, D. M., Asa, C. S. and Stover, J. 1987. Journal of Submicroscopic Cytology.
19(3): 489-494, Figs. 3-6.
posterior end may be a small annulus. In a transverse section of the

spermatozoon (Fig. 8.32C) mitochondria can be seen to surround the distal
centriole. A proximal centriole is not reported or figured.
Axoneme. In some transverse sections (Fig. 8.32B) there appears to be a very
narrow granular layer between the 9+2 axoneme and its plasma membrane.
This is possibly a vestigial amorphous sheath. However, Phillips et al. (1987)
state that an amorphous sheath is absent as are outer dense fibers.
Remarks. Phillips et al. (1987) mentions that 11 other species of crane were
found to have similarly pleomorphic sperm (Russman and Harrison 1981).
Gee and Temple (1978) found no correlation between the percentage of cell
types in the ejaculate and fertilizing capacity.
Phillips et al. (1987) considered the Grus vipio sperm to be the simplest
known non-passerine sperm. It is here considered that this simplicity is
probably due to reduction relative to paleognaths and, for instance,
galloanserans. A perforatorium similar to that of galloanserans has been
retained but the acrosome has lost its pointed form. The midpiece has become
reduced and simplified relative to the other groups and the absence or weak
development of the amorphous sheath and absence of dense fibers must be
considered further reductions. However, the reduction of the midpiece it not
constant for gruiforms as it is well developed in Fulica atra and Crex crex (Fig.
8.10). The button-like acrosome in Crex resembles that of Grus in external form
and there is perhaps a possibility that the needle like acrosome depicted for
Fulica by Retzius (1909) is an exposed perforatorium after loss of the acrosome
vesicle. If not, gruiforms must be considered unusual in having both
lanceolate and button-like acrosomes.
8.10.7 Order Charadriiformes

Harshman (Chapter 1) cites evidence that monophyly of both Pelecaniformes
and Ciconiiformes is falsified by the close relationship among the
pelecaniform family Pelecanidae (pelicans) and the two charadriiform families
Scopidae (hamerkop) and Balaenicipitidae (shoebill). However,
Charadriiformes is monophyletic when turnicids are added to the order. The
suborder Charadrii of the order Ciconiiformes is therefore restored to the
Charadriiformes in the present account.
8.10.7.1 Constituent families
Retzius (1909) illustrated the spermatozoon of the Common guillemot (Uria
aalge). It has a button-like acrosome, elongate fusiform nucleus, a midpiece
about one third the length of the nucleus and with approximately seven tiers
of mitochondria and a short free flagellum (Fig. 8.10).
McFarlane (1963) examined the sperm of six families of charadriiforms,
with a total of nine genera, by light microscopy (Fig. 8.33), though giving only
a fleeting account. He found the underlying feature of the sperm of this order
to be a cylindrical shape with a midpiece of slightly small diameter. The very
small acrosome vesicle was button-like or subspheroidal (Fig. 8.33).
Representative of the four families Recurvirostridae (stilts and avocets),
Charadriidae (plovers and lapwings), Laridae (skuas, gulls and terns) and
Alcidae (auks and puffins) showed only minor variations of this pattern. This
pattern, with small subspheroidal acrosome, was also demonstrated for Larus
canus, Common gull (Ballowitz 1888), L. fuscus, Lesser black-backed gull,
(Retzius 1909) (Fig. 8.10), and Vanellus vanellus, the Lapwing (Ballowitz 1888;
Retzius 1909) (Fig. 8.10).
Avian Spermatozoa: Structure and Phylogeny ""
Specimens of the Scolopacidae (sandpipers, phalaropes, and allies), though

true charadriiforms (see Fig. 1.2, Harshman, Chapter 1) exhibited a remarkable
contrast. In scolopacids, represented by Actitus macularia, Spotted sandpiper
(Fig. 8.33E), the spermatozoon has an elongate, spiral form very reminiscent of
the passerines. This form was seen in Actitus and Catoptrophorus by McFarlane
(1963) and was illustrated in the scolopacids Calidris (=Tringa), the Alpine
dunlin, Tringa (=Totanus) ochripus, the Green sandpiper, Scolopax rusticola, the
Woodcock, and in a somewhat attenuated form in Philomachus pugnax, the
Ruff, by Retzius (1909) (Fig. 8.21). While regarding similarity of scolopacid
and passerine sperm as convergence, McFarlane (1963) considered, perhaps
not entirely reasonably, that it might indicate a more recent origin for the
family than the remainder of the charadriiforms. We may note that
convergence is supported by the evidence from DNA hybridization (Sibley
and Ahlquist 1990) which indicates that scolopacids do not have passerine
affinities but lie in a larid through jacanid clade. Within this clade
Scolopacidae are distinct in grouping with Jacanidae and relatives and not
with Charadriidae and Laridae which occupy a second subclade. It remains
Fig. 8.33 Variation of spermatozoa among charadriiforms. A. Fratercula arctica,

Atlantic puffin. B. Gelochelidon nilotica, Gull-billed Tern. C. Himantopus mexicanus,
Black-necked stilt. D. Charadrius wilsonius, Wilson’s plover. E. Actitus macularia,
Spotted sandpiper. Relabeled after McFarlane, R. W. 1963. Proceedings of the XIII
International Ornithological Congress: 91-102, Fig. 6.
to be investigated what features of the reproductive tract or fertilization

biology are correlated with this superficially passerine-like sperm
morphology.
8.10.7.2 Jacana jacana
Spermiogenesis in Jacana jacana (Jacanidae) has been described by Saita et al.
(1983). Although the structure of the mature spermatozoon is not specifically
treated the chief features of its morphology are evident (Figs. 8.34). Tripepi et
al. (1991) have commented on, and illustrated the microtubules of the
spermatid of this species.
The spermatozoon or late spermatid has the typical charadriiform
morphology of button-like (subspheroidal) acrosome vesicle, elongate
cylindrical nucleus, and straight midpiece. As usual the proximal centriole is
Fig. 8.34 Jacana jacana. A. Longitudinal section (LS) of nucleus of advanced

spermatid capped by the subspheroidal acrosome vesicle. B. Same. C. Transverse
sections (TS) of nuclei and surrounding mitochondria of spermatids. D. LS
spermatozoon at junction of midpiece and principal piece. E. TS of spermatozoa,
showing circlet of several mitochondria and small dense fibers attached to the
doublets of the axoneme. F. TS posterior region of principal piece with fibrous
(amorphous) sheath less developed than further anteriorly. an, annulus; av,
acrosome vesicle; ax, axoneme; df, dense fiber; fs, fibrous sheath; n, nucleus.
Rearranged and relabeled after Saita, A., Longo, O. M. and Tripepi, S. 1983.
Accademia Nazionale dei Lincei. (Rendiconti della Classe di Scienze fisiche,
matematiche e naturali) 74: 417-430, Figs. 14-19.
Avian Spermatozoa: Structure and Phylogeny ""!
transverse to the long axis of the sperm and the distal centriole, which is
slightly longer, forms the basal body in this axis.
Further features which are possibly general for charadriiforms are the
absence of a perforatorium, the midpiece consisting of several mitochondria
arranged around the axoneme and terminating at an annulus, and the
presence of an amorphous sheath around the post-mitochondrial axoneme.
The arrangement of microtubules in the spermatid is as in Caprimulgus
(Tripepi et al. 1991).
Remarks. Persistence of the amorphous sheath, seen also, for instance, in
galliforms and anseriforms, is a plesiomorphic feature but the rounded
acrosome vesicle is clearly apomorphic as it is conical in those orders, in
ratites and in crocodiles.
The absence of a perforatorial rod is a major distinction from the sperm of
the gruiforms as exemplified by Grus vipio, described by Phillips et al. (1987)
but whether this is a constant difference between the two orders remains to be
ascertained. There is also no evidence that G. vipio sperm possess an
amorphous sheath but this also requires confirmation.
8.10.8 Order Falconiformes

Falcons and their near relatives were placed in the infraorder Falconides,
within the Suborder Ciconii, of the order Ciconiiformes by Sibley and Ahlquist
(1990). However, from analysis of molecular sequences placement within a
ciconiiform or charadriiform assemblage does not appear acceptable. There is
some morphological evidence for monophyly of Falconiformes but no
molecular analyses have so far succeeded in putting all families together. Nor,
however, is there strong evidence for relationship of any falconiform family to
any non-falconiform family (see Harshman, Chapter 1).
The only ultrastructural representation of falconiform sperm appears to be
a scanning electron micrograph of spermatozoa of the peregrine falcon by
Wagley (Anon. 1980) (Fig. 8.35).
Despite re-establishment of falconiforms as a separate order, the
morphology of the falcon sperm is consistent with placement of falcons in the
Charadriiformes so far as can be judged by our limited knowledge of these.
Thus the falcon sperm is very similar to the spermatozoa of the Lapwing
(Vanellus) and the Gull (Larus), both charadriiforms, as depicted from light
microscopy by Retzius (1912) (Fig. 8.10). Similarities are the short, domed
acrosome; the stout cylindrical nucleus many times the length of the acrosome;
and the midpiece consisting of a non-helical cluster of mitochondria and
much shorter than the nucleus. This structure is consistent with a drawing of
the whole sperm by light microscopy by Ballowitz (1888) (his Fig. 114), for the
Black kite (Milvus ater). It is, however, different from other charadriiforms such
as the Woodcock (Scolopax), the Dunlin (Calidris) and the Green sandpiper
(Tringa), in which the acrosome is a tortuous cone, the nucleus is somewhat
helical and the midpiece is more elongate (Fig. 8.10).
""" Reproductive Biology and Phylogeny of Birds
Fig. 8.35 Falco peregrinus. Scanning electron micrograph of spermatozoa.

Relabeled from Anonymous 1980. Western Release Program-1980. The Peregrine
Fund Newsletter. T. J. Cade and P. R. Dague (eds). 8, Fall 1980: 4. Photo L. Wagely.
8.10.9 Order Passeriformes

The non-passerine sperm is generally rather plain and elongate. The
passerine type has a pronounced spiral to the head region, approaching a
conic helix in some forms, an enormous acrosome, often 3-4 times the length
of the nucleus. Frequently an helical membrane extends some distance along
the axial filament (McFarlane 1963). This succinct definition in fact
characterizes the higher, non-corvidan passerines (Passeri). In suboscines
and, usually, Corvida the acrosome is shorter than the nucleus and in the
suboscines the ‘helical membrane’, at least in the form seen in oscine sperm, is
absent.
8.10.9.1 Suborder Tyranni (Suboscines), Parvorder Tyrannida
The spermatozoa of the Tyrannidae are very poorly known ultrastructurally.
Species that have been investigated are Western kingbird (Tyrannus verticalis,
McFarlane 1971 fide Koehler 1995); Eastern Kingbird (T. tyrannus, Feduccia
1979); Eastern wood peewee (Contopus virens, Feduccia 1979) and Great
crested flycatcher (Myiarchus crinitus (=M. griseisticta), McFarlane 1971 fide
Koehler 1995; Asa and Phillips 1987) but accounts are fragmentary.
Avian Spermatozoa: Structure and Phylogeny ""#
However, though the account was very brief, McFarlane (1971) examined
the sperm of six families and 23 genera of the suborder Tyranni by light
microscopy.
Feduccia (1979), in his innovative phylogenetic paper on avian ear
ossicles, showed that sperm of the suboscines Tyrannus tyrannus and Contopus
virens (Fig. 8.36) resemble those of oscines in occurring in the testes in sperm
bundles. However, the suboscine sperm differed from those of oscines in
important respects indicated below (see Tyrannus tyrannus). Asa and Phillips
(1987) added, for the suboscine Myiarchus griseisticta, that there are features
resembling both non-passerines and oscines.
Unlike most oscines, the acrosome and midpiece are shorter than the
nucleus and, according to McFarlane (1963), there are no spiral head
membranes or helical tail membranes in the Tyranni. However, an
‘undulating membrane’ of somewhat different construction occurs (see
sections 8.10.9.2-5, below). The ratio between head length and tail length is
conspicuously lower (1.3-3) than in all oscines (5.5-22) which McFarlane
(1963) examined, excepting the Corvidae and the Lanidae (2.2-3), which are
closely related (Christidis et al. 1996).
The woodhewers (Dendocalaptidae), exemplified by Deconychura typica
(Fig. 8.27G) and ovenbirds (Furnariidae) have a simple sperm that forms a
spiral which completes only one revolution from the acrosome to the
midpiece. A more highly coiled (helical) form occurs in the tyrant flycatchers
(Tyrannidae), exemplified by Tyrannus tyrannus (Fig. 8.27H), cotingas
(Cotingidae), manakins (Pipridae), and ant-thrushes (Formicariidae)
Fig. 8.36 Contopus virens, Eastern wood peewee. Transverse sections of principal
piece of several sperm. Relabeled after Feduccia, A. 1979. Proceedings of the
Biological Society of Washington 92(4): 689-696, Fig. 5.
""$ Reproductive Biology and Phylogeny of Birds
(McFarlane 1963). Sibley et al. (1988) reduced the Cotingidae and Pipridae to
subfamilial rank within the Tyrannidae; an arrangement here observed to
agree with sperm structure. However, the spermatologically similar
Formicariidae retained familial rank in a separate Parvorder Furnariida. In
this parvorder, the Furnariidae, in the superfamily Furnaroidea, contained the
Furnariinae and Dendrocalaptinae, both with the simple sperm form
mentioned above, and the Formicariidae, with more helical sperm, occupied
the separate superfamily Formicaroidea.
8.10.9.2 Tyrannus tyrannus
General morphology. By light microscopy the spermatozoon of Tyrannus
tyrannus (Fig. 8.27H) has been shown by McFarlane (1963) to have an helical
acrosome, nucleus and midpiece. The sperm is said to be similar to that of
corvids but to differ in that in corvids the midpiece is not included in the
spiral portion. Although this appears generally to be true, the midpiece in
Corvus splendens is, at least developmentally, spiral (Bawa et al. 1990).
Acrosome. Although the acrosome is shorter than the nucleus as in non-
passerines [but see also most oscine Corvida] it has lateral projections as in
oscines (Asa and Phillips 1987).
Nucleus. The nucleus is an helical cylinder as in oscines (Asa and Phillips
1987).
Undulating membrane. Asa and Phillips (1987) showed, for Tyrannus
tyrannus, that a bundle of singlet microtubules comprising what was
considered the oscine-like “helical membrane” is found either completely
encircling or clustered on one side of the midpiece and principal piece, the
latter condition presumably thought to resemble that in oscines (see also
Contopus virens, 8.10.9.5, below).
Neck region. The neck region has two centrioles as in non-passerines in
contrast with the single, distal centriole of oscines (Asa and Phillips 1987).
Mitochondria. In some sagittal sections of the midpiece, mitochondria encircle
the distal centriole but in others they appear more clustered on one side, a
combination of oscine and non-passerine features (Asa and Phillips 1987).
Dense fibers. In Tyrannus tyrannus, small dense fibers are present in the
proximal region of the principal piece only (Asa and Phillips 1987).
8.10.9.3 Tyrannus verticalis
Koehler (1995) cites some of the unpublished data of McFarlane (1971) for
Tyrannus verticalis, the Western kingbird: length sperm 55 µm, length acrosome
2.5 µm, length nucleus14 µm (therefore an acrosome:nucleus ratio of only 0.18,
contrasting with oscines), length midpiece 4 µm. The sperm is helical but no
helical membrane is reported. There is a shallow postnuclear fossa and
accessory dense fibers are present.
Avian Spermatozoa: Structure and Phylogeny ""%
8.10.9.4 Myiarchus crinitus

Koehler (1995) cites some of the unpublished data of McFarlane (1971) for
Myiarchus crinitus, Great-crested flycatcher: length sperm 50 µm, length
acrosome 2.5 µm, length nucleus 19.5 µm (therefore an acrosome:nucleus ratio
of only 0.14, contrasting with oscines), length midpiece 3.3 µm (again much
shorter than in Passerida). The sperm is helical; unusual for suboscines an
‘helical membrane’ is reported but is limited to the acrosome and is reduced
in size. The mitochondria are spiral but describe only one revolution. As
always in passerines, an annulus is absent. There is a shallow postnuclear
fossa and accessory dense fibers are present.
8.10.9.5 Contopus virens
Although having an ‘undulating membrane’ as in oscines, the suboscine
sperm, exemplified by Contopus virens (Fig. 8.36) and, according to Feduccia,
that of Tyrannus tyrannus, differs in the geometry of the microtubules that
surround the axoneme; the axoneme is said to be separated from the bundle of
microtubules by a double plasma membrane. In addition, the microtubules are
not arranged in an helical array with a mitochondrial component bound
around the axoneme for a mitochondrial component is said not to be
distinguishable. The undulating membrane of light microscopy in both oscine
and suboscine sperm is due in oscines to an helically wound tripartite
membrane in the latter (lapsus “former”) to an” undulating band” of singlet
microtubules that completely surround the axoneme (Feduccia 1979) though
here considered to characterize the spermatid.
8.10.10 Suborder Passeri (Oscines)
8.10.10.1 Introduction to oscine sperm ultrastructure

This introduction incorporates previous accounts of oscine sperm
ultrastructure (see Table 8.4) (notably Humphreys 1972; Asa and Phillips
1987; Henley et al. 1978; Vernon and Woolley 1999), the accounts from light
microscopy of McFarlane (1963), and observations, particularly those on
Myrmecocichla formicivora and Philetairus socius by Jamieson, Hodgson and
Spottiswoode (unpublished). Henley et al. (1978) made light and TEM
observations on Turdus migratorius, Pipilo erythophthalmus, Vireo olivaceus,
Zonotrichia albicollis, Piranga rubra, Parus bicolor, Cyanocitta cristatus and
Melanerpes carolinus but only distinguished between M. carolinus, P.
erythophthalmus, C. cristatus and T. migratorius by name.
Sperm bundles. Spermatogenesis in oscines (always?) occurs in a syncytial
mass. Thus a multinucleate spermatid normally differentiates into uninucleate
spermatozoa. In oscines a corollary of this syncytial development is that the
sperm in a bundle are very regularly arranged in precise register (Fig. 8.44). In
contrast, the sperm produced from the syncytial mass in the non-passerine
Melanerpes are not precisely arranged. The testicular sperm of oscines are not
motile in 0.9% saline whereas those of the piciform are (Henley et al. 1978).
""& Reproductive Biology and Phylogeny of Birds
Head. The acrosome and nucleus constitute the head. This was found to be
helical in 18 passeridan species examined by Birkhead et al. (2006) with the
exception of the rounded head of the Eurasian bullfinch. The number of head
gyres ranged from 2.3 in Song thrush (Turdus philomelos) to 4.6 in Sedge
warbler (Acrocephalus schoenobaenus); the corvids, Carrion crow (Corvus corone)
and Rook (C. frugilegus) had 3.5 and 3.6 turns. All of the 18 species illustrated
by Retzius have helical heads (6 Corvida, Figs. 8.37, 8.38; 12 Passerida, Figs.
8.41, 8.42).
Acrosome. The very long, tapering acrosome can be three or four times the
length of the albeit short nucleus (McFarlane 1963). Some acrosome:nucleus
ratios are given in Table 8.6. These may be compared with ratios for
paleognaths and non-passerines in Table 8.5. For references see species cited.
It is seen that the acrosome is much shorter than the nucleus in the
suboscines and in typical Corvida, though longer than the nucleus in Vireo
olivaceus. In contrast, most Passerida have an acrosome:nucleus ratio in excess
of 1, reaching 4 in the Summer tananger. The hirundins, other than the Violet-
green swallow, and possibly the blackbird, are exceptional in having a ratio
of less than 1.
Table 8.5 Ratio of acrosome length : nuclear length in some paleognaths and non-passerines
Taxon Common name Acrosome-nucleus ratio (mm)

Struthioniformes
Struthio camelus Ostrich 0.17
Dromaius novaehollandiae Emu 0.14
Galliformes
Coturnix japonica Japanese quail 0.13
Coturnix chinensis Blue-breasted quail 0.3
Meleagris gallopavo Turkey ca 0.16-0.25
Numida meleagris Guineafowl 0.14
Piciformes
Dendrocopos major Great spotted woodpecker 0.09
Apodiformes
Apus apus Common swift 0.03
Strigiformes
Strix aluco Tawny owl 0.03
Psittaciformes
Psittacus sp. Parrot 0.1
Melopsittacus undulatus Budgerigar 0.19
Nymphicus hollandicus Cockatiel 0.2
Columbiformes
Columba livia Domestic pigeon 0.1
Charadriiformes
Philomachus pugnax Ruff 0.04
Jacana jacana Jacana 0.07
Falconiformes
Falco peregrinus Peregrine falcon 0.23
Avian Spermatozoa: Structure and Phylogeny ""'
Table 8.6 Ratio of acrosome length : nuclear length in some suboscines and oscines
Taxon Common name Acrosome-nucleus ratio (mm)

Tyranni (Suboscines)
Tyrannus verticalis Western kingbird 0.18
Myiarchus crinitus Great crested flycatcher 0.14
Corvida
Perisoreus infaustus Siberian jay <0.1
Corvus frugilegus Rook <0.5
Vireo olivaceus Red-eyed vireo 1.5
Muscicapidae-Turdidae
Ficedula hypoleuca Pied flycatcher 1.6
Myrmecocichla formicivora Southern ant-eater chat 2.6
Sturnus vulgaris Starling 1.3
Turdus migratorius American robin 2.7
Turdus philomelos Song thrush 1.5
Turdus merula Blackbird 0.8?
Icteridae
Molothrus ater Brown-headed cowbird 2
Agelaius phoeniceus Red-winged blackbird 1.6
Quscalus quiscala Common grackle 1.75
Thraupidae
Piranga rubra Summer tananger 4
Hirundinidae
Tachycineta thalassina Violet-green swallow 3
Hirundo rustica Barn swallow <1
Riparia riparia Sand martin <1
Emberizidae
Cardinalis cardinalis Northern cardinal 1.65
Passeridae
Passer italiae Italian sparrow 1.3
Passer domesticus House sparrow 1-1.2
Ploceidae
Philetairus socius Social weaver 1.2
Paridae
Parus bicolor Tufted titmouse 1.2
Of all known oscine sperm, the American robin (Turdus migratorius)

examined by Henley et al. (1978), was exceptional in having a hooked
acrosome; however, the acrosome crest in the Social weaver (Philetairus socius)
(see below) could also be considered hooked.
Acrosome keel. An helical ridge on the acrosome is reported for the corvid
Corvus splendens (Bawa et al. 1990) and lateral projections or an helical
membrane have been reported for the acrosome of the suboscines Tyrannus
tyrannus (Asa and Phillips 1987) and Myiarchus crinitus (MacFarlane 1971,
Fide Koehler 1995).
"# Reproductive Biology and Phylogeny of Birds
The passeridan acrosome had formerly been regarded as an helical

structure with or without an encompassing helical keel. The distinctness of
this keel from the mitochondrial helix has often been unrecognized, the two,
with or without a nuclear keel, being termed the ‘helical membrane’. As
shown in this chapter, an helical keel (‘helical membrane’) on the acrosome
but not extending onto the nucleus has in fact been described, or is apparent
from illustrations, in ultrastructural works for many species: Piranga rubra
(Thraupidae); Tachycineta thalassina, the Violet-green swallow (Hirundinidae);
Turdus migratorius, American robin (Turdidae) (McFarlane 1971 fide Koehler
1995); Myrmecocichla formicivora, Southern ant-eater chat (Jamieson, Hodgson
and Spottiswoode, unpublished); Turdus merula, Blackbird (Turdidae) and
Passer italiae, Italian sparrow (Passeridae) (Furieri 1961); Sturnus vulgaris,
Starling (Sturnidae) (Vernon and Woolley 1999); Lonchura striata, ‘Loverbird’
(Kondo et al. 1988); Passer domesticus, House sparrow (Passeridae) (Furieri
1961), Passer diffusus, Grey-headed sparrow (Passeridae); Philetairus socius,
Social or Sociable weaver; Quelea quelea, Red-billed quelea; Euplectes orix, Red
bishop; Ploceus capensis, Cape weaver (Ploceidae) (Jamieson, Hodgson and
Spottiswoode, unpublished); and Parus bicolor, Tufted titmouse, (Paridae)
(Koehler 1995). Furieri (1961) clearly depicts the acrosomal keel of T. merula
and P. italiae as a lateral projection, differing in constitution from what is here
called the acrosome core, as also shown for Myrmecocichla formicivora and
Philetairus socius (Jamieson, Hodgson and Spottiswoode, unpublished).
The bipartite acrosome. The acrosome, in species as far apart phylogenetically
as Myrmecocichla formicivora and Philetairus socius is shown to be bipartite in
nature in a new interpretation of acrosome structure (Jamieson, Hodgson and
Spottiswoode, unpublished). An acrosome core is surmounted by an acrosome
crest, like a stock and scion, and the core is invested by a layer which is a
posterior extension of the crest, here termed the crest layer or sleeve. The
acrosome helix is a lateral extension of the crest layer with or without
inclusion of the acrosome core. The crest anterior to the core also shows lateral
extensions which constitute a continuation of the acrosome helix. Although
the crest is longer in M. formicivora than in P. socius, in both the helix of the
crest proper consists of only a single lateral extension or spur whereas the
acrosome core enveloped by the crest layer bears three extensions when
viewed in longitudinal section. The acrosome keel thus has only one full gyre
on the crest and three gyres on the core. It would be of great interest to
investigate development during spermiogenesis of the crest and the spur. It is
possible that the crest and its layer surrounding the core is the true acrosome
vesicle and that the core is subacrosomal or perforatorial material, in which
case loss of the perforatorium would not be an apomorphy of passeridans.
However, the crest and its posterior layer may merely be a modification of the
surface layer of an acrosome vesicle represented by the core. These alternative
possibilities would easily be resolved by study of the spermatid.
Nucleus. The nucleus of oscine sperm (Figs. 8.9B, 8.27K, 8.37, 8.39, 8.41, 8.55,
8.49, 8.53), twisted into an helical cylinder, is short compared with that of
non-passerines. Its length is in the order of 3-5 µm (e.g. Koehler 1995)

compared with 6.5-10 µm in psittaciforms, ca 13 µm in Struthio, ca 12 µm in
Dromaius, 21 µm in Coturnix japonica, 6.4 µm in C. chinensis, 7-9 µm in Turkey,
10-21 µm in Guineafowl and Rooster, i.e. 6-21 µm in galliforms.
Neck region. In contrast to the spermatozoa of non-passerines and, so far as
is know, suboscines, the neck region in oscines contains only one, the distal,
centriole. In all passerine species examined by Asa and Phillips (1987)
excepting the White-eyed vireo, Vireo griseus, electron-dense material
surrounds the neck anterior to the mitochondria of the midpiece (Fig. 8.9B), as
also reported for chaffinch sperm (Furieri 1961, 1962). This dense ring is well
developed in Myrmecocichla formicivora but is absent in the ploceid Philetairus
socius (Jamieson, Hodgson and Spottiswoode, unpublished).
Midpiece. No annulus has been detected in oscine sperm and therefore there
is no sharp distinction between midpiece and principal piece (that portion of
the axoneme surrounded by dense fibers). The mitochondria do not surround
the axoneme in a circlet but wind in a single helical strand along what is
defined as the midpiece (Figs. 8.9A, D, 8.40B, 8.46-8.49, 8.51, 8.56). The extent
of the mitochondria along the tail is very variable. For instance, in the White-
eyed vireo, as, we may add, in Corvidae (Fig. 8.37), Lanius (Fig. 8.37), and
Oriolus, the mitochondria extend only a short distance along the tail, whereas
in Taeniopygia guttata, Zebra finch, the mitochondrion is reported to extend
along 46 µm of the 73 µm flagellum (Vernon and Woolley 1999). However,
Birkhead, Pellatt et al. (2005) have shown much variation in the length of the
midpiece and other sperm components in Zebra finch. They showed that in
this species there is an extraordinary degree of inter-male variation in sperm
design that is independent of sperm swimming velocity. A quantitative
genetic study using data from over 900 zebra finches showed that sperm head,
midpiece and flagellum length are heritable, that negative genetic correlations
exist between sperm traits, and that significant indirect (maternal) genetic
effects exist. It was hypothesized that selection on the zebra finch sperm
phenotype may be low because sperm competition is infrequent in this
species and that this, in combination with negative genetic correlations and
maternal genetic effects, may account for the variation in sperm phenotype
between males.
In other Passerida, the straightened length of the helix, in mm, is at its
shortest at 1.86 in the Eurasian bullfinch (Pyrrhula pyrrhula), and 12.33 in
Beavan’s bullfinch (Pyrrhula erythaca) but the range in other species is 53.95 in
Song thrush (Turdus philomelos) to 138.48 in Linnet (Carduelis cannabina)
(Birkhead et al. 2006).
The recorded number of midpiece curves (gyres) in Passerida, excepting the
Eurasian bullfinch which lacks a mitochondrial helix, varies from 13.5 in
Song thrush) to 29.4 in Moustached warbler (Acrocephalus melanopogon). Thus
there is not a total correlation between helix length and numbers of gyres. In
contrast only a single gyre is reported for the corvidan Carrion crow (Corvus
corone) and Rook (Corvus frugilegus) (Birkhead et al. 2006) as is evident, with
"# Reproductive Biology and Phylogeny of Birds
small variations in length, from illustrations by Retzius (Fig. 8.37) for these
species and Magpie (Pica pica), Jackdaw (C. monedula), Siberian Jay (Perisoreus
infaustus), and Red-backed shrike (Lanius collurio).
After loss of the microtubular bundle in the excurrent ducts, the “helical
membrane” [a term used for an helical external structure of the spermatozoon
irrespective of its composition] distal to the acrosome is composed only of the
mitochondrial spiral (Asa and Phillips 1987) or may be accompanied by other
helical components (see below).
The mitochondrial and fibrous helices. The portion of the so-called helical
membrane surrounding the axoneme is what is here termed the mitochondrial
helix in passeridan species. It has been shown by Jamieson, Hodgson and
Spottiswoode (unpublished) that the muscicapid Myrmecocichla formicivora, a
second helix, termed the fibrous helix, intertwines with at least the more
proximal region of the mitochondrial helix. It is clearly the smaller structure
described for Sturnus vulgaris (also a muscicapoid) by Vernon and Woolley
(1999) and termed by them ‘(x)’, the larger structure being the single, helical
chain of (fused) mitochondria. It was earlier described by Furieri (1961) as a
small crest (i.e. keel) in Turdus merula and by Henley et al. (1978), as a fibrous
component, in T. migratorius (both also muscicapoids). Thus the fibrous keel is
absent at maturity not only in the ploceid Philetairus socius (Jamieson,
Hodgson and Spottiswoode, unpublished) but also in the Zebra finch
(Taeniopygia (=Poephila) guttata) (Fawcett et al. 1971); and Lonchura striata
(Kondo et al. 1988) and is not reported for the many other described passeridan
sperm with the exception, requiring confirmation, of Passer italiae where there
is said to be a large fiber helically surrounding the anterior region of the
axoneme in addition to the mitochondrial helix (Furieri 1961).
Loss of microtubular helix. The “helical membrane” has different
components in the testis and in the excurrent ducts. In testicular spermatozoa,
a single bundle of microtubules winds helically along the nucleus, midpiece
and principal piece. The helical bundle is said (Asa and Phillips 1987) never
to extend anteriorly beyond the nucleus. However, Bawa et al. (1990) state that
in Corvus splendens the microtubules extend around the acrosome, though with
no supporting micrograph, other than one (Fig. 8.39E) showing what are here
termed the spurs.
Contrary to the views of Fawcett et al. (1971) for the nucleus, Bawa et al.
(1990) and Asa and Phillips (1987) consider that the microtubular bundle may
be involved in disposition of the mitochondrial array. It will be shown below,
however, that in the Corvida, the microtubular bundle of the spermatid
(microtubular helix of the present author) continues independently posterior
of the mitochondria of the short midpiece.
After their release into the excurrent ducts, oscine sperm lose their external
microtubular helix (Nicander 1970b; Henley et al. 1978), although a portion of
it has been reported to remain on the spermatozoa of the American robin
(Henley et al. 1978), an observation requiring confirmation.
Avian Spermatozoa: Structure and Phylogeny "#!
Fig. 8.37 Drawing by light microscopy of corvidan sperm. Pica pica, Magpie;
Corvus corone, Carrion Crow. After Retzius, G. 1909. Biologische Untersuchungen,
Neue Folge 14(10): 89-122 Taf XXXII, Figs. 1, 12. Corvus monedula, Jackdaw;
Lanius collurio; Red-backed shrike. After Retzius, G. 1911. Biologische
Untersuchungen, Neue Folge 16: 89-92 Taf XXVII, Figs. 1, 2, 24. Perisoreus
infaustus, Siberian jay; Corvus frugilegus, rook. After Retzius, G. 1912. Biologische
Untersuchungen, Neue Folge 17: 95-99 Taf XIV, Figs. 21, 22, 30.
Origin of helical coiling. The structure and development of the microtubular

helix was elucidated by Fawcett et al. (1971). In the intermediate and later
stages of “finch” (Taeniopygia (=Poephila) guttata, Zebra finch, fide Asa and
"#" Reproductive Biology and Phylogeny of Birds
Phillips 1987) spermatids (Fig. 8.43B), a hundred or more microtubules occur

in a single bundle to which we have referred above which has an helical
course around the nucleus. The microtubules are arranged in rows (layers)
that alternate with pairs of parallel membranes bounding flat cisternae. The
bundle terminates anteriorly at one side of the base of the acrosome where the
microtubules end in the concavity of a caplike saccule which seems to be a
continuation of the outermost cistern of the microtubule bundle. This saccule,
with its microtubules, is here considered to be the equivalent of the spurs seen
at the base of the acrosome in Corvus splendens sperm. These authors argue
against the view that the microtubules are responsible for the helical shaping
the nucleus. On the other hand, they do consider them to be involved in
helical shaping of the mitochondria of the midpiece. When the mitochondria
first gather around the base of the flagellum in spermiogenesis they are
arranged end to end parallel with the axoneme (Fig. 8.43A). Concurrently with
the development of the microtubules bundle in the head region and its
extension caudad, the mitochondria take on an helical configuration
complementary to that of the microtubule bundle (Fig. 8.43B). Thus the
axoneme is enveloped by a double helix, one element of which is
mitochondrial and the other microtubular. The microtubules disappear as the
spermatid matures, as noted above, leaving a single helical mitochondrial
sheath with the same pitch as that of the helical nucleus (Fig. 8.43C). These
authors suggest that the microtubular helix, though a transient organelle that
participates in spermatid elongation, may determine the configuration of the
mitochondrial sheath by imposing upon it the same pitch as that of the
nucleus. The microtubules extend only around the base of the acrosome
vesicle and it is considered that the acrosome acquires is helical form without
their participation. Its helical form seems to be acquired as a result of
asymmetry of the contents of the acrosome vesicle much as coiling of the
nucleus is attributed to properties of the chromatin (Fawcett et al. 1971).
Tripartite helix. Henley et al. (1978) also demonstrated the tripartite nature of
the “undulating membrane” [helical membrane here termed the tripartite
helix] in oscine sperm but they showed that, in addition to the basic three
components of axoneme, mitochondrial sheath [mitochondrial helix] and
microtubular helix, there was [at least in Turdus] a fibrous component [fibrous
helix] associated with the mitochondrial sheath. The four components are
shown for Turdus migratorius, the American robin, in Fig. 8.45. They examined
eight oscine species, though only detailing T. migratorius and Cyanocitta
cristatus, and comparing these with the piciform Melanerpes. However, their
summary statement that the eight oscines possessed the tripartite structure,
including the helically wound strand of mitochondria, conflicts with their
suggestion that an “undulating membrane” was absent from the included
corvid, Cyanocitta, and the condition for the corvidan Vireo olivaceus was not
described. At maturity the tripartite helix is reduced to only the mitochondrial
helix, as shown by Fawcett et al. (1971; Fig. 8.43C), with or without other
components described here.
Avian Spermatozoa: Structure and Phylogeny "##
Granular body and helix. A major addition to our knowledge of the helical
components of the passerine sperm has been the demonstration by Tripepi
and Perrotta (1991) in several species of the Passeroidea and a certhioid of a
‘granular body’ (GB) which consists of many diffuse granules of medium size
in the early spermatid and becomes an helical structure, here termed the
granular helix (GH), investing the distal [and only] centriole and the proximal
region of the axoneme. Their view that the GB is constant in, and distinctive
of, the Passeroidea is not here confirmed as it has not been demonstrated, for
instance, in the estrildid Taeniopygia guttata (Fig. 8.43) or the ploceid
Philetairus socius (Fig. 8.53) and is present in the Treecreeper (Certhia
brachydactyla) now in the Certhioidea. However, in T. guttata (Fig. 8.43) the
mitochondrial helix can be seen to be embedded in a granular matrix which it
is here considered may be the homologue of the GB. Earlier, Humphreys (1972)
had very briefly referred to a ‘granular structure’ in Canary sperm. Koehler
(1995) referred to a spiral granular mass or granular body in the anterior
region of the midpiece of some passerine sperm in which it forms a spiral
mass located anterior to the mitochondrion, as observed in Passer domesticus,
with the anterior end of the mitochondrion being a few micrometres caudal to
the nucleus. This GB was also mentioned for Starling (Sturnidae), Brown-
headed cowbird (Icteridae), and Common Grackle (Icteridae)but not for Great
crested flycatcher (Tyrannidae), Cardinal (Emberizidae), or Red-winged
blackbird (Icteridae).
Further distally, at varying levels in the midpiece proper, the GH is
substituted by the mitochondrial sheath [mitochondrial helix], as mentioned.
It and the mitochondrial helix are invested by the transient microtubular helix
which is lost in the mature spermatozoon so that the fore part of the midpiece
spiral, as see in the Treecreeper (Fig. 8.56E), contains only the GH. The length
of the GH varies among species but it is generally shorter than the
mitochondrial helix.
This pattern of spermiogenesis is said by Tripepi and Perrotta (1991) to be
characterized by presence of the GB, acrosome: nuclear ratio >1, and sperm
length > 100 mm. It was reported for the Wren (Troglodytes troglodytes)
Troglodytidae (Fig. 8.51); Cirl bunting (Emberiza cirlus) Emberizidae (Fig. 8.56);
House sparrow (Passer domesticus), now in the Passeridae; Chaffinch (Fringilla
coelebs) and Greenfinch (Carduelis (=Chloris) chloris) Fringillidae, all of which
are Passeroidea; and Treecreeper (Certhia brachydactyla) Certhiidae,
Certhioidea.
In contrast, in the Corvida, Crow (Corvus corone), Magpie (Pica pica) and Jay
(Garrulus glandarius) Corvidae, Red-backed shrike (Lanius collurio) Laniidae,
and the sylvioids Nuthatch (Sitta europaea) Sittidae and House martin
(Delichon urbica) Hirundinidae, a GB is absent, acrosome: nuclear ratio <1, and
sperm length <100 mm. However, although these claims were made for the
families represented, it has been noted above that the sperm of the Violet-green
swallow (Tachycineta thalassina) has a length of 285 µm.
"#$ Reproductive Biology and Phylogeny of Birds
Function of helical coiling. The function of helical coiling of the sperm and of
the ‘helical tail membrane’ is uncertain. Helical coiling was considered by
McFarlane (1963) to intensify the rotary forward movement of the
spermatozoon. He reasonably considered that the helical tail membrane
(mitochondrial helix) might be instrumental in energy transfer to distal
portions of the axial filament comparable to columbiform sperm, also greatly
elongated, in which the midpiece extends far down the tail.
Passerine sperm typically spin rapidly while seeming to remain virtually
straight. Because there is no evidence for an helical wave on these flagella,
other possible means have been considered whereby rotation about the local
flagellar axis (self-spin) might be achieved. Sometimes, while maintaining
their spinning motion, they adopt a fixed curvature and it is considered that
this must be an instance of bend-transfer circumferentially around the
axonemal cylinder, though the mechanism is obscure (Vernon and Woolley
1999).
Axoneme. The axoneme has the typical 9+2 pattern of microtubules but in
contrast to non-passerines, the outer dense fibers associated with the doublets
are very prominent as shown for Thryothorus ludovicanus, Carolina wren (Fig.
8.9D), Grallina leucocephala, Magpie lark (Fig. 8.40B) Turdus migratorius,
American robin (Fig. 8.45). Myrmecocichla formicivora, Southern ant-eater chat
(Fig. 8.48N, O, P); Sturnus vulgaris, Starling (Fig. 8.49D, E); Philetairus socius,
Social weaver (Fig. 8.53J, K); Emberiza cirlus, Cirl bunting (Fig. 8.56D) and Tree
creeper (Sitta europaea) (Fig. 8.56E). They are uniform in shape at a given level
of transverse section, in contrast with those of mammalian sperm, but may
vary in form posteriad, for instance from comma-shaped to subcircular, as in
Sturnus vulgaris. In the suboscine Tyrannus tyrannus the dense fibers are small
(Asa and Phillips 1987).
A systematic account of passerine sperm follows.
8.10.11 Parvorder Corvida

8.10.11.1 Taxa investigated
Although several species of the Corvida have been examined for spermatozoal
ultrastructure, data provided are scanty and the light microscope accounts,
and particularly the illustrations, of Retzius therefore assume special
importance. Species which he investigated are: Hooded crow (Corvus corone);
Magpie (Pica pica) (Retzius 1909); Jackdaw (Corvus monedula) (Retzius 1911);
Corvus frugilegus (Rook); and Siberian jay (Perisoreus infaustus) (Retzius 1912)
(Figs. 8.37, 8.38). The earlier paper of Ballowitz (1888) also provides useful
information on the Corvida: Rook, Red-backed shrike (Lanius collurio), and
Golden oriole (Oriolus oriolus (=galbula)). Some data for Carrion crow and Rook
are given by Birkhead et al. (2006) (Table 8.7).
Brief ultrastructural accounts of spermatozoa or late spermatids exist for
Corvidae, Cyanocitta cristata, Blue jay (Henley et al. 1978); Corvus splendens,
Crow (Bawa et al. 1990); Grallinidae, Grallina cyanoleuca, Magpie lark
Avian Spermatozoa: Structure and Phylogeny "#%
Table 8.7 Species examined by light and scanning electron microscopy for pairwise
comparison. Slightly modified from Birkhead et al. (2006). Auk (In press). Measurements in
µm.
Species Straightened Length Total length Number Number

helix length flagellum sperm of of
(µm) (µm) midpiece head
(gyres) (gyres)
Corvidae
Carrion Crow Corvus corone 3.10 47.32 59.02 1.00 3.50
Rook Corvus frugilegus 3.71 48.06 59.78 1.00 3.60
Turdidae
Blackbird Turdus merula 55.75 70.82 82.82 14.00 2.50
Song Thrush Turdus philomelos 53.95 74.12 85.22 13.50 2.30
Sylviidae
Moustached Acrocephalus 84.34 92.04 105.03 29.40 4.20
Warbler melanopogon
Sedge Warbler Acrocephalus 70.21 76.62 90.01 23.40 4.60
schoenobaenus
Chiffchaff Phylloscopus collybita 96.76 98.01 116.13 25.30 4.20
Willow Warbler Phylloscopus trochilus 77.23 79.53 93.48 19.20 3.70
Blackcap Sylvia atricapilla 58.16 65.73 77.17 14.00 2.50
Lesser Sylvia curruca 70.98 73.64 86.19 18.60 3.00
Whitethroat
Paridae
WillowTit Parus montanus 57.27 67.01 80.32 15.60 4.00
Great Tit Parus maior 63.67 86.52 101.30 14.40 3.60
Fringillidae
Linnet Carduelis cannabina 138.47 146.70 162.16 25.80 2.10
Goldfinch Carduelis carduelis 111.50 119.44 132.79 23.90 2.70
Beavan’s Pyrrhula erythaca 12.33 35.48 49.11 0.00 2.60
Bullfinch
Eurasian Pyrrhula pyrrhula 1.86 38.78 43.96 0.00 0.00
Bullfinch
Emberizidae
Corn Bunting Emberiza calandra 131.46 138.68 153.63 28.60 2.80
Yellow Hammer Emberiza citrinella 107.63 116.90 130.97 22.80 2.80
Passeridae
House Sparrow Passer domesticus 72.77 87.51 101.56 15.00 3.00
Cape Sparrow Passer melanurus 64.53 76.87 90.13 13.90 3.10
(Jamieson 1995, 1999; present study); and Vireonidae, Vireo olivaceus, Red-eyed
vireo, McFarlane 1971 fide Koehler (1995); Henley et al. 1978) and V. griseus,
White-eyed vireo (Asa and Phillips 1987).
Data for species of Corvidae and of other oscine families examined by
Birkhead et al. (2006) are given in Table 8. 7.
The Corvida are of particular interest as they stand at the base of the oscines
(Sibley and Ahlquist 1990), or are paraphyletic to a (mostly) monophyletic
"#& Reproductive Biology and Phylogeny of Birds
Passerida (see Chapter 1), or are considered by some to contain the

Passeriformes. In either view, they may be expected to have spermatozoa
displaying a near primitive morphology and ultrastructure for the oscines.
From the limited information available it can be seen that the sperm of
Corvida retain some characteristics already developed in at least some
suboscines: the presence of a microtubular helix in the spermatid and
immature spermatozoon, ensheathing at least the nucleus and proximal
axoneme (Bawa et al. 1990; Jamieson 1999); and the helical form of the
acrosome vesicle and nucleus (Retzius 1909, 1911, 1912; Bawa et al. 1990).
They are less derived than and differ from typical Passerida in that the
acrosome, although elongate, is shorter than the nucleus (excepting Vireo
griseus and V. olivaceus) and the midpiece is shorter than the nucleus (Retzius
1909, 1911, 1912; McFarlane 1963; Bawa et al. 1990), whereas in Passerida at
maturity the acrosome is longer than the nucleus, sometimes several times
longer (with some exceptions, see Table 8.6), and the mitochondria spiral
around a large portion or the greater part of the axoneme (Retzius 1909;
McFarlane 1963). The sperm of the Corvidae and Lanidae are also very short
in contrast to the extreme elongation which occurs in the higher passerines
(McFarlane 1963). The data of Birkhead et al. (2006) (Table 8.7) for Carrion
crow and Rook confirm these characteristics.
In the Corvida and Lanius collurio, an helical rope winding around a great
length of the axoneme in immature sperm, shown in the exquisite drawings of
Retzius (1912), here exemplified, albeit from photocopies, by Siberian jay (Fig.
8.38A), and Rook (Fig. 8.38B), is independent of the short midpiece and
presumably consists of microtubules only. The microtubular helix in oscines
is said by Asa and Phillips (1987) not to extend around the acrosome and the
statement of Bawa et al. (1990) that is does in Corvus splendens requires
confirmation. These points will be further discussed in the accounts for
examined species, below.
8.10.11.2 Corvus splendens
Bawa et al. (1990) limited his account of the spermatozoon of Corvus splendens
(Fig. 8.39) to a consideration of the microtubular helix. He concluded that the
microtubules play a key role in effecting not only the elongation of the nucleus
but are also responsible for endowing the corkscrew-shaped appearance of
the acrosome and midpiece. This view was contrary to that of Fawcett et al.
(1971) who deduced that intrinsic forces of chromatin are solely responsible
for shaping of the nucleus though invoking microtubules for acrosomal
shaping. The observation of Bawa et al. (1990) that the acrosome and proximal
flagellum acquired an helical form after extension of the microtubular helix
along them does add weight to the view of the causative effect of microtubules
on spermatozoal form. The helix surrounding the acrosome, nucleus, midpiece
and proximal axoneme illustrated for corvids by Retzius (1912) (Fig. 8.38) is
here considered to be microtubular.
The plate provided by Bawa et al. (1990) allows some further observations.
Avian Spermatozoa: Structure and Phylogeny "#'
Fig. 8.38 Late spermatids of Corvida. A. Perisoreus infaustus, Siberian jay. B.

Corvus frugilegus, Rook. Both showing that in the Corvidae before maturity the
microtubular helix extends independently far posterior of the midpiece
mitochondria, as interpreted and labeled in the present study. After Retzius,
G. (1912). Biologische Untersuchungen, Neue Folge 17: 95-99, Taf XIV, Figs. 24
and 31.
Acrosome. The acrosome is helical and bears a distinct spiral ridge (acrosome
keel) (Fig. 8.39B, E). Two spurs visible at its base are presumably the anterior
limits of the regressing microtubular helix. It is shorter than the nucleus.
Nucleus. The moniliform condition of the nucleus as seen by SEM (Fig. 8.39E)
and the alternating nodes seen in longitudinal section (Fig. 8.39B) are
consistent with an helical form. The microtubular helix invests the helical
Fig. 8.39 Corvus splendens. A. Spermatid microtubular (MT) helix extending from
the nucleus (N) into the postnuclear region in the vicinity of mitochondria (M). B.
Spermatid, showing electron-dense nucleus (N). Microtubules (MT) are restricted
to the to the grooved region (asterisks) whereas ridges (arrows) are deficient of
microtubules. Microtubules ensheath at least the base of the acrosome. C. Freeze-
fracture replica of spermatid. Microtubules (MT) are disposed in an helical bundle.
D. Microtubules of the spermatid (MT) envelope and depress the midpiece which
spirals around the axoneme (AX). The mitochondrion (M) tentatively recognized
here was not labeled by Bawa. E. SEM of a spermatozoon in the duct. Relabeled
after Bawa, S. R., Kaur, R. and Pabst, M. 1990. Proceedings of the XIIth International
Congress for Electron Microscopy. Electron Microscopy, vol 3. Biological Sciences:
52-53, Figs. 1-5.
Avian Spermatozoa: Structure and Phylogeny "$
nucleus, apparently with some transient mitochondria. From analogy with a

finch (Fawcett et al. 1971) it is probable that these microtubules are lost when
the spermatozoon reaches the distal region of the male duct.
Midpiece. In the spermatid the mitochondria spiraling around the proximal
axoneme, and constituting with it the midpiece, are ensheathed by the
microtubular helix. The tripartite arrangement of axoneme, mitochondria and
helical microtubules is typical of developing oscine sperm.
8.10.11.3 Grallina leucocephala
Knowledge of the sperm of Grallina leucocephala, also a member of the Corvida,
is limited to two micrographs of transverse sections of late spermatids
(Jamieson 1999, and present study) (Fig. 8.40A, B). They give no indication of
the length of the midpiece (short in Corvida) but show clearly the typical
tripartite oscine arrangement in it: microtubular bundle, mitochondrion and
axoneme, in centripetal sequence. Nine uniform outer dense fibers, smaller
than those of Passerida, embrace the doublets of the axoneme.
Presence of a helix of densely packed microtubules investing the axoneme,
as in Grallina cyanoleuca, was stated by Jamieson (1999) to be a synapomorphy
of passerines. This remains the case but it should be noted that the
microtubules do not persist in the mature spermatozoon.
8.10.11.4 Lanius collurio
The spermatozoon of Lanius collurio, Red-backed shrike (Ballowitz 1888;
Retzius 1911) (Fig. 8.37), known only by light microscopy, closely resembles
that of Corvidae.
8.10.11.5 Oriolus oriolus
The spermatozoon of Golden oriole (Oriolus oriolus (=galbula)), described
optically by Ballowitz (1888) (his Fig. 63) is also closely similar to that of
Corvidae.
8.10.11.6 Vireo
Limited ultrastructural information is available for the corvidans Vireo
olivaceus, the Red-eyed vireo (McFarlane 1971 fide Koehler 1995; Henley et al.
1978) and Vireo griseus, the White-eyed vireo (Asa and Phillips 1987).
Although included in the study by Henley et al. (1978) the sperm was not
specifically described.
General morphology. A brief light microscope account for Vireo olivaceus is
given by McFarlane (1963) (Fig. 8.27J). He states that a ribbon-like membrane
extends from the nucleus to the acrosome and another helical membrane
extends a short distance down the tail. It is not stated whether the latter
membrane is an extension of the midpiece, and this is not clear in the
illustration but it seems likely that it is a purely microtubular helix of an
incompletely mature spermatozoon as in the Corvidae.
Koehler (1995), drawing from the unpublished thesis of McFarlane (1971),
gives the following apparently ultrastructural data for V. olivaceus. Sperm
Fig. 8.40 Grallina leucocephala, Magpie lark. Late spermatids in the testis. A.
Transverse section (TS) showing a section of the microtubular helix on one side of
Avian Spermatozoa: Structure and Phylogeny "$!
length 80 µm, acrosome length 8.0 µm, nuclear length 5.5 µm (giving a ratio of
1.5 more characteristic of Passerida), midpiece length 10 µm, shape helical,
‘helical membrane’ [helical acrosome keel] restricted to the acrosome, anterior
and posterior nuclear fossae absent, accessory dense (axonemal) fibers
present’, one helical mitochondrion present.
8.10.12 Parvorder Passerida
8.10.12.1 Introduction
Distinctive features of the Passerida are the persistence of a mitochondrial
helix extending for long distances around the axoneme and elongation of the
acrosome so that it is (with some exceptions, see Table 8.6) longer than the
nucleus. The latter feature is also seen in the corvid Vireo olivaceus.
The passeridan sperm type (as it is here termed), with helical, pointed
acrosome, helical nucleus, shorter than the acrosome, and mitochondrial helix
extending far along the tail is well demonstrated in the drawings of Ballowitz
(1888) and particularly of Retzius (1909) (Figs. 8.41, 8.42) and McFarlane
(1963) (Figs. 8.27K, 8.50) (though the mitochondrial nature of the posterior
helix could not be demonstrated at the time) in the following families and
species:
Muscicapidae: Muscicapa striata (=Muscicapa grisola), Spotted flycatcher
(Ballowitz 1888); Turdus philomelos (= musicus), Song thrush (Fig. 8.41); Luscinia
luscinia (=Aedon luscinia), Thrush nightingale; Ficedula hypoleuca (=Muscicapa
atricapilla), Pied flycatcher (Retzius 1909) (Fig. 8.42).
Sturnidae: Sturnus vulgaris, Starling (Retzius 1909) (Fig. 8.42).
Sylviidae: Phylloscopus sibilatrix (=Phylloscopus sibilator), Wood warbler
(Retzius 1909) (Fig. 8.42).
Hirundinidae: Hirundo rustica, Barn swallow (Ballowitz 1888; McFarlane
1963); Chelidon urbica, House martin (Ballowitz 1888); Petrochelidon pyrrhonota,
Cliff swallow; Riparia riparia, Bank swallow; Iridoprocne bicolor, Tree swallow
(McFarlane 1963) (Fig. 8.50).
Alaudidae: Alauda arvensis, Skylark (Retzius 1909) (Fig. 8.42).
Fringillidae: Fringilla coelebs, Chaffinch (Ballowitz 1888; Retzius 1909) (Fig.
8.41); Carduelis spinus (=Chrysomitris spinus) (Retzius 1909) (Fig. 8.41),
Cardeulis (=Spinus) pinus, Pine siskin (McFarlane 1963); Carduelis chloris

the condensing nucleus. B. TS of four spermatids. 1) through the nucleus and
ensheathing microtubular helix. 2) and 3) TS through a continuous (?) midpiece
mitochondrion invested on one side by the microtubular helix and in turn
ensheathing the axoneme. The nine prominent outer dense fibers of the axoneme
are well developed and are uniform in size and shape. 4) TS of the anterior end of
the midpiece through the (distal) centriole. Original.
"$" Reproductive Biology and Phylogeny of Birds
Fig. 8.41 Spermatozoa of Passerida by light microscopy. Turdus philomelos,

Songthrush; Fringilla coelebs, Chaffinch; Passer domesticus, House sparrow, also
showing sperm bundle; Carduelis chloris, Greenfinch; Carduelis spinus, Siskin.
After Retzius, G. 1909. Biologische Untersuchungen, Neue Folge 14(10): 89-122
Taf. XXXIII Fig. 1, XXXIV Fig. 3, XXXV Figs. 1, 9, XXXVI Figs. 1, 12.
Avian Spermatozoa: Structure and Phylogeny "$#
Sturnus
Fig. 8.42 Spermatozoa of Passerida by light microscopy, continued: Alauda
arvensis, Skylark Phylloscopus sibilatrix, Wood warbler Anthus spinoletta, Rock
pipit; Ficedula hypoleuca, Pied flycatcher Emberiza citrinella, Yellowhammer;
Sturnus vulgaris, Starling; Luscinia luscinia, Nightingale. After Retzius, G. 1909.
Biologische Untersuchungen, Neue Folge 14(10): 89-122 Tafel XXXVII, Figs. 1, 3,
5, 7, 9, 13, 15, 17, 19, 20.
"$$ Reproductive Biology and Phylogeny of Birds
Fig. 8.43 Three successive stages in differentiation of the mitochondrial sheath of

the finch sperm tail. (The example in A has an abnormal double flagellum but the
relations are the same as in a normal spermatid.) From Fawcett, D. W., Anderson,
W. A. and Phillips, D. M. 1971. Developmental Biology 26: 220-251, Figs. 30-32.
With permission from Elsevier.
Avian Spermatozoa: Structure and Phylogeny "$%
(=Chloris chloris), Greenfinch (Fig. 8.41); Emberiza citrinella, Yellowhammer

(Retzius 1909) (Fig. 8.42).
Passeridae: Passer domesticus, House sparrow (Figs. 8.41, 8.52C); Anthus
spinoletta (=Anthus obscurus), Rock pipit (Retzius 1909) (Fig. 8.42).
Additional families for which the passeridan sperm type is implied or
stated by McFarlane (1963) are: Paridae (1 gen., 4 spp.); Sittidae (1 gen., 2
spp.); Troglodytidae (2 gen., 2 spp.); Mimidae (3 gen., 3 spp.); Bombycillidae
(1 gen., 1 sp.); Coerebidae (1 gen., 1 sp.); Parulidae (13 gen., 27 spp.);
Ploceidae (1 gen., 1 sp.); Icteridae (8 gen., 8 spp.); and Thraupidae (7 gen., 8
spp.)
Passeridan families for which additional details are available will now be
considered.
8.10.12.2 Muscicapoidea, Muscicapidae (and Turdidae)
Descriptions of muscicapid sperm by light microscopy are: Muscicapa striata
(=Muscicapa grisola) (Ballowitz 1888); Turdus philomelos (= musicus), Song
thrush (Fig. 8.41); Luscinia luscinia (=Aedon luscinia), Thrush nightingale;
Ficedula hypoleuca (=Muscicapa atricapilla), Pied flycatcher (Retzius 1909) (Fig.
8.42). Harshman (Chapter 1) reviews strong evidence that Turdidae and
Muscicapidae are sister groups.
They are classical passeridan sperm with the exception that the acrosome
is not always longer than the nucleus; some acrosome:nucleus ratios,
including ultrastructural accounts, are estimated here as about 2.7 in Turdus
migratorius, 2.7 in Myrmecocichla formicivora, 1.6 in Ficedula hypoleuca and 1.5
in Turdus philomelos but only 0.8 in T. merula. The amplitude of the nuclear
helix is unusually large in Muscicapa striata (=Muscicapa grisola) (Ballowitz
1888).
Ultrastructural accounts of muscicapid sperm are limited to a TEM whole
mount showing the anterior end of the sperm of Turdus grayi, Clay-colored
robin (McFarlane 1963); a detailed account for T. merula, Blackbird (Furieri
1961); brief reference to T. migratorius, American robin (Henley et al. 1978); and
details given here for the Southern ant-eater chat, Myrmecocichla formicivora.
8.10.12.3 Turdus migratorius
The sperm bundles of oscines, as exemplified by Turdus migratorius, have been
discussed above. A micrograph of sperm bundle is illustrated in Fig. 8.44 and
a section through a bundle in Fig. 8.45.
Negative staining of the helical membrane is illustrated in Fig. 8.46 (Henley
et al. 1978).
Some data are given by McFarlane (1971 fide Koehler 1995). Dimensions
are: length spermatozoon 70 µm; acrosome 6.7 µm; nucleus 2.5 µm; midpiece
46 µm. The spermatozoon is helical but only the acrosome bears the so called
helical membrane. There are no anterior or posterior nuclear fossae. Dense
fibers are present and the midpiece is helical and large.
It is here considered that the helical membrane restricted to the acrosome is
the same structure as the small crest (here termed the helical acrosome keel)
"$& Reproductive Biology and Phylogeny of Birds
Fig. 8.44 Turdus migratorius, the American robin. Bright-field light micrograph of a
single feulgen-stained sperm bundle. The sperm nuclei are still embedded in a
common cytoplasmic mass and the remaining parts of the spermatozoa are in
sharp register with one another. Relabeled after Henley, C., Feduccia, A. and
Costello, D. P. 1979. The Condor 80: 41-48, Fig. 2.
on the acrosome of Turdus merula and Myrmecocichla formicivora (see below).

The large amplitude of the helical midpiece is presumably due to presence of
a fibrous keel as demonstrated in the latter species by Furieri (1961).
8.10.12.4 Turdus merula
General morphology. Furieri (1961) provides useful diagrammatic sketches
of the whole mature spermatozoon accompanied by corresponding cross
sections (Fig. 8.47A). The spermatozoon has a length of 85 µm, of which the
conical acrosome occupies 4 µm and the cylindrical nucleus 5 µm.
Acrosome. The acrosome is solid and somewhat electron-dense, contained in
the plasma membrane. It is circular in cross section but bears a small crest
Avian Spermatozoa: Structure and Phylogeny "$'
Fig. 8.45 Turdus migratorius, American robin. TEM of a transverse section through
a bundle of spermatozoa at a level posterior to the region of the nuclei. Each
spermatozoon is invested in a plasma membrane (PM) and has dense fibers (DF)
surrounding a central axoneme (A). There is a single mitochondrion (M) in each
and the plane of section through the sheath of singlet microtubules (SI MT) is
almost exactly the same for each. Part of the residue of the cytoplasmic mass (CM)
in which the bundle originated is peripheral to and between the spermatozoa.
There is a fourth component (IV), roughly triangular in section, of unknown nature
[probably the fibrous helix] next to the mitochondrion. From Henley, C., Feduccia, A.
and Costello, D. P. 1979. The Condor 80: 41-48, Fig. 5.
[helical acrosome keel], 0.1-0.2 µm high and a little wider at its base. It is clear
from Furieri’s drawing that this crest does not continue onto the nucleus and
therefore that the fibrous keel (as it is here termed) on the midpiece is a
separate entity. In longitudinal section the acrosome is a solid, helical cone.
Superficially the acrosome and nucleus are gently curved, the acrosome
convexly, and the nucleus concavely.
Nucleus. Following the acrosome, the nucleus is an helical cylinder, the
chromatin of which is strongly electron-dense but contains vacuoles. The two
ends of the nucleus are concave, slightly at the apex, more strongly caudally.
Axoneme and appurtenances. The tail is inserted into the basal nuclear fossa.
On one side of the flagellum is the commencement of an helical structure
[mitochondrial and fibrous helix] that is wrapped around the greater part of
the flagellum while on the other side there is an elongated formation, parallel
to the flagellum and containing fine amorphous trabeculae, the nature of
"% Reproductive Biology and Phylogeny of Birds
Fig. 8.46 Turdus migratorius, American robin. TEM of negatively stained

spermatozoa. A. The investing plasma membrane has been digested away
revealing the three components: straight axoneme (A), helical strand of
mitochondria (M) and helical array of singlet microtubules (MT). Artifacts (B). B. The
same three components in a slightly different orientation with respect to one
another. Note the complex interweaving of the microtubules at the arrow. Relabeled
after Henley, C., Feduccia, A. and Costello, D. P. 1979. The Condor 80: 41-48,
Fig. 6.
which is unclear. The axoneme has the usual 9+2 arrangement and each of
the nine doublets is reinforced peripherally by a large dense fiber. These fibers
are assumed to be responsible for limiting the flexibility of the axoneme.
The helix consists of two parallel superimposed structures. consisting of a
mitochondrion overlain by a large cytoplasmic fiber [fibrous helix] of similar
appearance but larger which extends far along the flagellum. The
mitochondrion embraces about half the circumference of the flagellum. The
cytoplasmic fiber has the form of an isosceles triangle in transverse section
and occupies about two thirds of the width of the mitochondrion on which it
is superimposed.
The cristae of the mitochondrion are digitiform and orientated orthogonally
relative to the long axis of the flagellum, disposed in quincunx (see further
details in Furieri 1961).
Fig. 8.47 Diagram of the whole spermatozoon with corresponding transverse

sections. A. Turdus merula, blackbird. B. Passer italiae, Italian sparrow. a,
acrosome; b, nucleus; c, transverse section (TS) through the tail, showing the
flagellum, 9 dense fibers, the helical crest formed, basally, by a mitochondrion to
which, in T. merula, is appended a large triangular cytoplasmic fiber [fibrous helix];
d, TS of the tail at the end of the helical crest which here consists only of the
mitochondrion; e, TS of the endpiece. After Furieri, P. 1961. Archivio Zoologico
Italiano (Napoli) 46: 123-147, Figs. 32 and 33.
"% Reproductive Biology and Phylogeny of Birds
Further posteriorly, the keel formed by the dual helix progressively

diminishes until the flagellum is naked. The peripheral dense fibers of the
axoneme also diminish and disappear.
Remarks. It is apparent that of the three components of the helical membrane
identified by Henley et al. (1978) in oscine sperm, only the fibrous keel and the
mitochondrial helix persist in the mature Turdus merula spermatozoon, the
microtubules (presumed to be present in the spermatid) being lost.
8.10.12.5 Muscicapidae, Myrmecocichla formicivora
A study of the spermatozoon of Myrmecocichla formicivora, the Southern ant-
eater chat, has clarified hitherto imperfectly understood aspects and revealed
previously unrecognized features of passerine spermatozoon ultrastructure
(Jamieson, Hodgson and Spottiswoode, unpublished). This filiform
spermatozoon demonstrated par excellence the passeridan features of an
acrosome longer than the nucleus and great prolongation of the midpiece as a
single mitochondrion wound helically around much of the length of the
axoneme. A new finding is the bipartite nature of the acrosome.
Acrosome. The acrosome is an helical structure 2.7 times the length of the
nucleus which it surmounts (Fig. 8.48L). It is inserted into the nucleus, at the
acrosomal-nuclear junction (Fig. 8.48F, G, H), in an asymmetrical fossa. The
acrosome is bipartite. Its distal, longer portion, consists of an electron-dense
gently helical column of three gyres, here termed the acrosome core, ca 5 µm
long, which bears a prominent keel seen in profile as three prominent spurs.
The dense core is drawn out towards the keel as can be seen in longitudinal
(Fig. 8.48F, G, L) and in transverse section (Fig. 8.48K). The proximal (anterior)
portion, here termed the acrosome crest, consists of a narrower spirally
angular electron-pale shaft, 3 µm long, tapering to a narrow tip (Fig. 8.48L). Its
substance is also drawn out towards its angular projections, as seen in
longitudinal (Fig. 8.48F, L) and transverse (Fig. 8.48J) section. Although the
acrosome keel is limited to the region of the acrosome core, it consists of
electron-pale material which is continuous with that of the acrosome crest and
must be considered to be part of the crest. The acrosome crest abuts on the core
at an oblique very slightly concave junction (Fig. 8.48F, L). The morphological
and developmental implications of the bipartite constitution are explored
above in section 8.10.10.1.
Nucleus. The nucleus, like the acrosome core, is strongly electron-dense. It
forms a very slightly sinuous stout cylinder (Fig. 8.48L), 2.9 µm long, of
subcircular transverse section (Fig. 8.48H). Its greatest width, 0.9 µm, is
shortly below the acrosomal nuclear junction. Its base closely abuts the
pericentriolar dense ring (Fig. 8.48A, B, F, I, L) against which it forms a slight
convexity or shallow, slightly protuberant double concavity.
Centriolar complex. There is no proximal centriole. The distal centriole is
surrounded by and fused with a dense ring outside which is a ring formed by
the proximal end of the mitochondrion of the midpiece (Fig. 8.48D). At least
Avian Spermatozoa: Structure and Phylogeny "%!
some of the centriolar microtubules have been shown to form triplets (Fig.
8.48M). It is short and appears to be penetrated by the two central axonemal
singlets or material continuous with these. At its junction with the axoneme,
the dense ring has given way to 9 large dense fibers, but it is still encircled by
the mitochondrial ring (Fig. 8.48N).
Helical components of the axonemal region. Longitudinal sections of the
base of the nucleus and adjacent centriolar complex and midpiece reveal two
components spiraled around the axoneme: a very elongate mitochondrion
which proximally forms the continuous mitochondrial ring and a strongly
electron dense component here termed the fibrous helix. Dense fibers are also
seen in glancing longitudinal profiles encircling the axoneme (Fig. 8.48A, B, F,
I). Further distally, for the greater length of the axoneme, the only helical
component is the mitochondrial helix (Fig. 8.48C).
Mitochondria. As noted, a mitochondrial ring encircles the junction of the
distal centriole and the axoneme, the latter with its large dense fibers (Fig.
8.48N). This mitochondrial ring is continuous with the single, extremely
elongate mitochondrion which spirals along the axoneme for the greater part
of the length of the latter. The course of this mitochondrial helix is seen in
longitudinal section in Fig. 8.48A, B, C, F, I. In transverse section the
mitochondrion is seen to accompany the portion of the axoneme which has
large dense fibers where initially it lies external to the helical fiber (Fig. 8.48O)
and more distally, in the absence of the helical fiber, is in direct contact with
the dense fibers (Fig. 8.48P).
Fibrous helix. A well developed electron-dense helix intervenes between the
mitochondrial helix and the axoneme in the proximal region of the latter, as
seen in longitudinal (Fig. 8.48A, B, I) and transverse section in which its
crescentic form is seen (Fig. 8.48O).
Axoneme. The axoneme has the conventional 9+2 arrangement of
microtubules. For much of its length each doublet is accompanied by a dense
fiber which is circular in cross section except for a small prolongation which
joins each A microtubule near the junction of the latter with the B subtubule
(Fig. 8.48O, P). The dense fibers greatly reduce in size distally (Fig. 8.48Q).
Judging from the small number of transverse sections, the endpiece of
flagellum is short, lacking dense fibers and mitochondrial helix (Fig. 8.48R).
The extreme posterior end of endpiece has a disrupted arrangement of
doublets and singlets (Fig. 8.48S).
Remarks. Examination of the sperm of Myrmecocichla formicivora (with the
ploceid Philetairus socius) allowed a new interpretation of the structure of the
passeridan acrosome and clarification of the structure of the so-called helical
membrane (see section 8.10.10.1 above).
8.10.12.6 Sturnidae, Sturnus vulgaris

The spermatozoon of a further muscicapoid, Sturnus vulgaris has been briefly
characterized by Koehler (1995) and Vernon and Woolley (1999). Koehler
"%" Reproductive Biology and Phylogeny of Birds
Fig. 8.48 Myrmecocichla formicivora. TEM of spermatozoon. A, B, F, I. Longitudinal

section (LS) of the base of the nucleus and adjacent centriolar complex, midpiece
and anterior axoneme. Note two components spiraled around the axoneme: a very
elongate mitochondrion which proximally forms a continuous ring and a strongly
electron dense component here termed the fibrous helix. Dense fibers are also
seen encircling the axoneme. C. LS of the longest portion of the flagellum around
Avian Spermatozoa: Structure and Phylogeny "%#
gives a sperm length of 50 µm, acrosome length 2.5 µm and nuclear length 3.0
µm; the sperm is helical, has an helical membrane, and has granular material
at the midpiece.
The passeridan form of the sperm is well illustrated by Retzius (1909) for
Sturnus (Fig. 8.42) but it will be noted that a departure from the usual
passeridan condition is the relatively short acrosome, though longer relative
to the nucleus than in non-passerines. Although Koehler gives its absolute
length as shorter than the nucleus in the drawing by Retzius it appears that
the acrosome:nucleus ratio in Sturnus is about 1.3. Other characteristics of
passeridan sperm which it displays are the helical forms of the sperm and the
spiral and very extensive midpiece.
The brief account of Vernon and Woolley (1999) is particularly useful as it
correlates external morphology as revealed by SEM with transverse sections
by TEM. The acrosome, nucleus and the single chain of (fused) mitochondria
all contribute to giving the proximal part of the cell an helically grooved
surface (resembling a carpenter’s auger bit). The helical groove is sinistral,
with approximately four gyres on the sperm head and 14 gyres on the
midpiece, where the mean pitch is 3.3 µm (Fig. 8.49A-C). In the neck region,
and for two gyres distally an extra helical structure is present (Fig. 8.49B,D).
The sperm head is 10.3 µm long. The flagellum is 73.4 µm long, with the
mitochondrion wound around the proximal 46 µm. There are nine uniform
dense fibers around the axoneme (Fig. 8.49D, E). These fibers diminish
distally, where they become round instead of comma-shaped in transverse
which spirals the mitochondrial helix. D. Transverse section (TS) of the distal (and
only) centriole, surrounded by a dense ring and the mitochondrial ring. E. LS of the
nucleus surmounted by the acrosome. The acrosome is bipartite, consisting of a
long, electron-dense acrosome core, which bears a prominent keel, and a distal
spirally angular acrosome crest. The acrosome core fits into an oblique fossa at
the tip of the nucleus, more clearly seen in G. H. TS nucleus. J. TS acrosome crest.
K. TS acrosome core through helical keel. L. LS of the entire length of the
acrosome crest and core and of the short nucleus, followed by a glancing section
of the mitochondrial circlet. M. Detail of D, showing the triplets of the distal
centriole. N. TS mitochondrial ring encircling junction of distal centriole and
axoneme with large dense fibers. O. TS near proximal end of axoneme, showing
9+2 pattern with 9 dense fibers, crescentic section of fibrous helix and, external to
this, the mitochondrion. P. TS axoneme with dense fibers and section of the
mitochondrial helix. Q. TS distal region of the flagellum with no mitochondrial helix
and reduced dense fibers. R. TS endpiece of flagellum lacking dense fibers and
mitochondrial helix. S. Extreme posterior end of endpiece with disrupted
arrangement of doublets and singlets. acr, acrosome crest; ac, acrosome core; ak,
acrosome keel; anj, acrosome-nuclear junction; ax, axoneme; dc, distal (only)
centriole; df, dense fiber; dr, dense ring around centriole; fh, fibrous helix; m,
mitochondrion; mh, mitochondrial helix; n, nucleus. From Jamieson, Hodgson and
Spottiswoode, unpublished.
"%$ Reproductive Biology and Phylogeny of Birds
Fig. 8.49 Sturnus vulgaris, Starling. A. SEM of the sperm head (h) and proximal
flagellum. The surface has the form of a sinistrally helical keel that is continuous
from the acrosome, through the nucleus and into the midpiece. Bar = 1 µm. B.
From the neck and into the proximal two gyres of the midpiece, the mitochondrial
helix (m) is accompanied by another helical structure (x), as shown also in D. Bar
= 1 µm. C. The remainder of the midpiece; the helical keel is now simply
mitochondrial as shown also in E. D. TEM of a transverse section (TS) through the
proximal midpiece, corresponding to B. There is a mitochondrial helix (M) and
another helix (x) of unknown origin. The axoneme is surrounded by nine accessory
dense fibers. Bar = 0.25 µm. E. TS of more distal midpiece, corresponding to C.
Bar = 0.25 µm. From Vernon, G. G. and Woolley, D. M. 1999. Cell Motility and the
Cytoskeleton 42: 149-161, Figs. 16 and 17.
section; they are absent from the distal 7-8 µm of the axoneme. Freeze-etch
replicas reveal that the outer dynein arms are typical for vertebrate
spermatozoa (Vernon and Woolley 1999). Although the spermatozoon is
Avian Spermatozoa: Structure and Phylogeny "%%
described as grooved it is clear that the grooving is the complement of two, or

more distally one, rope-like helical structures investing the axoneme. The
larger of these is the mitochondrial helix and the smaller is a structure of
unknown origin (x in Fig. 8.49B and D). This smaller helix is clearly the
homologue of the fibrous keel seen in, for instance, Turdus merula (Fig. 8.47A)
and described here for Myrmecocichla formicivora (Fig. 8.48). It is possible that
it is derived from the transient microtubular helix characteristic of passeridan
spermatids.
8.10.12.7 Sylvioidea, Hirundinidae, Petrochelidon, Hirundo, Riparia,
Iridoprocne and Sylviidae
McFarlane (1963) used the Hirundinidae as an example of quantitative
variation in sperm morphology within a family, for the four genera
Petrochelidon, Hirundo, Riparia and Iridoprocne (Fig. 8.50). While some overlap
occurs in comparative lengths of the head and midpiece (their combined
length), the total lengths of the sperm of the four genera are completely
different.
The exceedingly great length of the helical element around the axoneme,
leaving only a small fraction naked as the endpiece, is shown for Hirundo
rustica by Ballowitz (1888) (his Fig. 96).
Fig. 8.50 Variation of sperm in the family Hirundinidae. A. Petrochelidon

pyrrhonota. B. Hirundo rustica. C. Riparia riparia. D. Iridoprocne bicolor. E. Full view
of spermatozoon of Iridoprocne bicolor; an helical membrane, not apparent in the
drawing, extends to within 6 µm of the tail. From McFarlane, R. W. 1963.
Proceedings of the XIII International Ornithological Congress: 91-102, Fig. 3.
"%& Reproductive Biology and Phylogeny of Birds
Koehler (1995) cites the unpublished TEM description of the sperm of

Tachycineta thalassina, the Violet-green swallow, by McFarlane (1971). It
reaches the great length of 285 µm. The acrosome is 13.5 µm long and the
nucleus is much shorter, at 4.5 µm. The sperm is helical but the helical
membrane [probably merely an helical keel] is said to be limited to the
acrosome. There is an anterior nuclear fossa, 0.7 µm deep, and a posterior
fossa is absent. Only a distal centriole is present. One or two mitochondria are
present of which the long one is helical but the length of the midpiece is not
given. Dense peripheral fibers are present at the anterior end of the axoneme.
As in all investigated passeridans, a perforatorium and an annulus are
absent.
Dimensions and other morphological data are given by Birkhead et al.
(2006) (Table 8.7) for the sylviids Moustached Warbler (Acrocephalus
melanopogon); Sedge Warbler (Acrocephalus schoenobaenus); Chiffchaff
(Phylloscopus collybita); Willow Warbler (Phylloscopus trochilus); Blackcap
(Sylvia atricapilla) and Lesser Whitethroat (Sylvia curruca).
8.10.12.8 Certhioidea: Troglodytidae and Certhiidae
Troglodytidae. Aspects of the ultrastructure of the spermatid of the Wren
(Troglodytes troglodytes) have been briefly described by Tripepi and Perrotta
(1991) (Fig. 8.51A, B). In this important paper these authors recognize, for the
Wren and several other species, a ‘granular body’ (GB) the nature and
development of which have been discussed above in section 8.10.10.1. In the
advanced spermatid (Fig. 8.51B), the helix is formed internally by the GB,
which coils around the proximal axoneme and its dense fibers, and externally
by the transient microtubular bundle. In the remainder of the midpiece, the GB
is substituted by the mitochondrial sheath [mitochondrial helix].
TEM sections of the sperm of the Carolina wren (Thryothorus ludovicanus)
have been illustrated by Asa and Phillips (1987) (Fig. 8.9B, D). The
micrographs show the acosome with a keel, the spiral nucleus, and the 9 + 2
axoneme with large dense fibers external to which is the mitochondrial helix.
Electron-dense material of the neck possibly represents a granular helix.
Certhiidae. For the Treecreeper (Certhia brachydactyla) Tripepi and Perrotta
(1991) have demonstrated the presence of a granular helix persisting after
regression of the microtubular helix (Fig. 8.56E). The granular helix may,
therefore, be characteristic of the Certhiidae in which the subfamily
Troglodytinae is sometimes included.
8.10.12.9 Passeroidea, Thraupidae, Piranga rubra
The spermatozoon of Piranga rubra, Summer tananger, is 170 µm long, with an
acrosome 12 µm long, nucleus much shorter, at 3 µm, and a very long, 146 µm
midpiece (McFarlane 1971, fide Koehler 1995). The spermatozoon is helical but
the ‘helical membrane’ (here considered to be only the helical keel) is restricted
to the acrosome. Anterior and posterior nuclear fossae are absent.
Avian Spermatozoa: Structure and Phylogeny "%'
Fig. 8.51 Wren (Troglodytes troglodytes). A. Early spiralized spermatid. The

granular body encircles the proximal part of the axoneme. Mitochondria are
accumulating in a spiral array. B. Elongating spermatid. The granular body has
become a granular helix, distal to which is the mitochondrial helix. Both of these
are invested by the transient microtubular helix. A, relabeled after Tripepi, S. and
Perrotta, E. (1991). Pp. 1021-1023. In Baccetti B. (ed.), Comparative Spermatology
20 Years After. Serono Symposia Publications, vol. 75. Raven Press, Rome. Fig. 2.
B, micrograph courtesy of Dr. Sandro Tripepi.
8.10.12.10 Passeroidea, Passeridae, Passer domesticus

Passeridae are often reduced to subfamilial rank within the Ploceidae but are
here retained. Nevertheless, they lie within a ploceid clade (see Chapter 1, Fig.
2.2B).
Koehler (1995) tabulates features of the spermatozoon of Passer domesticus,
the House sparrow, derived from SEM and TEM examinations and gives an
SEM of the acrosome (Fig. 8.52C). A detailed account of spermiogenesis in this
species is given by Góes and Dolder (2002) which is summarized in Chapter
7 of this volume. It does not, however, recognize the bipartite nature of the
passerine acrosome revealed in the present chapter for Myrmecocichla and
Philetairus. Birkhead et al. (2006) present a scanning electron micrograph of
the head of a House sparrow sperm, noting the helical head and pointed
acrosome and the mitochondrial helix (from Table 8.7, having 15 curves).
The spermatozoon is said to be 77 µm long (but contrast 101.56 µm,

Birkhead et al. 2006, see Table 8.7), with an acrosomal length of 6-7 µm, a
nuclear length of 6 µm and a midpiece length of 55 µm. There is a small
anterior nuclear fossa, 0.7-1.08 µm deep; a posterior nuclear fossa is absent.
The acrosome has an electron dense core surrounded by a more electron
lucent region but lacks a perforatorium. It is said that the helical membrane
(here considered a keel) is limited to the acrosome. This spiral keel is
illustrated by SEM (Fig. 8.52C), here considered equivalent to that of Passer
italiae. However, from the length of midpiece given and the illustration of
Retzius (1909) (Fig. 8.41) it is clear that the midpiece forms a helix extending
far along the axoneme, though with a smaller amplitude than in most
passerines. A distal but no proximal centriole is present. Accessory [dense]
fibers are present in the midpiece (Koehler 1995). The sperm of this species
was also examined but not individually described by Humphreys et al. (1972)
and Asa and Phillips (1987).
8.10.12.11 Passer italiae
General morphology. Furieri (1961) provides useful diagrammatic sketches of
the whole mature spermatozoon of Passer italiae, the Italian sparrow,
accompanied by corresponding cross sections (Fig. 8.47B).
The acrosome and nucleus are helical and the tail is surrounded by an
helical crest (apparently a true helical membrane consisting of the spiral
mitochondrion). The spiral nature of the head is more evident than in Turdus
merula, the blackbird, but the ‘spiral crest’ of the tail is less evident.
The spermatozoon has a length of 110 µm (cf 101.56 µm for P. domesticus,
Birkhead et al 2006); the maximum width of the head is 1.5 µm; the length of
the acrosome is 9 µm and that of the nucleus 7 µm, thus demonstrating the
usual oscine feature of an acrosome:nucleus ratio greater than 1.
Acrosome. The acrosome is an elongate gently spiraled cone consisting of
homogeneous, strongly electron-dense material. The spiral crest of the
acrosome is better developed than in the blackbird and arises from a larger
base. It extends perpendicularly to the long axis of the acrosome and differs in
constitution from the acrosome.
Nucleus. The nucleus resembles that of the blackbird.
Flagellum. Unlike the blackbird, the flagellum is not implanted directly in the
long axis of the spermatozoon. Whereas the mitochondrial component of the
helical crest extends the whole length of the crest in the blackbird, in Passer,
the first tract appears to be formed by a large fiber [presumably the granular
helix], as in the blackbird, but here preceding the mitochondrion in which the
first cristae are evident at the end of the first gyre of the helix. The other
difference is the absence in Passer of the large structureless fiber (fibrous keel).
8.10.12.12 Passer diffusus
A scanning electron micrograph of the spermatozoon of the Grey-headed
sparrow (Passer diffusus) is given in Figure 8.54A. A well developed helical
acrosome keel, and a mitochondrial helix, are seen.
Avian Spermatozoa: Structure and Phylogeny "&
Fig. 8.52 Scanning electron micrographs of acrosomes of the sperm of four

species of Passerida. Some species have a nearly linear acrosomal axis while
others have a spiral shaped axis. All have an helical membrane. A. Quiscalus
quiscalus. ¥ 6800. B. Agelaius phoeniceus, Red-winged blackbird. ¥ 8700. C.
Passer domesticus, House sparrow. ¥ 9300. D. Cardinalis cardinalis, Cardinal. ¥
9300. After Koehler, L. D. 1995. Mémoires du Muséum National d’Histoire
Naturelle, Paris. 1995; 166: 437-444, Fig. 1.
Fig. 8.53 Social weaver (Philetairus socius), Ploceidae. A. LS of the entire

acrosome, nucleus and centriolar region. As in D and I, at least the first gyre of the
helix consists of the granular helix. B. Transverse section (TS) of acrosome core
through the helical keel. C. Acrosomal-nuclear junction showing anterior nuclear
fossa receiving the base of the acrosome core. D. LS base of nucleus and the
centriolar region. E. Longitudinal section (LS) of acrosome and anterior nucleus. F.
Detail of acrosome crest and anterior acrosome core. G. LS acrosome crest and
anterior acrosome core at right angles to F. H. TS nucleus. I. LS base of nucleus,
and centriolar and anterior axonemal region surrounded by the mitochondrial helix
which appears to commence with a granular helix. J. TS axoneme and
mitochondrial helix, showing nine dense fibers associated with the axonemal
doublets. K. TS axoneme behind posterior to the mitochondrial helix. L. TS
posterior region of axoneme with reduced dense fibers. acr, acrosome crest; ac,
acrosome core; ak, acrosome keel; anj, acrosome-nuclear junction; ax, axoneme;
dc, distal (only) centriole; df, dense fiber; mh, mitochondrial helix; n, nucleus.
Jamieson, Hodgson and Spottiswoode unpublished.
Avian Spermatozoa: Structure and Phylogeny "&!
8.10.12.13 Ploceidae, Philetairus socius

Acrosome. The acrosome of Philetairus socius sperm is a helical structure 1.2
times the length of the nucleus which it surmounts (Fig. 8.53A). It is inserted
into the nucleus, at the acrosomal-nuclear junction (Fig. 8.53A, C, E), in an
approximately symmetrical V-shaped fossa. The acrosome is bipartite. Its
distal, much longer portion, consists of an electron-dense gently helical
column of three gyres, the acrosome core, 5.8 µm long, which bears a
prominent keel seen in profile as three prominent spurs. Unlike Myrmecocichla
formicivora, the dense core is not drawn out towards the keel which is formed
solely from the crest layer which invests the core, as seen in longitudinal (Fig.
8.53A, E, G) and transverse section (Fig. 8.53B). The proximal (anterior)
portion of the acrosome, the acrosome crest, as seen in longitudinal section,
consists of a short electron-pale shaft, 1.3 µm long, tapering to a narrow tip
(Fig. 8.53G) and bearing near the level of the core, a single spur representing
the keel. However, the crest extends posteriorly as the crest layer or sleeve to
the base of the acrosome and bears along its length three spurs representing
the continuation of the acrosome keel (seen in cross section in Figure 8.53B).
In longitudinal sections approximately at right angles to this (Fig. 8.53A, E, F),
the short acrosome crest has the form of an inverted shoe of which the first
spur forms the heel. The tip of the acrosome core, fits into a deep asymmetrical
fossa formed by the base of the crest (Fig. 8.53A, E, F).
Nucleus. The nucleus, like the acrosome core, is strongly electron-dense. It
forms a very slightly sinuous stout cylinder (Fig. 8.53A, D, E, G), 6.1 µm long,
of subcircular transverse section (Fig. 8.53H). Its greatest width, 0.8 µm, is
basal but there is little variation throughout its length. Its base closely abuts
the centriolar region (Fig. 8.53A, D).
Centriolar complex. There is no proximal centriole. A dense ring surrounding
the distal centriole, seen in M. formicivora, has not been found and there is no
mitochondrial ring. However, dense convoluted masses are present within the
lumen of this centriole and the proximal region of the axoneme (Fig. 8.53I).
Helical components of the axonemal region. A longitudinal section of the
base of the nucleus and adjacent centriolar complex and midpiece (Fig. 8.53I)
reveals a very elongate mitochondrion wound helically around the axoneme.
However, at least the first gyre of this helix is amorphous and granular and is
presumed to be identifiable as a granular helix. No fibrous helix, seen in M.
formicivora, is present. Glancing sections of nine dense fibers are visible
between the mitochondrion and the axonemal doublets, as confirmed from
transverse sections (Fig. 8.53J).
Axoneme. The axoneme has the conventional 9+2 arrangement of
microtubules. For much of its length each doublet is accompanied by a dense
fiber which is circular in cross section except for a small prolongation which
joins each A microtubule near the junction of the latter with the B subtubule,
at the level of the mitochondrial helix (Fig. 8.53J) and posterior to this (Fig.
8.53K). The dense fibers greatly reduce in size distally (Fig. 8.53L). Sections of
the endpiece have not been obtained (Jamieson, Hodgson and Spottiswoode
unpublished).
"&" Reproductive Biology and Phylogeny of Birds
Fig. 8.54 Scanning electron micrographs of Spermatozoa of Passeridae and

Ploceidae. A. Grey-headed sparrow (Passer diffusus), Passeridae. B-D. Red-billed
quelea (Quelea quelea), Ploceidae. acr, acrosome crest; ac, acrosome core; ak,
acrosome keel; anj, acrosome-nuclear junction; ax, axoneme; mh, mitochondrial
helix; n, nucleus. Courtesy of A. Hodgson, relabeled.
Other ploceids. The spermatozoa of Red-billed quelea (Quelea qualea) (Fig.

8.54B), Red bishop (Euplectes orix) (Fig. 8.55A-C) and Cape weaver (Ploceus
capensis) (Fig. 8.55D, E) are illustrated by scanning electron microscopy. All
show an helical acrosome keel and a mitochondrial helix. However, in one
specimen of the Red bishop (Fig. 8.55C) the midpiece displays a short,
projecting mitochondrion resembling the midpiece of a corvid. The
significance of the variant is not clear.
Avian Spermatozoa: Structure and Phylogeny "&#
Fig. 8.55 Scanning electron micrographs of Spermatozoa of Ploceidae, continued.

A, B, C. Red bishop (Euplectes orix). D, E. Cape weaver (Ploceus capensis). acr,
acrosome crest; ac, acrosome core; ak, acrosome keel; ax, axoneme; m,
mitochondrion; mh, mitochondrial helix; n, nucleus. Courtesy of A. Hodgson,
relabeled.
8.10.12.14 Paridae, Parus bicolor, P. major and Cyanistes caeruleus

Koehler (1995) cites the unpublished optical description of the sperm of Parus
bicolor, the Tufted titmouse. It is 90 µm long, with an acrosome 7.1 µm long
and a shorter nucleus, at 5.8 µm. The sperm is helical but the helical
membrane (keel) is said to be limited to the acrosome. There is no anterior
"&$ Reproductive Biology and Phylogeny of Birds
nuclear fossa but a slight posterior fossa is present. The midpiece is 50 µm

long. Dense fibers, of unspecified location, are present.
Birkhead et al. (2006) (Table 8.7) give dimensions and other morphological
data for the Great tit (Parus major) and Willow tit (Parus montanus).
Comparable measurements for the Blue tit (Cyanistes caeruleus) are straightened
helix length 81.80 µm, length flagellum 92.86 µm, total sperm length 109.61
µm, number of midpiece gyres 19.60, number of head gyres 4.00 (T. R.
Birkhead, pers. comm.)
8.10.12.15 Icteridae, Icterus galbula, Agelaius phoeniceus, Molothrus
ater, and Quiscalus quiscula
Spermatozoal ultrastructure in the Icteridae has been briefly treated for Icterus
galbula, Northern oriole (Asa and Phillips 1987); Agelaius phoeniceus, Redwing
(Asa and Phillips 1987; Koehler 1995) (acrosome, Fig. 8.52B); Molothrus ater,
Brown-headed cowbird (Koehler 1995); and Quiscalus quiscula, Grackle
(Koehler 1995) (acrosome, Fig. 8.52A).
Data for the last three species are given in Table 8.8.
Table 8.8 Characters of spermatozoa of the Icteridae (from Koehler 1995)
Species Molothrus ater Agelaius phoeniceus Quiscalus quiscala

Common name Brown-headed cowbird Red-winged blackbird Common grackle
Sperm length 62 µm 110 µm 48 µm
Acrosome length 6.0 µm 8.0 µm 7.0 µm
Nuclear length 3.0 µm 5.0 µm 4.0 µm
Ratio acrosome:nucleus 2 1.6 1.75
Granular body at midpiece yes yes
Midpiece length 24 µm
Helical shape yes yes
Helical membrane yes yes
Anterior nuclear fossa shallow shallow
Posterior nuclear fossa yes yes
Centrioles distal only
Accessory dense fibers yes yes
Mitochondria spiral
These data for icterids, though incomplete, are characteristic of passeridan

sperm: sperm helical, acrosome longer than the nucleus (ratio 1.6-2), midpiece
spiral and very extensive (in Molothrus ater almost half the length of the
axoneme); distal centriole, only, present. The presence of dense peripheral
axonemal fibers is shared with non-passerines, though they are larger (size
not demonstrated for icterids) in passerines. The passeridan nature of the
sperm is confirmed by Asa and Phillips (1987) for Northern oriole and
Redwing, in their generalized account of oscine sperm.
Allen et al. (1967) gave detailed measurements for the sperm if 12 icterid
species or subspecies (Table 8.9). Their data for the species described above by
Koehler (1995) differ significantly from those of the latter author. Statistical
Table 8.9 Dimensions of spermatozoa of 12 taxa of Icteridae. Relabeled after Allen et al. (1967). Proceedings of the Indiana Academy of
Science 77: 434-441,Table 1
Species and subspecies Sperm head Acrosome Midpiece

— — —
X t = Std. error Xt = Std. error X t = Std. error
1. Sturnella neglecta confluenta Western meadowlark 14.95 ± .0704 9.90 ± .0641 1.93 ± 0.332
2. Sturnella magna argutula Southern meadowlark 15.73 ± .0491 10.39 ± .0837 2.00 ± .0232
3. Quiscalus quiscula versicolor Bronzed grackle 14.20 ± .688 9.55 ± .0896 2.13 ± .0194
4. Dolichonyx oryzivorus Bobolink 14.18 ± .0701 9.23 ± .0872 2.24 ± .0289
5. Icterus bullockii bullockii Bulock’s oriole 15.51 ± .0724 10.62 ± .1331 2.24 ± .0291
6. Icterus spurius Orchard oriole 14.75 ± .0543 10.48 ± .0408 1.99 ± .0291
7. Icterus parisorum Scott’s oriole 14.54 ± .0683 9.35 ± .0456 2.31 ± .0311
8. Agelaius phoeniceus phoeniceus Eastern red-winged blackbird 16.11 ± .0407 10.75 ± .0763 2.13 ± .0272
9. Agelaius phoeniceus mearnsi Florida red-winged blackbird 15.97 ± .0982 10.48 ± .0761 2.30 ± .0253
10. Agelaius phoeniceus nevadensis Grinnell red-winged blackbird 16.12 ± .0970 11.09 ± .0723 2.09 ± .0349
11. Agelaius phoeniceus floridanus Maynard’s red-winged blackbird 15.67 ± .0850 10.17 ± .0682 2.35 ± .0287
12. Cassidix mexicanus major Eastern boat-tailed grackle 13.74 ± .0763 8.80 ± .0566 1.88 ± .0270
Total length Principal piece End piece
1. Sturnella neglecta confluenta Western meadowlark 77.94 ± .1939 42.84 ± .2287 17.46 ± .1363
2. Sturnella magna argutula Southern meadowlark 85.97 ± .2461 49.11 ± .1035 18.57 ± .0946
3. Quiscalus quiscula versicolor Bronzed grackle 90.73 ± .3010 51.96 ± .2873 25.02 ± .1569
4. Dolichonyx oryzivorus Bobolink 142.02 ± .3832 111.49 ± .2133 17.59 ± .1258
5. Icterus bullockii bullockii Bullock’s oriole 110.45 ± .2974 70.47 ± .3224 23.59 ± .1632
6. Icterus spurius Orchard oriole 105.92 ± .2295 75.07 ± .2030 17.75 ± .1289
7. Icterus parisorum Scott’s oriole 105.93 ± .2337 68.95 ± .1644 20.21 ± .0862
8. Agelaius phoeniceus phoeniceus Eastern red-winged blackbird 148.59 ± .4166 106.84 ± .2623 24.02 ± .1183
9. Agelaius phoeniceus mearnsi Florida red-winged blackbird 146.48 ± .3623 107.85 ± .2326 23.24 ± .1233
Avian Spermatozoa: Structure and Phylogeny
10. Agelaius phoeniceus nevadensis Grinnell red-winged blackbird 139.64 ± .3327 104.12 ± .2304 19.52 ± .1753
11. Agelaius phoeniceus floridanus Maynard’s red-winged blackbird 146.95 ± .3929 111.13 ± .2607 18.51 ± .1449
"&%
12. Cassidix mexicanus major Eastern boat-tailed grackle 109.12 ± .3340 71.45 ± .2320 23.92 ± .1184
"&& Reproductive Biology and Phylogeny of Birds
analyses showed that the measurements ‘provide criteria for distinguishing

between most the twelve blackbird forms studied’. In a line drawing of a
typical icterid sperm an helical ‘undulating membrane’ is shown extending
throughout the length of the acrosome, nucleus and principal piece. The
differentiation of this membrane, in the present chapter, into acrosome keel,
nuclear keel and mitochondrial helix was not recognized by light microscopy.
8.10.12.16 Emberizidae, Cardinalis cardinalis and Emberiza spp.
The Cardinal (Cardinalis cardinalis) was included in the oscine study of Henley
et al. (1978) but was not specifically referred to. Koehler (1995) has given an
SEM of the acrosome (Fig. 8.52D) and the following data: length sperm 76 µm,
length acrosome 6.6 µm, length nucleus 4.0 µm, length midpiece 35 µm, sperm
helical, helical membrane present. The large acrosome:nucleus ratio of 1.65
and the long midpiece are typical passeridan features.
Tripepi and Perrotta (1991) have charted the development of the granular
body (GB) in the Cirl bunting (Emberiza cirlus) (Fig. 8.56A-D; see also section
8.10.10.1). The GB is evidenced in the early stages of spermatogenesis (Fig.
8.56A) as the spermatid begins to elongate and the midpiece is forming.
Subsequently, when the microtubular helix appears and the spermatid
assumes an helical form, the GB constitutes the proximal part of what is here
interpreted as an helical band around the axoneme, the granular helix (GH).
In the advanced spermatid (Fig. 8.56B, C) the helix is formed internally by the
GH, which coils around the axoneme and its dense fibers, and externally by
the transient microtubular bundle (Fig. 8.56B, C, D). In the remainder of the
midpiece, the GH is substituted by the mitochondrial sheath [mitochondrial
helix] (Fig. 8.56B).
Tripepi et al. (1991) have commented on, and illustrated, the microtubules
of the spermatid of Emberiza cirlus, the Cirl bunting. The arrangement is con-
sidered to be at a fourth, and final, level above the “reptilian” arrangement.
As in other passeriforms, Sertoli cell microtubules are absent but cytoplasmic
microtubules reappear in the spermatid. They are arranged in an helicoidal
bundle which is eliminated during the last phase of spermiogenesis.
Fig. 8.56 A-D. Cirl bunting (Emberiza cirlus). A. Spermatid at commencement of
elongation. The granular body has formed near the base of the uncondensed
nucleus. A large acrosome vesicle surmounts the nucleus which is still
subspheroidal. B. Elongating spermatid with helical components. The nucleus is
condensed and the granular body has become a granular helix around the
proximal region of the axoneme and is overlain by the microtubular helix. Further
distally, the granular helix gives way to the developing mitochondrial helix. C. Detail
of same. D. Two transverse sections of the proximal region of the axoneme,
showing the granular helix overlain by the microtubular helix. Dense fibers
surround the axoneme. E. Tree creeper (Certhia brachydactyla). Transverse section
of the proximal region of the axoneme and dense fibers through the granular helix,
after regression of the microtubular helix. A and E. Relabeled after Tripepi, S. and
Perrotta, E. (1991). Pp. 1021-1023. In Baccetti B. (ed.), Comparative Spermatology
20 Years After. Serono Symposia Publications, vol. 75. Raven Press, Rome, Figs.
1 and 4. B–D. From micrographs courtesy of Sandro Tripepi.
Avian Spermatozoa: Structure and Phylogeny "&'
Fig. 8.56
"' Reproductive Biology and Phylogeny of Birds
Birkhead et al. (2006) have tabulated data for the Corn bunting (Emberiza
calandra) and the Yellow Hammer (E. citrinella) (Table 8.7).
8.10.12.17 Fringillidae
Fringillids are briefly referred to in 8.10.12.1 above where they are shown to
have typical passeridan sperm. However, Birkhead et al. (2006) have
demonstrated a remarkable and profound departure from passeridan sperm
structure in the Eurasian bullfinch (Pyrrhula pyrrhula).
The Eurasian bullfinch sperm are relatively short: 46.87 µm ± 3.83 SD (N =
11) (captive-bred males: 47.19 µm ± 4.28 SD (N = 8) and wild males 46.01µm
± 2.75 SD (N = 3)). The sperm have a rounded acrosome and head, and no
obvious mitochondrial helix. The sperm of Beavan’s Bullfinch, like those of
the Eurasian bullfinch are short (length 49.11µm) but instead of a rounded
head they have a more pointed, spiral-shaped head. Both Pyrrhula species
have an extremely small midpiece, in contrast to the elongated mitochondrial
helix that forms the midpiece in almost all other passerines.
A pair-wise study using principle components analysis (PCA) to combine
quantitative and qualitative sperm morphology traits, together with a
phylogenetic correlation, comparing the sperm morphology of the Eurasian
bullfinch and Beavan’s bullfinch Pyrrhula erythaca with nine other pairs of
congeneric passerines revealed that the Eurasian bullfinch was a dramatic
outlier in terms of its sperm morphology and also more different from the
closely related Beavan’s Bullfinch than were any other pair of species.
Excluding the Eurasian bullfinch from the analysis showed that most
variation in sperm morphology in the other species was attributable to
phylogeny (a correlation long espoused by the present author). The Eurasian
bullfinch has extremely small testes for its body size, indicating that sperm
competition is infrequent in this species; the possibility was discussed that
relaxed selection via a lack of sperm competition may have contributed to
unusual sperm morphology in this species. The sperm structure in this
species resembles that of a spermatid in the early stages of elongation and
suggests a suppression of late spermiogenesis resulting in what could be
termed spermatozoal neoteny.
Fig. 8.57 Lonchura striata domestica Lovebird (Bengalese finch). Late maturation
phase of spermatids. A. Longitudinal section (LS), through the head showing the
posterior one gyre of the acrosome (A) with the same pitch as the nucleus (N). A
spiral keel of the acrosomal ridge is indicated by arrows. MTB, microtubule bundle.
B. Oblique section through the posterior region of the acrosome (A), showing the
Sertoli cell process (arrow) protruding between the acrosome and the microtubule
bundle (MTB). S. Sertoli cell. SER. Smooth endoplasmic reticulum. C. Transverse
section (TS) through the microtubule bundle (MTB) on the ridge of the nucleus (N),
showing the alternating layers consisting of microtubules and flattened cisternae
of endoplasmic reticulum (sER). D. Ts through anterior half of the midpiece,
showing the layered structure of the microtubule bundle. MS. Mitochondrial sheath.

SER. Smooth endoplasmic reticulum. E. LS through the posterior half of the
midpiece, showing few cisternae of smooth endoplasmic reticulum in the
microtubule bundle (MTB). AN. Annulus. MS. Mitochondrial sheath. F. LS through a
mature spermatid, showing disappearance of the microtubule bundle. A.
Acrosome. MS. Mitochondrial sheath. N. Nucleus. Scales: A. ¥ 14700. B. ¥ 31000.
C. ¥ 108400. D. ¥ 62000. E. ¥ 11600. F. ¥ 9000. After Kondo, T., Hasegawa, K. and
Uchida, T. 1989. Journal of Ultrastructure and Molecular Structure Research 98:
158-168, Figs. 18-23. With permission from Elsevier.
"' Reproductive Biology and Phylogeny of Birds
An exception such as the Eurasian bullfinch is not here considered to

substantially militate against the value of sperm ultrastructure for taxonomy.
One would not argue that we cannot morphologically recognize and classify
the Crustacea because Sacculina resembles a fungal mycelium. The statement
(Birkhead et al. 2006) that ‘most variation in sperm morphology in the other
species was attributable to phylogeny’ supports use of spermatozoal
morphology in phylogeny and therefore classification. Sperm morphology is
only one tool but a useful complement to other means of determining
relationship in addition to its interest per se. In fact the passerine status of the
Eurasian bullfinch sperm has since been confirmed by recognition (Birkhead,
Giusti, Immler and Jamieson, in preparation) in it of a microtubular helix, a
hallmark of passerine sperm, differing from the manchette of any other animal
spermatid.
Passerines examined by Birkhead et al. (2006) by light and scanning
electron microscopy for pairwise analysis of spermatozoal dimensions are
listed in Table 8.7. These authors also give the following data on the sperm of
Fig. 8.58 Lonchura striata, Lovebird (Bengalese finch). Scanning electron

micrographs of spermatozoon. A. Sperm head and midpiece of the flagellum
surrounded by the loosely coiled helical membrane. ¥ 4400, B. Detail of the head.
¥ 34500. After Yasuzumi, G. 1974. International Review of Cytology 37: 53-119, Figs.
10-11. With permission from Elsevier.
Avian Spermatozoa: Structure and Phylogeny "'!
the Pine Grosbeak (Pinicola enucleator). The sperm are similar to those of other
passerines in length (162.7 µm ± 9.63 S.D.; N = 30 sperm, N = 1 male) and
resemble typical finch sperm in morphology unlike Pyrrhula.
8.10.12.18 Estrildidae, Lonchura striata
The spermatozoon of Lonchura striata, the ‘Loverbird’, also named the
Bengalese finch, Society finch, White-rumped mannikin, or White-rumped
munia, is of the classical passeridan type, with helical acrosome, nucleus and
midpiece, the midpiece extending far along the axoneme (Yasuzumi and
Sugioka 1966; Fawcett et al. 1971; Yasuzumi and Sugioka 1971; Yasuzumi
1974; Kondo et al. 1988) (Figs. 8.57, 8.58). From TEM, the helical membrane
(Fawcett et al. 1971; Kondo et al. 1988) (Fig. 8.57C, D) is (axoneme included) of
the tripartite type and lacks the additional fibrous keel seen in Turdus.
However, the helical membrane on the acrosome is clearly a keel distinct and
separate from the helical membrane of the tail, both being absent from the
nucleus as here shown for Philetairus socius.
8.10.12.19 Estrildidae, Taeniopygia (=Poephila) guttata
Vernon and Woolley (1999) give some data on the spermatozoon of the Zebra
finch (Taeniopygia gutatta). By light microscopy and TEM the sperm are said to
be very like those of the starling. The mean head length is 11.3 ± 1.0 µm and
flagellar length 64.1 ± 5.7 µm (n=16 from 1 individual). The mean pitch of the
mitochondrial helix is 3.13 µm. There is a highly significant negative
correlation (r = – 0.85) between the lengths of the mitochondrial and non-
mitochondrial regions of the flagellum. The mitochondrial helix is sinistral, as
judged from serial sections (Vernon and Woolley 1999).
8.10.12.20 Prunellidae, Prunella collaris
Chiba and Nakamura (2001) give a TEM view of part of the head of the
spermatozoon of Prunella collaris, the Alpine accentor, on the wall of a sperm
storage tubule (SST) and SEM of the spermatozoon on the utero-vaginal part
of the oviduct and a group of sperm on the wall of a SST. The utero-vaginal
micrograph well displays the auger-like form of the passerine spermatozoon.
8.11 PHYLOGENETIC SUMMARY OF AVIAN SPERMATOZOA
Introduction. A subset of the spermatozoal characters discussed in this

chapter (see Table 8.10) is here used for a preliminary phylogenetic analysis
of taxa which have been examined ultrastructurally. Many spermatozoal
descriptions are fragmentary and, because of the resulting missing characters,
do not support a full parsimony analysis using exhaustive or branch and
bound analyses. Resultant consensus trees are highly polytomous. A single
nearest neighbor (NJ) tree is therefore presented (Fig. 8.59A). It was obtained
using PAUP (Swofford 2001). Although neighbor-joining is essentially
phenetic, unambiguous character state changes were plotted on the tree using
MacClade (Maddison and Maddison 2000). Some clearly synapomorphic
"'" Reproductive Biology and Phylogeny of Birds
characters which were not computed as unambiguous, probably because of

missing states in various taxa, are added to the tree in parentheses. The
analyses were conducted on 29 avian species (only Corvus and Passer being
composite taxa), plus the outgroups Crocodylus (Jamieson et al. 1997) and a
composite Chelonia (Healy and Jamieson 1997). The character matrix is
available from the author on request. It has been claimed (Johnson 2001, citing
Huelsenbeck and Hillis 1993) that the performance of neighbor joining (NJ) in
recovering the correct phylogeny is similar to that of parsimony. A portion of
a 50% majority rule, maximum parsimony (MR) tree is, nevertheless, shown in
Fig. 8.59B.
The tree obtained and character transformations indicated can only be
taken as heuristic in the broad sense, the chief aim of the analyses being to
graphically present the spermatozoal character states of the various taxa.
Nevertheless, monophyly is supported for Neognathae, Psittaciformes,
Columbiformes, Passeriformes, Passeri (oscines) and Passerida.
Spermatozoal evidence rejects basal passerines. A major recent controversy
in avian molecular phylogenetics has centered on the conflicting hypotheses
that the Passeriformes constitute a basal group, the sister-group of all other
birds or the traditional hypothesis that paleognaths (ratites and tinamous) are
the sister group of all other birds.
The basal passerine hypothesis has derived from analysis of mitochondrial
DNA sequences (Mindrell et al. 1997, 1999; Johnson 2001). However, Braun
and Kimball (2002) have shown that mtDNA sequences are capable of
supporting the traditional classification, with basal paleognaths, depending
on the type of analysis, number of sites analyzed and taxon sampling.
Phylogenetic analysis of nuclear DNA sequences has consistently supported
the traditional hypothesis (Sibley and Ahlquist 1981, 1990; Sibley et al. 1988)
Table 8.10 Characters used in phylogenetic analysis of evolution of bird spermatozoa
Characters (All unordered)

1 Acrosome1 shorter 2 equal to 3 longer than nucleus
2 Acrosome 1 conical 2 round tip 3 button-like
3 Perforatorium 1 present 2 absent
4 Endonuclear canal 1 deep 2 short 3 absent
5 Proximal centriole 1 present 2 absent
6 Distal centriole 1 very long 2 shorter 3 short
7 Mitochondria 1 several per TS 2 one per TS 3 small group
8 Midpiece 1 short 2 very long
9 ‘Helical membrane’ 1 present 2 absent
10 Singlets in distal centriole 1 present 2 basal 3 absent
11 Fibrous sheath 1 ribbed 2 amorphous 3 absent
12 Annulus 1 present 2 absent
13 Dense fibers 1 small 2 moderate 3 large
14 Tip granule 1 present 2 absent
15 Nuclear rostrum penetrating acrosome 1 deeply 2 slightly 3 negligibly 4 absent
Avian Spermatozoa: Structure and Phylogeny "'#
Fig 8.59 A. Neighbor-joining tree of major groups of birds from a PAUP analysis
derived from spermatozoal ultrastructure for characters listed in Table 8.10, with
Chelonia and Crocodylus johnstoni as the outgroup. Neighbor-joining search
settings: ties (if encountered) broken systematically; distance measure = mean
character difference. Character changes are those which are unambiguous and
were plotted using Macclade. Some deduced synapomorphies which were not
plotted as ambiguous have been added in parentheses to the tree. B. Portion of a
50% majority rule most parsimonious tree. Settings used in computation of the
tree: heuristic search settings; optimality criterion = parsimony; 15 total characters,
all unordered, with equal weight, all parsimony-informative; starting tree(s)
obtained via stepwise addition; addition sequence: random; 10 replicates; 1 tree
held at each step during stepwise addition; branch-swapping algorithm tree-
bisection-reconnection (TBR); steepest descent option not in effect; Initial
‘MaxTrees’ setting 900 (auto-increased by 100); other parameters at PAUP default
settings.
"'$ Reproductive Biology and Phylogeny of Birds
and recently Garcia Moreno et al. (2003) have shown that combined mt and
nuclear DNA analysis supports the traditional view. A study of a-crystalline
sequences also supported the basal position of ratites (Stapel et al. 1984).
The NJ tree (Fig. 8.59A), as do intuitive considerations, unequivocally
supports a basal position for paleognaths when Chelonia and Crocodylia are
used as outgroups. A basal position for passerines is highly unparsimonious
and, computationally and intuitively, would involve most implausible
pathways for spermatozoal evolution.
Are paleognaths paraphyletic? Monophyly of the Palaeognathae has
previously been accepted on the basis of spermatozoal studies, as in the
molecular ‘Tapestry’ (Sibley et al. 1988; Sibley and Ahlquist 1990), though the
position of Tinamou within these has remained unresolved (Asa et al. 1986;
Asa and Phillips 1987; Baccetti et al. 1991; Soley 1993). It must be stressed that
the spermatozoal characters which appear to unite Palaeognathae are in fact
symplesiomorphies: the conical acrosome, ribbed fibrous sheath, elongate
centriole, and 9 dense fibers of ratite sperm are all seen in Chelonia and
Crocodylia, and do not prove avian or paleognath monophyly, despite the fact
that the first three features were considered to unify paleognaths (Baccetti et
al. 1991). In the NJ tree monophyly of paleognaths is not supported (Fig.
8.59A). It is not here proposed that paraphyly of paleognaths indicated by
spermiocladistics should be considered unquestionable but it does warrant
further consideration of paraphyly for the group.
The struthioniform spermatozoon is crocodiloid (see section 8.6.1.3),
departing from the condition in crocodile sperm in an apparent reduction in
size of the outer dense fibers of the axoneme and the deeper, more anterior
extension of the nuclear rostrum into the subacrosomal space of the acrosome,
neither of which characters computed as unambiguous in the NJ tree. Like the
crocodilian (and chelonian) spermatozoon the elongate distal centriole (and
the midpiece) is penetrated throughout its length by the central singlets of the
axoneme. By deduction, withdrawal of the central singlets from the distal
centriole appears to be an apomorphy, but homoplasy, of the Crested tinamou
(Eudromia elegans), the Emu (Dromaius novaehollandiae) and the remainder of
the birds. However, in the NJ tree withdrawal of singlets from the centriole
computes as basic to Neornithes with a, perhaps unlikely, repenetration in the
Struthio+Rhea clade. Tinamou has a unique autapomorphy (not computed),
the presence of glycogen around the axoneme, but this is phylogenetically
uninformative. Emu is distinctive among the paleognaths in loss of the
endonuclear canal and of the perforatorium (two probably correlated
characters).
If the Palaeognathae were truly paraphyletic, the strict application of
Hennigian phylogenetics would demand that Rhea+Struthio, Eudromia and
Dromaius were each given separate and successively reduced ranks as
collectively they would constitute a grade rather than a clade. The author is
doubtful, however, that any useful purpose is served by thus distinguishing
taxa which have arisen in immediate succession from a basal lineage, here of
Avian Spermatozoa: Structure and Phylogeny "'%
the Aves, and bear a distinctive morphological and molecular facies.

Nevertheless, intervention of Tinamou between the Ostrich and Emu branches
(Fig. 8.59A), if borne out by further studies, would be of considerable interest.
A possible sister relationship of Emu to the Neognathae is highly questionable
as it would require reacquisition of the perforatorium and endonuclear canal
in the Galloanserae rather than the more probable retention of these from an
ancestor shared with paleognaths.
The summary tree for molecular data of Harshman (Chapter 1, Fig. 1.1)
indicates monophyly of the Palaeognathae.
Galloanserae. Any member of the Galloanserae, consisting of the Galliformes
and Anseriformes, could instantly be diagnosed by a combination of features
of sperm ultrastructure: acrosome shorter than nucleus: stout perforatorium in
a shortened endonuclear canal; both centrioles present; distal centriole long
though (unlike paleognaths) not fully penetrating the midpiece; mitochondria
in a circlet around the axoneme; midpiece not elongate; ‘helical membrane’
absent; singlets basal in the distal centriole; fibrous sheath amorphous, not
ribbed as in paleognaths; annulus present; dense fibers of moderate diameter;
nuclear rostrum slightly penetrating the acrosome. However, among these
character states there is no distinctive synapomorphy for the group and the
NJ tree presents an unresolved assemblage of seven separate galloanseran
branches, together with three non-galloanseran clades.
Nevertheless, spermatozoal ultrastructure certainly does not contraindicate
monophyly of the Galloanserae which is supported by molecular analyses
(Harshman, Chapter 1, Fig. 1.1). The Anseriformes and Galliformes can be
linked by the synapomorphy slight penetration of the nuclear rostrum into the
subacrosomal space and the Galliformes can possibly be separated from the
Anseriformes by presence of singlets basally in the distal centriole, both of
which are ambiguous characters and do not therefore appear on the NJ tree.
The latter (singlet) character is uncertain for the Anseriformes and requires re-
examination.
It is likely that when more complete spermatozoal data are obtained
considerable resolution of the Galloanserae will be obtained. For instance, the
presence of a ‘tip granule’ unites Coturnix japonica and Gallus gallus. It is
uncertain that its absence from other taxa is real but the thorough examination
of Turkey and Guineafowl (Thurston et al. 1982; Thurston and Hess 1987)
suggests that it is absent at least in these two species.
A notable difference of the spermatozoal analyses from the Tapestry (Sibley
et al. 1988) is that the Galloanserae do not form the sister taxon of the ‘Ratitae’.
Sibley and Ahlquist (1990), did, however, repudiate this sister taxon
relationship in their classification. A sister relationship of Galloanserae and
Neoaves is endorsed by Harshman (Chapter 1, Fig. 1.1).
Metaves. Three orders of the Metaves have been investigated for spermatozoal
ultrastructure: Apodiformes, Caprimulgiformes and Columbiformes. There is
no clear spermatozoal support for the grouping Metaves in the computer
analyses (Fig. 8.59A).
"'& Reproductive Biology and Phylogeny of Birds
Apodiformes. The midpiece-length distal centriole of Apus apus (Fig. 8.22)

unless (as seemed most unlikely) a reversal from the ancestral condition
retained in crocodiles and paleognaths, could be considered (Jamieson and
Tripepi 2006) to place the apodids very basally in the avian phylogenetic tree
but the loss of the perforatorium is derived relative to the Galloanserae. The
long centriole of A. apus, if not a reversal, is inconsistent with placement of the
Apodiformes above (i.e. at a more derived node than) the Psittaciformes from
DNA-DNA hybridization by Sibley and Ahlquist (1990). It is not inconsistent,
however, with placement in the Metaves (see Harshman, Chapter 1). The
centriolar condition might suggest that apodiforms are the plesiomorphic
sister taxon of all other Metaves and that they have independently lost the
perforatorium. However, the computer analysis (Fig. 8.59A) does not support
this position and Apus has a relatively ‘high’ position, lying in a clade which
includes Piciformes, Cuculiformes, Charadriiformes, and Columbiformes +
Passeriformes at a node above Psittaciformes.
It seems necessary to consider a scenario in which a long (midpiece-length)
centriole existed at three major nodes: the Palaeognathae-Neognathae node,
the Galloanseran-Neoaves node, and though not evident in the present
analyses, a Metaves-Coronaves node. The centriole would then become
shorter, though still elongate, in the ancestral galloanseran and would be
retained in the Neoaves only (so far as is known) in Apus, whether in the
Metaves or the Coronaves.
Caprimulgiformes. The phylogenetic position of Caprimulgus, as evidenced by
spermatozoal ultrastructure (Fig. 8.23), has been partly discussed in section
8.9.2.1 above.
The caprimulgid spermatid (and clearly the spermatozoon) is closely
similar in structure to that of psittaciforms. In contrast, the Apodidae, placed
in the same clade as the Caprimulgidae in the DNA-DNA hybridization
analyses of Sibley and Ahlquist (1990), differ from Caprimulgus in the long
distal centriole of the spermatozoon (Jamieson and Tripepi 2006). Other
members of the unnamed clade of Sibley and Ahlquist (1990) which contains
the caprimulgids (Hemiprocnidae through Eurostopodidae) have not been
examined for sperm ultrastructure. Although the Trochilidae and (here
removed to the Coronaves) the Strigidae in this clade, are listed as having been
examined by light microscopy (McFarlane 1963), no data are given.
The NJ tree (Fig. 8.59A) places Caprimulgus in the Neognathae but gives no
indication of their affinities other than that they do not belong in a
columbiform+passerine clade. In the MR tree (Fig. 8.59B), although
Caprimulgus lies in the same clade as Apus, this clade also contains members
of the Coronaves and therefore the grouping Metaves cannot be considered to
be supported.
Columbiformes. In the molecular Tapestry (Sibley et al. 1988; Sibley and
Ahlquist 1990) the Passeriformes branch off first in the tree dealing with the
Passerimorphae and form the sister-group of the Columbiformes +
(Gruiformes + ‘Ciconiiformes’), although the classification does not call
Avian Spermatozoa: Structure and Phylogeny "''
attention to this order of branching as the Columbiformes is the first order

listed in it.
In the NJ tree (Fig. 8.59A) columbiforms are the sister of the passerine clade.
Association of columbiforms with passerines is marked by the loss of the
fibrous or amorphous sheath (a change which we could alternatively envisage
as homoplastic). The mutual presence of an elongate midpiece maps as a
homoplasy. Homoplastic elongation of the midpiece is acceptable as detailed
ultrastructure of the midpiece (not computed) in columbiforms differs from
that of passerines.
If the Metaves were a valid grouping loss of the amorphous sheath in
Columbiformes would be homoplastic with taxa in the Coronaves.
Nevertheless, the location of columbiforms by Sibley and Ahlquist (1990) in a
clade which would now be attributable to the Coronaves is not
contraindicated by spermatozoal ultrastructure. The phylogenetic position of
the Columbiformes and the validity of the Metaves are far from settled. The
molecular consensus of Harshman (Chapter 1, Fig. 1.1) places Columbiformes
in the Metaves far removed from the Passeriformes.
Coronaves. Orders of the Coronaves that have been investigated for
spermatozoal ultrastructure (see accounts in previous sections) are:
Piciformes, Cuculiformes, Psittaciformes, Gruiformes, Charadriiformes,
Falconiformes and Passeriformes. As usual in avian studies, the passerines
have received the most attention in terms of numbers of taxa investigated. The
grouping Coronaves is not supported by sperm ultrastructure.
Psittaciformes. In the molecular analyses of Sibley et al. (1988) and Sibley and
Ahlquist (1990), the psittaciforms are preceded hierarchically only by the
Coliiformes and Cuculiformes at the base of the Passerae. Psittaciforms have a
highly unresolved position in the Coronaves in the consensus phylogenies of
Harshman (Chapter 1, Fig. 1.2).
In the NJ tree psittaciforms lie in the same unresolved assemblage
(containing both Metaves and Coronaves) as Caprimulgus. They share with
columbiforms a homoplastic reduction in the size of the dense periaxonemal
fibers. Psittaciforms show a homoplastic loss of the amorphous sheath, a loss
shared with cuculiforms and passeriforms though this character did not map
unambiguously on the NJ tree.
The distinctive feature, and autapomorphy, of psittaciform sperm compared
all other Aves is the condition of the nuclear-acrosomal junction (see section
8.10.5) though, as an autapomorphy, this does not clarify their relationship
with other orders. The unique acrosome-nuclear disjunction was not scored
in the matrix but has been added to the tree, thus uniting Melopsittacus and
Nymphicus.
In the portion of a 50% majority rule (MR), maximum parsimony tree shown
in Fig. 8.59B Psittaciformes have a sister group relationship with Grus+Falco
and successively more basal sister groups are Caprimulgus and Apus.
Piciformes. The Piciformes were placed by Sibley et al. (1988) and Sibley and
Ahlquist (1990) at the base of the Neoaves (ignoring the questionably placed
Turniciformes), i.e. as the sister group of all other Neoaves, a view supported,
again from DNA-DNA hybridization, by Bleiweiss et al. (1994). In the NJ tree
(Fig. 8.59A) Melanerpes has an unresolved position as part of a polytomy
consisting also of a charadriiform (Jacana), a cuculiform (Crotophaga),
Columbiformes+Passeriformes. This clade is united by loss of the
perforatorium, and endonuclear canal, a feature which could, alternatively,
have occurred homoplastically.
Features of piciforms differing from oscines and shared with other non-
passerines (asterisks indicate characters that were not computed) are the
small acrosome: nucleus ratio, the acrosome being shorter than the elongate
nucleus whereas it longer in at least the higher passerines; absence of an
helical membrane; distribution of mitochondria in a circlet around the
axoneme of the principal piece, as seen in transverse section; the presence (so
far as can be deduced from a micrograph, Fig. 8.26) of an amorphous sheath
around the principal piece; the stated absence of dense axonemal fibers; and
the irregular distribution* of spermatozoa in the testes (a difference from
Passerida but possibly not from corvids). On the other hand, there is no
mention of presence of a perforatorium seen in Galloanserae and most other
non-passerines, nor of the elongate distal centriole seen in paleognaths, Apus
and, with some shortening, in Galloanserae. Absence of outer dense axonemal
fibers requires confirmation.
This uncertainty as to the position of the Piciformes is reflected in the
molecular consensus (Harshman, Chapter 1, Fig. 1.2) in which a clade
containing the Piciformes has an unresolved position in the Coronaves.
Cuculiformes. The sperm of Crotophaga ani as described by Saita et al. (1982b)
and in this study, has the following characteristics: acrosome conical, shorter
than the nucleus; perforatorium and endonuclear canal absent; short distal
centriole, short midpiece with several mitochondria in transverse section,
helical membrane absent, central axonemal singlets absent from the centriole,
amorphous sheath present; and presence of an annulus. It is thus a distinctly
non-passerine sperm.
In the molecular consensus of Harshman (Chapter 1, Fig. 1.2) cuculiforms
are part of a huge polytomy in the Coronaves. In the NJ tree (Fig. 8.59A)
Crotophaga is part of a large polytomy, other members of which are Melanerpes,
Jacana, the columbiform+passeriform clade and Apus. Determination of the
phylogenetic position of the Cuculiformes thus still requires morphological
and molecular clarification.
Passerimorphae. The Passerimorphae (sensu Sibley et al. 1988; Sibley and
Ahlquist 1990) contains the orders Columbiformes, Gruiformes, the greatly
expanded order ‘Ciconiiformes’ and the Passeriformes. Passerimorphae
cannot be said to be supported in the spermatozoal analyses. There is,
similarly, no equivalent of the Passerimorphae in the consensus phylogenies
of Harshman (Chapter 1) as the constituent families (or orders) lie in the
Metaves (Columbiformes) and Coronaves (Gruiformes, orders of the former
Ciconiiformes and the Passeriformes).
Avian Spermatozoa: Structure and Phylogeny #
Charadriiformes, Gruiformes and Falconiformes. Charadriiformes,

subsumed in the Ciconiiformes by Sibley et al. (1988) and Sibley and Ahlquist
(1990), are represented for sperm ultrastructure only by Jacana jacana. As
shown in section 8.10.7.2, a button-like acrosome characterizes this species
and many other charadriiform species examined by light microscopy whereas
the Scolopacidae have elongate, spiral sperm. The button-like acrosome fails
to group Jacana with Grus and Falco, all of which are ciconiiforms sensu Sibley
and Ahlquist, in the NJ and MR trees (Fig. 8.59A, B). Grus and Falco have an
unlikely sister-group relationship on the basis of the shared button-like
acrosome and midpiece consisting of a small group of mitochondria. It is
uncertain how much credence can be placed on this grouping as the
possibility exists that this acrosomal form and midpiece morphology are
homoplastic. This must indeed be the case in the passeridan Eurasian
bullfinch in the sperm of which Birkhead et al. (2006) have demonstrated a
button-like acrosome (and, we may add, a falconiform-like external sperm
morphology), the only known exception for passerines. Furthermore, Jacana
has only an unresolved relationship with Grus + Falco in the MR tree (not
illustrated). With regard to the spiral sperm of scolopacids it is pertinent that
molecular analyses place them firmly in a charadriiform clade (Paton et al.
2003). Falco has an unresolved position, though also in the Coronaves in the
molecular consensus (Harshman, Chapter 1, Fig. 1.2).
Suboscines. The sperm of suboscines (Tyranni), represented in the trees only
by Tyrannus tyrannus, are poorly known. Nevertheless, they occupy their
traditional place at the base of the Passeriformes in the NJ tree (Fig. 8.59A) in
a passeriform clade. Monophyly of passeriforms and the basal position in
these of suboscines is endorsed by molecular studies (Sibley et al. 1988; Sibley
and Ahlquist 1990; Harshman, Chapter 1, Fig. 1.4). In the NJ tree (Fig. 8.59A)
the only unambiguous synapomorphy for the Passeriformes is the presence of
the ‘helical membrane’ (but see below). Computation of unambiguous changes
(not shown) on the MR tree gave none for Tyrannus but grouped all oscines by
enlargement of the periaxonemal dense fibers.
It is uncertain whether in suboscines the mitochondrial component is
unilateral, as it is in Passerida, and there appears to be more than one
mitochondrion; the dense outer axonemal fibers have either not been
illustrated or, in Tyrannus, are smaller than those of oscines; absence of a
nuclear rostrum requires confirmation; and accounts are contradictory as to
whether there is an helical membrane, though where described for Tyranni
this differs in structure from that of oscines. As noted, an apomorphy (not
computed) shared with oscines is the helical nucleus. A plesiomorphic feature
not seen in oscines is the (constant?) presence of two centrioles.
Passeri (oscines). The Corvida are represented by Corvus splendens but with
some reference to the light microscopy of Ballowitz (1888) and Retzius (1911,
1912) and the account of Birkhead et al. (2006). In the NJ tree they were not
resolved by any unambiguous character state from Tyrannus. However, they
are shown as the plesiomorph sister group of the Passerida in Fig. 8.59A as
one can confidently add two Corvida+Passerida synapomorphies: loss of the

proximal centriole and enlargement of the dense fibers. Tyrannus has both
centrioles and small dense fibers. These features require confirmation in most
suboscines. Corvida have the oscine synapomorphy of a true helical
membrane (imperfectly known but at least present as a microtubular bundle
in the spermatid) but remain plesiomorphic in the short acrosome and
midpiece, relative to the length of the nucleus. However, some exceptions to
the typical acrosome: nuclear ratios, below unity in Corvida and above in
Passerida are noted in section 8.10.10.1 and Table 8.6.
Passerida. Two synapomorphies for the Passerida are well established from
the large number of light microscope descriptions reviewed in this chapter
and from new information, notably for Myrmecocichla formicivora and
Philetairus socius. The most distinctive synapomorphy is the very long spiral
midpiece differing from that of the other group with a long midpiece, the
columbiforms (and possibly Coturnix japonica), in consisting of a single, spiral
mitochondrial strand. The second is the elongation of the acrosome relative to
the nucleus, though exceptionally, as recorded in Table 8.6, it remains shorter
than the nucleus as in the suboscines and most Corvida.
A more discriminating analysis should be attempted when a greater
knowledge of sperm ultrastructure in a much larger range of species accrues.
For instance, a detail of the mitochondrial helix, the presence of a fibrous helix,
might well diagnose the Muscicapoidea and presence of a granular helix may
have taxonomic value.
8.12 CONCLUSION
The author has attempted in this chapter to review works on the ultrastructure
of avian spermatozoa, with some reference to light microscopical studies, with
a view to demonstrating the diversity which exists in sperm structure in birds
and phylogenetic patterns which may be discerned. A very preliminary
phylogenetic analysis using PAUP has been presented. It is uncertain, when a
more complete matrix for all groups is obtained, whether the considerable
homoplasy which exists in avian sperm characters will permit the production
of highly resolved trees. Nevertheless, the taxonomic and phylogenetic value
of sperm characters is indisputable. Thus we have seen that even the highly
peculiar, ‘neotenous’ sperm of the Eurasian bullfinch yielded up its identity
as a passerine sperm when closely examined.
Spermatozoal characters of paleognaths have confirmed crocodiloid and
basal amniote relationships. They also allow confident identification of a
spermatozoon with its ordinal or subordinal grouping in many cases and
increasing discernment will presumably become possible as descriptions are
improved. Thus it is already possible to recongize a distinctive sperm type for
each of the groups Struthioniformes, Galloanserae, Galliformes, Anseriformes,
Psittaciformes, Apodiformes, Columbiformes, Passeriformes, Corvida and
Passerida.
Avian Spermatozoa: Structure and Phylogeny #!
It is hoped that this compendium will prove a stimulus and aid to other
researchers on avian spermatozoal morphology and will provide a
background for those interested in sperm function and general biology.
The author gratefully acknowledges facilities provided by the School of
Integrative Biology, University of Queensland, and particularly the assistance,
in procedures for electron microscopy, of Lina Daddow and Dr. David
Scheltinga, who is also thanked for commenting on the manuscript. Professor
Sandro Tripepi and Professor Alan Hodgson kindly provided unpublished
micrographs and Dr. Claire Spottiswoode collected material in Africa. Dr.
John Harshman provided many insightful comments on the manuscript and
aided with cladistic computations. Professor T. R. Birkhead kindly provided
some references and commented on the ms. Thanks are also due to the many
publishers who have allowed reproduction of illustrations.

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n n
CHAPTER
9
Testis Size, Sperm Size and
Sperm Competition
James V. Briskie1 and Robert Montgomerie2
9.1 INTRODUCTION
Almost since the moment animal semen was first viewed under the
microscope, it was clear that the shape and size of spermatozoa varied from
one species to the next (Leeuwenhoek 1697). Early comparative biologists
similarly noted striking differences in the size of the testis across different
species (Ray 1678). Although the general features of the male reproductive
system are rather conservative across the 10,000 or so extant species of birds,
recent work has confirmed large interspecific variation in the sizes of both
testes and sperm. For example, the combined mass of the left and right testes
of breeding Alpine accentors (Prunella collaris) is 3.0 g and comprises about
8% of adult body mass, while that of the Zebra finch (Taeniopygia guttata) is
only 0.05 g or 0.4% of its body mass (Birkhead et al. 1991; Nakamura 1990).
Likewise, a single spermatozoon of the Yellow warbler (Dendroica petechia)
averages 277 µm in length, while that of the Red-backed shrike (Lanius collurio)
is only 43 µm long, a six-fold difference in size (Briskie et al. 1997). Why
should testes mass and sperm size differ so much from one species to the
next?
In this chapter we examine some of the reasons for variation in both testis
size and sperm size in birds. A major turning point in our understanding of
this variation both within and between species, came with the seminal paper
by Parker (1970) in which he introduced the concept of sperm competition.
Parker (1970) defined sperm competition as occurring whenever a female
mated with more than one male during the course of a single breeding attempt.
1
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch,
New Zealand, E-mail: Jim.Briskie@canterbury.ac.nz
2
Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6, Canada. E-mail:
montgome@biology.queensu.ca
For birds, this might occur when a female engages in an extrapair copulation
with a neighboring male, or when her social mating system involves some
degree of polyandry. Sperm storage by female birds (Van Drimmelen 1946;
Hatch 1983; Briskie and Montgomerie 1993) increases the risk of sperm
competition as inseminations even weeks apart can be involved in the
fertilization of a single clutch of eggs (Oring et al. 1992; Birkhead 1998a). Thus
the intensity of sperm competition varies across species and it soon became
clear that differences in the reproductive morphology of birds could be linked
to this variation (Møller 1991; Birkhead and Møller 1992a; Briskie and
Montgomerie 1992, 1993, 1997). The advent of modern genetic profiling
techniques has further invigorated the field as variation in the morphology of
reproductive structures was shown to be related to differences in the sexual or
genetic (rather than social) mating strategies of males and females, both
within and between species (Møller and Briskie 1995; Briskie et al. 1997).
Our objective in this chapter is to take an evolutionary approach to the
gross morphologies of avian testes and sperm, particularly with respect to
variation in size (detailed morphological description of the avian testis is
provided in Chapter 2, the process of sperm production in Chapter 7, and
sperm ultrastructure in Chapter 8). To do this, we review mainly studies that
have used the comparative method (Harvey and Pagel 1991) to investigate
adaptive patterns. Thus our approach focuses on deriving explanations for
the evolution of the wide diversity of testes and spermatozoa sizes in birds
(i.e., the “ultimate” explanations for traits). The quest for such explanations
involves searching for potential explanatory variables that correlate with the
trait of interest, and then testing (either through experiments or further
comparative analyses) whether these explanations can be generalised to other
taxa. Despite more than a century of detailed anatomical and physiological
work on avian reproduction, modern comparative methods with appropriate
statistical rigor have been applied to understanding the evolution of testis and
sperm size only during the last two decades, and experimental studies are just
beginning (Birkhead et al. 2005). As the application of these approaches to the
study of avian reproduction is still in its early stages, we start by describing
variation in the number, position and size of the avian testis, and then review
the adaptive hypotheses that have been proposed to explain this variation. We
also survey variation in sperm size and review the evidence explaining the
evolution of this variation. We conclude by outlining gaps that remain in our
knowledge and suggesting where future workers can make a contribution.
9.2 EVOLUTION OF THE AVIAN TESTIS

9.2.1 Location and Number of Testes
Unlike the male genitalia of most mammals, those of birds are not obvious
externally. Instead, the testes of birds are positioned deep inside the body
cavity, visible only after dissection and removal of the viscera (Fig. 9.1). No
species of bird is known to have their testes in an external scrotum, and there
Testis Size, Sperm Size and Sperm Competition ##
Fig. 9.1 Urogenital tract of a male House sparrow during (right) and outside (left)
the breeding season, as revealed by dissection. On the right-hand drawing, the
right testis is indicated by a dashed line to reveal the location and size of the
epididymal duct. Presumably drawn to scale but no scale bars shown in the
original. Modified after Witschi, E. 1961. Pp. 115-168. In A. J. Marshall (ed.), Biology
and Comparative Physiology of Birds, Vol. II. Academic Press, New York, Fig. 18.
is no known variation in the position of the testes internally, unlike the

situation in mammals (Freeman 1990; Werdelin and Nilsonne 1999). External
genitalia could be disadvantageous in flight by reducing streamlining and
this may have prevented any adaptive change in testicular positioning in
birds. On the other hand, the position of the testes in birds (Fig. 9.1) is similar
to that in reptiles and other lower vertebrates, and the testes may have been
retained in this ancestral position simply in the absence of any selective
pressure to change.
The inhibition of sperm production at high body temperatures has often
been cited to explain the externalization of the testes in some mammals (Moore
1926; Werdelin and Nilsonne 1999). However, this cannot be a general
explanation as birds also maintain high body temperatures, often exceeding
that found in mammals (McNab 1966; Prinzinger et al. 1991), and avian testes
are not kept cooler than other internal organs (Beaupré et al. 1997).
Interestingly, Wolfson (1954) found that the cloacal protuberance of passerine
birds (Chapter 9) can store sperm at cooler than body temperatures. He
proposed this structure functions in an analogous manner to the externalized
testes of mammals, thus improving sperm storage and the later stages of
sperm maturation. The absence of a cloacal protuberance in non-passerines
appears consistent with their generally lower body temperature than
passerines (Lasiewski and Dawson 1967). Unfortunately, little work has been
done on sperm storage in relation to temperature in birds since Wolfson’s
(1954) pioneering work, and experimental studies are needed to confirm the
effects of high temperatures on the storage of sperm in both passerine and
non-passerine birds.
With few exceptions, male birds have two testes, whereas in females the
right ovary and oviduct are usually rudimentary. A few cases each of single
and supernumerary testes have been reported but these are generally
considered to be developmental abnormalities (e.g., Ferris 1933; Hocking
1992). The only exception to males having two testes appears to be the
Centropus coucals, in which the left testis is normally undeveloped or absent
altogether (Rand 1933; Chapin 1939; Ligon 1997). Ligon (1997) suggests that
the loss of the left testis in the coucals is related to lower testosterone levels in
these sex-role reversed species. However, it is difficult to see why hormone
titres could not be lowered by reducing the quantity of endocrine tissue in
each testis rather than by the loss of an entire gonad with, presumably, a
reduction in sperm production. The absence of a left testis in male coucals is
also unlikely to be homologous to the loss of the right oviduct in female birds,
as different sides of the body are involved (Ligon 1997). The loss of the right
oviduct in female birds has been considered an adaptation to the cost of flight
(Witschi 1935), but it is not known if flight is particularly costly for coucals or
would have been made less costly by the loss of the left testis. Alternatively, if
sex-role reversal has led to a reduction in the risk of sperm competition, this
may have favored the reduction in testis size. Thus, perhaps the entire loss of
one testis simply reduces the costs of maintaining two gonads. Further
Testis Size, Sperm Size and Sperm Competition #%
information on the levels of promiscuity in coucals relative to other species

with and without sex-role reversal is needed before this idea can be assessed.
9.2.2 Asymmetry in Testis Size

Differences in the size of the left and right testes are widespread among
vertebrates (Yu 1998). In birds, the most common pattern is for the left testis to
be larger than the right, but there are exceptions, with no difference in size in
some species and a larger right testis in others (Friedmann 1927; Lake 1981;
Kimball et al. 1997; Birkhead et al. 1998; Merilä and Sheldon 1999). For
example, the left testis in the Sedge warbler (Acrocephalus schoenobaenus)
averages 0.087 g, and is significantly heavier than the right testis, which
weighs only 0.074 g (Birkhead et al. 1997). In contrast, the left (0.326 g) and
right (0.328 g) testes of the Tree swallow (Tachycineta bicolor) do not differ
significantly in size (Kempenaers et al. 2002).
Stanley and Witschi (1940) suggested that the widespread right/left
asymmetry in testis development was a by-product of selection for asymmetry
in the female reproductive tract, in which only the left side develops. The
proximate basis for such an asymmetry may lie in the uneven migration of
primordial germ cells to the left and right sides of the body during the
development of the embryo (Witschi 1935). As the germ cells migrate to each
side of the body before sexual differentiation occurs, the differential attraction
of the germ cells to the left side may be so advantageous to females that it may
persist despite any disadvantage accrued to the development of the right testis
of males (Witschi 1935). However, it is not clear whether the number of
primordial germ cells directly affects the size of the mature gonad, especially
considering the large seasonal fluctuations in avian testis size that occur
through repeated mitosis of the germ cells. Furthermore, the development of
the left reproductive tract in female birds is nearly universal whereas there is
considerable variation in presence or absence of testis size asymmetry in
males, and the magnitude of this asymmetry varies widely both within and
among bird species. Thus, in at least some species, testicular asymmetry
cannot be a simple by-product of strong selection on female reproductive
asymmetry or germ cell number. As a result, a number of authors have sought
an adaptive explanation for this pattern.
Based on patterns of testicular asymmetry in Barn swallows (Hirundo
rustica) and House sparrows (Passer domesticus), Møller (1994) proposed that
the smaller right testis plays a compensatory role, developing to a similar size
as the normal left testis only when the latter does not develop properly (see
also Domm and Juhn 1927). He reasoned that males in poor condition might
be more likely to have a malfunctioning left testis and as a result compensate
by developing a larger right testis. Thus the degree of testicular asymmetry
could be seen as a measure of developmental homeostasis, with the highest
quality males sporting the most asymmetrical testes and lower quality males
with smaller, more symmetrical testes. An assumption of this hypothesis is
that it is more costly to support two similar-sized testes than two testes of
unequal size. It is not clear what these costs might be but they could include
increased flight costs (see below). In support of his compensation hypothesis,
Møller (1994) found that the degree of asymmetry in testis size varied
significantly with male quality as measured by tail streamer length in the
swallow, and black bib size in the sparrow. In both species, testis asymmetry
was largest in individuals having the most elaborate plumage ornaments, and
thus presumed to be of highest quality.
A number of subsequent studies across a variety of species have tested
Møller’s (1994) compensation hypothesis, but to date no support has been
found (Birkhead et al. 1997; Kimball et al. 1997; Merilä and Sheldon 1999;
Kempenaers et al. 2002; Graves 2004). Thus, it seems unlikely that this
hypothesis can provide a general explanation for testicular asymmetry in
birds. Indeed, in the only experimental test of this hypothesis, Birkhead et al.
(1998) found the opposite pattern: the difference between the masses of the left
and right testes was even greater in Zebra finches whose condition was
experimentally reduced. Thus, the patterns shown by Møller (1994) are a
curious artefact that deserve further investigation.
Despite the general lack of relation between male quality and the degree of
testicular asymmetry, a number of studies have found that testicular
asymmetry increases with age (Birkhead et al. 1997; Kimball et al. 1997, Merilä
and Sheldon 1999). Birkhead et al. (1997) suggested that males with greater
testicular asymmetry might survive better, although it is not clear how this
would occur unless directional asymmetry was related to male quality (as
suggested by Møller 1994) or if testicular asymmetry was more costly to
maintain. Graves (2004) found that older Black-throated blue warblers
(Dendroica caerulescens) exhibited a higher level of testicular asymmetry than
yearling males, as a result of the disproportionate enlargement of the left testis
with age. As older males in this species also have larger testes than younger
males, variation in the absolute and relative sizes of the left and right testes
may be related to variation in mating strategy (Graves 2004; see also Hill 1994;
Merilä and Sheldon 1999). For example, in some species, older males appear
to be preferred as mates and as extrapair copulation partners (Weatherhead
1984), and engaging in copulations with more than one female may require
higher rates of sperm production (Cartar 1985) and thus a greater investment
in testis tissue. Despite this, it is not obvious why selection for increased testes
size would necessarily favor an increased testicular asymmetry.
It is possible that testicular asymmetry functions to reduce the combined
testes mass, thereby reducing the costs of flight (as has been suggested for the
loss of the right oviduct in females; Witschi 1935). Flightless species, or those
that fly only occasionally, might then be expected to show less testicular
asymmetry than strongly aerial species (Kimball et al. 1997). Kempenaers et al.
(2002) did not find any support for this hypothesis in the highly aerial Tree
swallow which, contrary to expectations, had relatively symmetrical testes.
However, a wider comparative study is needed to test this idea more
thoroughly. It is worth noting that this cost of flight hypothesis might not just
Testis Size, Sperm Size and Sperm Competition #'
involve a reduction in total mass, but also a redistribution of mass to increase

overall balance along the longitudinal axis of the bird. For example, most of
the liver resides on the right side of the body in birds and other organs (e.g.,
gizzard, spleen) are similarly placed asymmetrically, potentially creating a
mass asymmetry between the left and right sides of the body. It is not clear
why these organs are positioned asymmetrically in the first place, but a larger
left testis might compensate any imbalance that could cause roll during flight.
We are not aware that this idea has been tested.
Finally, in some passerines, it has been observed that males exhibit a left-
side bias when mounting during copulation (Chapters 3 and Volume 6B,
Chapter 6). As only the left ovary of the female is functional in most species
(Chapter 4), and the left oviduct opens on the left side of the cloaca, it could be
advantageous for males to mount from the left if this results in sperm being
placed closer to this opening. In turn, this might favor differential investment
in sperm production by the two testes, resulting in a left-biased testicular
asymmetry if sperm from the left testis and vas deferens is more likely to reach
the left oviductal opening. Unfortunately, the dynamics of ejaculation are not
known in sufficient detail to test this idea quantitatively. However, as some
species have a right-side bias during copulation (e.g., American avocet,
Recurvirostra americana, and Black-necked stilt, Himantopus mexicanus; Sordahl
2001), it should be possible to compare patterns of testis asymmetry with
copulation behavior to evaluate this hypothesis.
9.2.3 Sperm Production and Testis Size

Although the testes play a role in the production of both hormones and sperm
(Chapter 7), the vast majority of tissue in the testes is devoted to the latter
function. Thus it is not surprising that larger testes are capable of producing
more sperm (Fig. 9.2; Møller 1988, 1989; Schärer et al. 2004). Sperm production
rates have been measured in only a few bird species, but rates appear to vary
interspecifically (Fig. 9.2). For example, sperm production in the socially
monogamous Zebra finch averages 1.9 ¥ 106 sperm/day (Birkhead et al. 1995),
while males in the promiscuous White-winged fairy-wren (Malurus leucopterus)
produce an average of 646 ¥ 106 sperm/day (Tuttle and Pruett-Jones 2004).
Sperm production can also vary among males within a species. Birkhead et
al. (1994) found that testes mass in one population of House sparrows ranged
from about 0.45 g to 1.0 g, and was correlated with the number of sperm stored
in the seminal glomera, suggesting that individuals with larger testes produce
more sperm. A similar pattern was found between males with different testis
sizes in the Zebra finch (Birkhead et al. 1993a). It should be noted that most
data on rates of sperm production by individual males have been collected
from domesticated and captive birds, and that a number of different
techniques were used. More information from wild birds is needed to confirm
that studies of captive birds provide realistic rates of sperm production, and
to determine why sperm production rates vary among different males within a
population.
Fig. 9.2 Relation between daily sperm production and testes volume in birds
(Model II regression, log(sperm production per day) = 8.11 + 0.69 log (testis volume).
r2 = 0.52, n = 9 species. Data from Tuttle, E. M. and Pruett-Jones, S. 2004. Animal
Behaviour 68: 541-550, Tables 1 and 4.
Species with large testes not only produce more sperm overall, but
apparently also produce more sperm per ejaculate (Møller 1988). Actual
ejaculate size (the number of sperm per ejaculate) is known for only a few
species, and Møller’s (1988) analyses were based on data from early studies of
ejaculate size in domestic birds that used collection methods (e.g., electro-
ejaculation, abdominal massage) likely to provide biased estimates of sperm
number. More recently, dummy models fitted with false cloacas have been
used to collect ejaculates deposited directly by males, and have provided
estimates that are more realistic with respect to natural copulations (Pellatt
and Birkhead 1994). For example, ejaculates collected in this manner average
3.89 ¥ 106 sperm in Zebra finches, 12.5 ¥ 106 sperm in Red-winged blackbirds
(Agelaius phoeniceus; Westneat et al. 1998), and 124 ¥ 106 in Peafowl (Pavo
cristatus; Birkhead and Petrie 1995).
In all studies to date, ejaculate size has been found to be highly variable,
both among males and within males. In the Peafowl, for example, ejaculates
contained from 4.2 ¥ 106 to 248 ¥ 106 sperm (Birkhead and Petrie 1995).
Reasons for this variation are not clear, although at least some of it may be
adaptive. For example, Nicholls et al. (2001) found that male Bank swallows
(Riparia riparia) increased the size of their ejaculates when faced with rival
males (i.e., more intense sperm competition). Ejaculate sizes also decline with
time since last copulation when successive copulations occur within a short
period of time, such that sperm depletion may limit the rate of insemination
Testis Size, Sperm Size and Sperm Competition #
(Birkhead et al. 1995). This in turn should lead to selection favoring increased
testis size, and thus increased sperm production, in species with high
copulation rates and high levels of promiscuity (Birkhead et al. 1993b).
9.2.4 Seasonal Change in Testis Size

Most bird species undergo cyclical patterns of testicular activity and inactivity
(Murton and Westwood 1977), with a period of rapid testicular re-growth or
recrudescence in mature males just prior to the start of the breeding season.
After breeding, the testes regress and remain inactive until the next breeding
season, when they undergo another phase of recrudescence (e.g., Fig. 9.3). The
difference in testis size between the non-breeding and breeding seasons can
be dramatic (Fig. 9.3). In the House crow (Corvus splendens), for example, the
combined mass of both testes in the breeding season is more than 75 times
that in the non-breeding season (Dang and Guraya 1978). Increases of 300-
500 fold between the non-breeding and breeding season have been recorded
in the Eurasian tree sparrow (Passer montanus; Lofts and Murton 1973),
Common magpie (Pica pica; Erpino 1969), and Brambling (Fringilla
montifringilla; Marshall 1961).
Changes in testis size over the season are well documented among
temperate species in the Northern Hemisphere, but cyclical changes in testis
size also occur in tropical environments (Fig. 9.3C, D; Wikelski et al. 2003).
Birds living in relatively unpredictable environments, on the other hand, may
maintain their testes in a relatively developed state to take advantage of rapid
improvement in breeding conditions (Immelmann 1971; Zann et al. 1995). The
testes may also remain enlarged for the entire breeding season in species with
multiple breeding attempts per season, although sperm production may
increase during the periods that coincide with the fertility of their mates (Lofts
and Murton 1973).
Studies on patterns of testis recrudescence and regression typically focus
on a species-specific pattern, but there can also be considerable variation
among individuals within a population on the timing of the testicular cycle.
For example, Dawson (2003) found that older European starlings (Sturnus
vulgaris) initiated testicular recrudescence 3-4 weeks earlier than younger
males and did not enter the regression phase until 2 weeks later than younger
males. This translated into a 50% longer period of spermatogenic activity in
older males. In the Winter wren (Troglodytes troglodytes), males that underwent
recrudescence earlier also nested earlier than males that delayed testicular
growth (Evans and Goldsmith 2000). Among species breeding year round (e.g.,
some tropical species), each individual may continue to cycle through periods
of recrudescence and regression such that at any given time some males have
active spermatogenesis while others do not (Lofts and Murton 1968).
In contrast to their wild counterparts, domestic fowl (Gallus g. domesticus)
and some domesticated birds have little seasonal change in testis size and are
thus capable of producing sperm year round. In the domestic fowl, this pattern
has likely been the product of selective breeding to maximize their rate of
Fig. 9.3 Seasonal varition in testis size in some temperate zone (A, B) and tropical
(C, D) bird species. A European blackbird (Turdus merula): testis width (mm) of
urban birds. Modified after Partecke, J., Van’t Hof, T. and Gwinner, E. 2004.
Proceedings of the Royal Society B 271: 1995-2001, Fig. 1(i). B House sparrow:
testis mass (mg); shaded zones indicate three successive broods in this rural
population. Modified after Hegner, R. E. and Wingfield, J. C. 1990. Pp 123-135. In J.
Pinowski and D. Summers-Smith (eds), Granivorous Birds in the Agricultural
Landscape. INTECOL, Warsaw. C Red-billed quelea (Quelea quelea): maximum
testis diameter (mm); birds maintained on constant 12-h photoperiod over two
years. Modified after Lofts, B. and Murton, R. K. 1973. Pp. 1-107 In D. S. Farner,
J. R King and K. C. Parkes (eds). Avian Biology, volume 3. Academic Press, New
York, Fig. 7. D Red-vented bulbul (Pycnonotus cafer): testis volume (mm3); birds
maintained in captivity on natural light cycle. Modified after Lal, P. and Thapliyal, J.
P. 1982. General and Comparative Endocrinology 48: 98-103, Fig. 1.
reproduction (Sossinka 1982). Wyndham et al. (1981) suggested that the

abundance of food in captivity might also explain why domesticated
Budgerigars (Melopsittacus undulatus) undergo reduced periods of testicular
regression relative to their wild progenitors.
The initiation of testicular recrudescence appears to be under
photoperiodic control, at least for temperate-breeding passerines, although it
can be modified by temperature, weather, food supply, and behavioral
interactions (Lofts and Murton 1968; Lal and Thapliyal 1982; Follett 1984).
The histological and physiological changes that take place during the
Testis Size, Sperm Size and Sperm Competition # !
testicular cycle are well documented in a variety of bird species (Chapters 2

and 7).
9.2.5 Body Size and Testis Size

Within species, larger males tend to have larger testes (Fig. 9.4A; Rising 1987;
Møller and Erritzøe 1988; Merilä and Sheldon 1999; Evans and Goldsmith
2000; Brown and Brown 2003), though body size often accounts for a small
proportion of variation in testis size. In 48 populations across the range of
Savannah sparrows (Passerculus sandwichensis), for example, only 7% of the
variation in testis size among individuals (n = 1157) was explained by
variation in body mass, though 29% of the variation in mean testis mass
among populations was explained by mean body mass (Fig. 9.4A; Rising
1987). Older males are sometimes larger than young males, either due to
growth or differential mortality, but variation in age alone cannot always
explain the positive relation between testis size and body size. For example,
older male Black-headed grosbeaks (Pheucticus melanocephalus) had relatively
larger testes in an analysis controlling for body size (Hill 1994). The greater
relative size of testes in larger males may thus be related to a high cost of
testicular recrudescence and maintenance that only males in good condition
are able to bear.
Testis size is also positively correlated with body size across species
(Møller 1991; Pitcher et al. 2005). For example, in a sample of 1010 bird species,
Pitcher et al. (2005) found that almost half of the inter-specific variation in
testis mass was explained by body mass (Fig. 9.4B). In a similar study, Møller
(1991) noted that the slope of the regression of testes mass on body mass was
significantly less than one (see also Fig. 9.4B). Thus larger-bodied species
have relatively smaller testes than smaller species, though the reasons for this
are unknown. Because of the strong effect of body mass on testis size, most
studies of testis size variation have controlled for body size either by
examining residual testis size, or by entering body mass as an independent
variable in multiple regression analyses.
9.2.6 Age-related Changes in Testis Size

Birds of many species mature and initiate reproduction during the first
breeding season after they hatch. However, among bird species for which data
are available from the breeding season, the testes are generally larger in adult
birds (2 years and older) than among males breeding for the first time (Wright
and Wright 1944; Davis 1958; Selander and Hauser 1965; Foster 1987;
Deviche et al. 2000). In Cliff swallows (Petrochelidon pyrrhonota), testes size was
smallest in birds aged 1 and 2 years but did not vary with age in males 3 years
old or more (Fig. 9.5; Brown and Brown 2003). As mentioned above, the size of
the left and right testes may also change differentially with age, such that the
degree of testicular asymmetry differs between younger and older males.
In the Black-headed grosbeak, males breeding at the end of their first year
do not develop adult breeding plumage and thus their smaller testes may be
# " Reproductive Biology and Phylogeny of Birds
Fig. 9.4 Relation between testes size and body size in birds; model II regressions
are plotted. A Intraspecific variation among 48 populations (n = 9-45 males per
population) of Savannah sparrows [log(testis volume) = –0.81 + 1.25 log(femur
length), r2 = 0.29]; single testis volume calculated from length and width measured
on freshly-killed specimens. Data from Rising, J. D. 1987. Wilson Bulletin 99: 63-
72, Table 1. B Interspecific variation among 1010 bird species [log(testes mass) =
–2.04 + 0.89 log(body mass), r2 = 0.47]. Testes mass calculated as the combined
mass of both testes averaged over at least five breeding males per species;
estimated from linear measurements, mainly from museum specimens. Redrawn
from data in Pitcher, T. E., Dunn, P. O. and Whittingham, L. A. 2005. Journal of
Evolutionary Biology 18: 557-567, Fig. 1.
Testis Size, Sperm Size and Sperm Competition # #
Fig. 9.5 Age-related changes in testes size in the Cliff swallow. Testes size is
measured as residuals from the regression of the combined testes volume
(calculated from length and width measurements) on a multivariate measure of
body size. Numbers near data points are sample sizes. Modified after Brown, C. R.
and Brown, M. B. 2003. Behavioral Ecology 14: 569-575, Fig. 1.
part of a more general, adaptive delay in reproduction (Hill 1994). As rates of

sperm production are related to testis size (Fig. 9.2), younger males would not
produce as much sperm as older males. Young males in the lek-breeding
Sharp-tailed grouse (Tympanuchus phasianellus) also have smaller testes than
older males and are more likely to occupy peripheral territories on the edge of
a lek, while older males dominate the central territories (Tsuji et al. 1992).
Nitchuk and Evans (1978) confirmed that peripheral males in this species do
produce less sperm than central males, but it is not clear that females would
obtain fewer sperm by mating with younger males, nor if this would reduce
the female’s fertility relative to mating with older males. Although some
studies of parentage have found that younger males were more likely to be
cuckolded (e.g., Wetton et al. 1995), this could be due to female choice or the
failure of young males to protect mates from extrapair copulation attempts,
rather than fewer sperm being inseminated by younger males. In some
polygynous species (e.g., Graves 2004), younger males are also less likely to
attract multiple mates and thus may have smaller testes as an adaptation to
the lower number of sperm required to fertilize the ova of a single female
(Cartar 1985).
9.2.7 Geographic Variation in Testis Size

Until recently, geographic variation in testis size within species has either
been ignored, or has been assumed to be small relative to that observed among
species (Pitcher and Stutchbury 1998). For some species the latter appears to
# $ Reproductive Biology and Phylogeny of Birds
be true. For example, Graves (2004) found no geographic variation in testis

size among 25 populations of the Black-throated blue warbler across its entire
North American range. However, for most other species examined to date,
testis size appears to vary with geographic location, with the most common
pattern an increase in testis size with latitude (Fig. 9.6A-C; Pitcher and
Stutchbury 1998; Merilä and Sheldon 1999). Such an increase in testis size
with latitude has been suggested to result from either more intense sperm
competition with latitude (Pitcher and Stutchbury 1998; Merilä and Sheldon
1999) or the shortened breeding season at high latitudes, which may require
high rates of daily sperm production (Kenagy and Trombulak 1986). There are
few intraspecific data available on the concomitant levels of sperm
competition, breeding seasonality and testis size that would be needed to test
either of these ideas. The opposite pattern, seen in Savannah sparrows (Fig.
9.6D), is harder to explain but may be influenced by the larger number of
broods and thus longer breeding season at lower latitudes (Rising 1987).
Fig. 9.6 Relation between relative testis size (RTS) and latitude in different bird
species. A House finch (Carpodacus mexicanus): 8 populations. B Red-eyed vireo:
19 populations. C Greenfinch (Carduelis chloris): 8 populations. Modified after
Merilä, J. and Sheldon, B. C. 1999. Behavioral Ecology and Sociobiology 45: 115-
123, Fig. 2. D Savannah sparrow: 24 populations. Modified after Pitcher, T. E. and
Stutchbury, B. J. M. 1998. Canadian Journal of Zoology 76: 618-622, Fig. 1a, c, d. In
A, B and D, RTS was measured as the ratio (¥ 103) of testis mass (calculated from
length and width) to body mass; in C, RTS is testis length in mm, correcting for age
and body size. Model II regression lines shown to illustrate trends; all relations
were significant in the original analyses.
Testis Size, Sperm Size and Sperm Competition # %
Broader patterns of geographic variation in testis size have also been

examined among species. In a comparative study analyzing data from a
variety of temperate and tropical species, Stutchbury and Morton (1995) found
that testis size was generally smaller in a small sample of Neotropical birds.
Such a pattern might be expected if sperm competition is less intense in
tropical environments. Further work is needed to determine whether the
intensity of sperm competition varies geographically.
In a much larger sample of species, Pitcher et al. (2005) confirmed that testis
size varies across geographic regions, with species living on the Eurasian and
North American landmasses having larger relative testis size than species in
South America and Australasia. It is unclear, however, why residence in a
particular continental region should influence testis size. It seems more likely
that such geographic differences are due to confounding variables that vary
between the regions (e.g., differences in taxonomic representation, climate,
body size, mating systems, etc.).
9.2.8 Testis Size and Mating System

Even when differences in season, age, latitude and body size are taken into
account, testis size varies dramatically among bird species. Harcourt et al.
(1981) suggested a simple explanation for this pattern based on sperm
competition theory (Parker 1970), and found that primate species in which
females copulate with more than one male had relatively larger testes than
monogamous species. As the rate of sperm production increases with testis
size (Fig. 9.2), Harcourt et al. (1981) argued that an evolutionary increase in
testes mass is favored whenever a male’s sperm competes with the sperm of
other males to fertilize a female’s ova. The advantage of producing more
sperm in such situations has been likened to a lottery in that the more sperm
produced, the greater the probability of securing fertilizations; increased
sperm numbers could function either to dilute or displace the sperm of rival
males. Subsequent studies have confirmed positive associations between testis
size and the intensity of sperm competition across a range of taxa, including
birds, in general (Fig. 9.7A; Birkhead and Møller 1998), and waterfowl (order
Anseriformes), in particular (Fig. 9.7B: Coker et al. 2002).
Testis size is particularly large in species with polygynandrous mating
systems such as Dunnock (Prunella modularis; Birkhead et al. 1991), Smith’s
longspur (Calcarius pictus; Briskie 1993), and Superb fairy-wren (Malurus
cyaneus; Mulder and Cockburn 1993). Combined testes mass in these species
varies from 4-8% of body mass, compared to only about 1% for birds in
general (Pitcher et al. 2005). In polygynandrous species, both males and
females regularly mate with multiple partners during the span of a single
breeding attempt. Although large testes may partly be an adaptation to avoid
sperm depletion (see below; Cartar 1985), the high levels of polyandry by
females in these species also means that sperm competition is particularly
intense. Thus, species in which females have highly promiscuous mating
behavior tend to have larger testes than species with single-male mating
# & Reproductive Biology and Phylogeny of Birds
Fig. 9.7 Relative testes mass of bird species with different social mating systems.
Social mating systems are shown from left to right in order of increasing expected
intensity of sperm competition; relative testes mass measured as residuals from
the regression of combined testes mass on body mass; number of species is
shown at the base of each bar. A Social mating systems in 1002 bird species
(MON = monogamy, CPR = cooperative breeding, MPG = monogamy with some
males polygynous, PGY = polygyny, LEK = lekking, PAN = polyandry). Modified after
Pitcher, T. E., Dunn, P. O. and Whittingham, L. A. 2005. Journal of Evolutionary
Biology 18: 557-567, Fig. 4b. B Social mating systems in 29 waterfowl species
(MON = monogamous, IFC = infrequent forced extrapair copulations, FFC =
frequent forced extrapair copulations, PRM = promiscuous). Modified after Coker,
C. R., McKinney, F., Hays, H., Briggs, S. V. and Cheng, K. M. 2002. Auk 119: 403-
413, Fig. 4A.
Testis Size, Sperm Size and Sperm Competition # '
systems (Fig. 9.7A; Møller 1991; Birkhead and Møller 1992a; Rising 1996;
Pitcher et al. 2005).
This result is further supported by analyses using levels of extrapair
parentage from studies of genetic paternity in birds. Thus species with high
proportions of extrapair young in their nests have significantly larger testes
than expected for their body size (Fig. 9.8; Møller and Briskie 1995;
Garamszegi et al. 2005). However, using data from 34 species, Garamszegi et
al. (2005) found that residual testes size alone was not a significant predictor
of the rate of extrapair paternity (Fig. 9.8) and suggested that testosterone
production might mediate this relation. Using path analysis they compared
statistically three models for the interaction between these two variables on
the evolution of testes size, as follows. First, they suggested that extrapair
paternity (EPP) and residual peak testosterone level (RPT) might affect each
other and both directly influence the evolution of testis size. Second, they
proposed that testis size might evolve first, under the influence of intense
sperm competition, resulting directly in higher levels of EPP and a secondary,
indirect effect on RPT. Finally, they suggested that testis size might evolve first
under sperm competition, but then have a primary effect on RTP and a
secondary one on EPP. Their analysis provided most support for the second
model, explaining 28% of the variation in the data.
Species that nest in colonies or at high density have also been suggested to
experience higher levels of sperm competition than solitarily nesting species
Fig. 9.8 Rate of extrapair paternity in relation to relative testes size in 34 bird
species. Rate of extrapair paternity is measured as the percent of offspring sired
by extrapair males; relative testes size is measured as the residuals from the
regression of combined testes mass on body mass, both log-transformed; model
II regression is plotted to show trend. Data from Garamszegi, L. Z., Eens, M.,
Hurtrez-Bousses, S. and Møller, A. P. 2005. Hormones and Behaviour 47: 389-409,
Table 1.
#! Reproductive Biology and Phylogeny of Birds
(Gladstone 1979; Møller 1991; Birkhead and Møller 1992a). This may occur
either because of the proximity of potential extrapair copulation partners or
due to increased opportunities for multiple copulation partners in those
species in which one member of the pair must stay and defend the nest site,
thereby preventing males from guarding their mates. As expected, colonial
species have significantly larger testes than solitarily nesting species (Møller
1991; Pitcher et al. 2005). Moreover, even within a species, there is evidence
that the increased risk of sperm competition in colonial nesters can favor
increased testis size. Brown and Brown (2003) found that Cliff swallow males
nesting in larger colonies had testes masses almost three times that of males
nesting in small colonies or solitarily. Although this pattern could be a
facultative response to the perceived risk of sperm competition (i.e., males
invest more in sperm production when nesting in big colonies), Brown and
Brown (2003) suggested there may also be a genetic component to the
difference as the preference for individuals to nest in different-sized colonies
was heritable.
Although testis size correlates with the intensity of sperm competition, an
alternative hypothesis was proposed by Cartar (1985) based on the frequency
of matings that a male obtains (the fertilization frequency hypothesis). He
suggested that selection should favor larger testis size in species in which
males pair and copulate with more than one female, simply because they
require more sperm to fertilize the additional ova. Under this hypothesis,
males in polygynous species are expected to have relatively larger testes than
males in a monogamous species, even in the absence of sperm competition. In
support of this hypothesis, Cartar (1985) found that polygynous waders
(family Scolopacidae) had larger testes than monogamous species. It is now
clear that sperm competition is widespread in birds (Birkhead and Møller
1992a; Birkhead 1998a,c): studies of mating behavior in a number of species
have shown high levels of mate infidelity, and the use of DNA profiling
techniques has demonstrated that extrapair paternity is common (Griffith et al.
2002). Thus, Cartar’s (1985) results were likely confounded by differences in
the levels of sperm competition between socially monogamous and socially
polygynous waders, though there are few data currently available to test this
idea.
Recently, in a large comparative study with data from 934 bird species,
Pitcher et al. (2005) found that testis size increased with clutch size, suggesting
that species with larger clutches actually require larger testes (Fig. 9.9). Large
testes might be required to produce enough sperm to fertilize a greater number
of ova and avoid sperm depletion during the fertilization period. The fitness
costs of sperm depletion could thus favor increased testis size not only in
species in which males require more sperm to fertilize a larger number of
females (as originally proposed by Cartar 1985), but also in situations where
each female has more eggs to fertilize. In addition, sperm depletion has been
suggested to play a role favoring increased testis size in species in which
breeding is restricted to a few days or weeks, such as at high latitudes (Kenagy
Testis Size, Sperm Size and Sperm Competition #!
Fig. 9.9 Relation between residual testes mass (RTM; calculated as in Fig. 9.7)
and clutch size (CS) in 934 bird species. Model II regression line is shown (RTM =
–0.81 + 0.22 CS, r2 = 0.02, P <0.0001). Redrawn from data in Pitcher, T. E., Dunn,
P. O. and Whittingham, L. A. 2005. Journal of Evolutionary Biology 18: 557-567, Fig.
4c and appendix.
and Trombulak 1986). The increase in testis size with latitude found in some
species (Pitcher and Stutchbury 1998; Merilä and Sheldon 1999) is consistent
with this idea, but further work is required to tease apart the influences of
variation in the intensity of sperm competition with both shortened breeding
seasons and geographic range. Moreover, the relation between clutch size and
relative testis size is very weak, with clutch size explaining only about 1% of
the variation in testis size. This may suggest that some confounding variable
is actually responsible for the reported relation (Fig. 9.9).
9.2.9 Costs of Large Testis Size

Despite the seeming benefits of increased testis size when sperm competition
is intense, it is not clear what ultimately limits testis size in birds. In other
words, are there costs to larger testes and can these costs explain some of the
variation in testis size across species? Studies on the costs associated with
relatively large testes have lagged far behind those on the proposed benefits,
but a few hypotheses have been suggested. For example, Zuk et al. (1990)
proposed that increased testis size might increase the risk of parasitemia
brought on by the immunosuppressive effects of higher androgen levels (see
also Folstad and Karter 1992; Salvador et al. 1996; Merilä and Sheldon 1999).
However, it is not clear whether increased testis size necessarily brings about
an equivalent increase in androgen production (Weatherhead et al. 1993;
Wikelski et al. 2003). In theory it should be possible to increase spermatogenic
#! Reproductive Biology and Phylogeny of Birds
tissue without increasing endocrine tissue but, across species, larger testes
generally result in higher circulating levels of testosterone (Wingfield and
Moore 1987; Garamszegi et al. 2005). Brown and Brown (2003) were able to
increase testis size experimentally in Cliff swallows by applying insecticidal
powder to their nests, thereby reducing their ectoparasite burden. One
interpretation of this result is that exposure to parasites elevates levels of
plasma corticosterone, which in turn alters the adrenal response to stress,
resulting in both a suppression of testosterone and a reduced testes mass
(Dunlap and Schall 1995). It is also possible that a reduced parasite load frees
up resources for investment in testis tissue that otherwise would be needed
for immune responses.
Larger testes might also be costly energetically. This could include costs of
production and maintenance, as well as the increased flight costs that result
from carrying more mass. In most species, the mass of both testes combined is
only 1-3% of body mass, so would seem unlikely to result in an appreciable
increase in flight costs. However, in species subject to intense sperm
competition, with relatively large testes as a result, testes mass can range from
4-8% of body mass (Briskie 1993). Studies that have experimentally attached
weights to birds have found significant effects of the extra burden at levels
similar to this testes mass (e.g., Wright and Cuthill 1989). The fact that
younger males have smaller testes than older males, and that the testes of
males of all ages typically undergo a period of regression in the non-breeding
season, suggests there is a cost to maintaining enlarged testes. Perhaps
studies that experimentally induce earlier or larger recrudescence with gonad-
stimulating hormones (and thereby result in a male with a larger pair of testes
than normal) can be used to examine the presumed costs of large testis size.
9.3 EVOLUTION OF AVIAN SPERM

9.3.1 The Avian Spermatozoon
The avian spermatozoon has two main structural parts, the head and the tail.
The sperm head consists of an anterior acrosome and a posterior nucleus,
while the tail consists of an anterior midpiece (plesiomorphically just
posterior to the nucleus) that surrounds an axoneme comprised of an anterior
principal piece and a posterior endpiece. In the parvorder Passerida, the
midpiece mitochondria typically extend for most of the length of the tail
(Chapter 8). Extensive variation in the structure of sperm among different
species of birds was described by early workers (Ballowitz 1888; Retzius 1909,
1911), and more recent studies have particularly uncovered extensive
interspecific variation in the lengths of sperm and their component parts
(McFarlane 1963; 1971; Briskie and Montgomerie 1992; Koehler 1995). The
ultrastructure of avian sperm has been described across a wide range of
species and is reviewed comprehensively in Chapter 8.
The greatest differences in avian sperm morphology are found when
comparing passerine (order Passeriformes) and non-passerine birds (all
Testis Size, Sperm Size and Sperm Competition #!!
remaining extant orders). The spermatozoa of non-passerines are similar to

some reptilian sperm and are linear (or slightly undulating) in the head
region, while the sperm of passerine birds are coiled in a helical fashion
(McFarlane 1963, 1971; Chapter 8). Passerine sperm can vary among species
from being helical only in the acrosomal region to having a helical acrosome,
nucleus and midpiece (Koehler 1995). At the ultrastructural level, passerine
sperm lack the perforatorium, endonuclear canals, distinct centrioles, and
annulus found in non-passerines, while they possess a series of nine
accessory fibers that is absent or less developed in non-passerines (Asa and
Phillips 1987; Koehler 1995). Passerine sperm also have an ‘undulating
membrane’ that arises from the apex of the acrosome and is said to extend for
most of the length of the sperm (Humphreys 1972; Henley et al. 1978; but see
Chapter 8). Species in the orders Charadriiformes and Procellariformes also
possess a helical sperm membrane but this is thought to have evolved
independently of the similar membrane found in passerines (Koehler 1995).
The only exception to the distinctive nature of passerine sperm was
recently reported by Birkhead et al. (2006) in the Eurasian bullfinch (Pyrrhula
pyrrhula). Unlike other passerines, the sperm of this species has a rounded
head, a tiny midpiece, and no obvious, external helical structure. It does,
however, retain the passerine hallmark of a microtubular helix (Birkhead,
Giusti, Immler and Jamieson, pers. comm.). This sperm structure may be
unique to this species, as the congeneric Beavan’s bullfinch (P. erythaca) has
sperm closer to that of other passerines (Birkhead et al. 2006). As sperm
morphology has been examined microscopically in only about 100 bird
species (Chapter 8), it is too early to say if the Eurasian bullfinch is the only
exception to the generally conservative structure of passerine sperm.
9.3.2 Variation in Sperm Size

Sperm length is highly variable in birds (McFarlane 1963, 1971; Allen et al.
1968; Humphreys 1972; Briskie and Montgomerie 1992; Briskie et al. 1997;
Johnson and Briskie 1999). Total sperm length varies six-fold among
passerines species that have been examined, from 42.7 µm in the Red-backed
shrike (Briskie et al. 1997) to 292.4 µm in the Reed bunting (Emberiza
schoeniclus; Dixon and Birkhead 1997). Among waders (families Scolopacidae,
Charadriidae and Jacanidae), sperm length varies from 57.0 µm in the
Semipalmated plover (Charadrius semipalmatus) to 133.2 µm in the Ruff
(Philomachus pugnax; Johnson and Briskie 1999).
Even within a family, sperm length can be quite variable. Allen et al. (1968)
measured the sperm lengths of 9 species (and 4 subspecies) of passerines in
the family Icteridae (New World blackbirds) and found that sperm length
varied from 77.9 µm in the Western meadowlark (Sturnella neglecta) to 148.6 µm
in eastern populations of the Red-winged blackbird (A. p. phoeniceus).
Although published information on sperm length is available for only
about 120 bird species, there are no striking differences between the sperm
#!" Reproductive Biology and Phylogeny of Birds
lengths of passerines (maximum 292.4 µm in the Reed bunting) and non-

passerines (maximum ~230 µm in the Japanese quail, Coturnix japonica;
Woolley 1995). However, more information on a wider variety of species is
needed to determine how sperm size varies with taxonomic affiliation and to
what degree such phylogenetic effects might constrain evolutionary changes
in sperm size.
Across species, the relative size of each region of the spermatozoon can also
vary. For example, the size of the midpiece varies from only 3.3 µm in the Great
crested flycatcher (Myiarchus crinitus; Koehler 1995) to 161.4 µm in the
Japanese quail; Woolley 1995). Among 17 species of passerines studied by
Briskie and Montgomerie (1992), tail length varied from 36 µm in the Least
flycatcher (Empidonax minimus) to 262 µm in the Yellow warbler. In this
sample of birds, differences in the length of the tail explained more than 99%
of the variation in total sperm length (Briskie and Montgomerie 1992). In
contrast, the size of the nucleus among passerines varies only from 2.5 µm in
the American robin (Turdus migratorius) to 18.5 µm in the Great crested
flycatcher (Koehler 1995), and the size of the acrosome only from 2.5 µm in the
European starling to 13.5 µm in the Violet-green swallow (Tachycineta
thalassina; Koehler 1995). Even though there is relatively little interspecific
variation in the size of the nucleus and acrosome, the lengths of the various
components of the spermatozoon appear to be correlated across species
(Johnson and Briskie 1999). For example, Birkhead et al. (2005) found that
flagellum length was positively correlated with midpiece length in a sample
of 29 passerine species. As the midpiece extends for most of the length of the
flagellum in most passerines, this result is perhaps not unexpected (see
Chapter 8), though it does suggest that genetic correlation plays a role in
sperm design (Birkhead et al. 2005).
Most comparative studies of sperm size variation have assumed that there
is little variation within a species. Although interspecific variation is usually
much larger than variation within a species (Briskie and Montgomerie 1992;
Briskie et al. 1997), recent work has revealed that variation in sperm length
(and the size of each component part) can also vary significantly among
males of a single species (Tuttle et al. 1996; Morrow and Gage 2001a; Birkhead
et al. 2005). Thus, in the Zebra finch, variation in sperm flagellum length and
midpiece length between males was even greater than variation in an external
morphological trait such as tarsus length (Birkhead et al. 2005). Total sperm
length in this species varied from 45 to 78 µm (Birkhead et al. 2005) but, within
an individual male, sperm length was highly consistent, both within an
ejaculate and over time (Birkhead and Fletcher 1995). Intraspecific variation
in the size of sperm and its component parts are heritable and negatively
correlated in Zebra finches (Birkhead et al. 2005), confirming that this
variation is not simply due to environmental effects.
9.3.3 Sexual Selection and Sperm Morphology

To date, understanding variation in sperm size between species has focused
mostly on identifying the likely benefits to increased sperm length as an
Testis Size, Sperm Size and Sperm Competition #!#
adaptation to variation in the female reproductive tract. As all birds have

internal fertilization, it is the female’s reproductive tract that forms the arena
in which sperm must function and in which selection will favor changes in
sperm size. Two factors that have attracted the most attention in this regard
are the relations between sperm length and the forces of sperm competition
(Birkhead and Møller 1992a) and the effects of cryptic female choice (Eberhard
1996).
Given a limited budget for expenditure on gamete production, males subject
to intense sperm competition are expected to increase the number of sperm
produced (i.e., ejaculate size, sperm concentration, copulation frequency) at
the expense of sperm size (Parker 1982). In other words, males face a trade-off
between the number and size of sperm, and sperm competition is expected to
favor males that produce large numbers of small sperm in order to dilute or
displace the ejaculates of rival males. However, this expected pattern has
generally not been supported in birds and most other groups of animals
(Gomendio and Roldan 1991; Briskie and Montgomerie 1992; Gage 1994;
Briskie et al. 1997; Johnson and Briskie 1999). Instead, bird species subject to
intense levels of sperm competition generally have longer sperm (Fig. 9.10;
Briskie et al. 1997). Species with large testes also generally produce longer
sperm (Johnson and Briskie 1999), as would be expected if testis size is a
reasonable index of the intensity of sperm competition, as outlined earlier in
this chapter. This suggests that males do not directly trade-off sperm size for
sperm number but instead invest proportionately more into gamete
Fig. 9.10 Sperm length in relation to the rate of extrapair paternity in 21 species of
passerines. Model II regression line is shown (sperm length = 52.4 + 1.74 EPP
rate, r2 = 0.31, P = 0.01). The relation remains significant when controlling for
phylogeny. Modified after Briskie, J. V., Montgomerie, R. and Birkhead, T. R. 1997.
Evolution 51: 937-945, Fig. 2.
#!$ Reproductive Biology and Phylogeny of Birds
production (via increased testis and sperm size) in species subject to more
intense sperm competition.
Early studies of sperm size variation proposed that increased sperm length
was an adaptation that resulted in faster swimming sperm (Gomendio and
Roldan 1991; Briskie and Montgomerie 1992). Among a small sample of
domesticated mammal species, Gomendio and Roldan (1991) found a positive
relation between sperm length and swimming speed, and they suggested that
increased levels of sperm competition would favor increased swimming
speeds in the race to fertilize a female’s ova. Thus longer flagella would make
sperm swim faster and generate more thrust (Katz et al. 1989; Katz and
Drobnis 1990). However, birds differ from mammals in that sperm are
typically stored by the female in specialized sperm storage tubules (SSTs)
before they move up the oviduct to fertilize an ovum in the infundibulum
(Hatch 1983; Shugart 1988; Briskie and Montgomerie 1993; Chapters 6 and
10). This led Briskie and Montgomerie (1992) to propose that increased sperm
length in birds might be an adaptation to increase swimming speed in order
to reach SSTs before the sperm of rival males. Indeed, wader species with more
promiscuous mating systems had longer midpieces (the site of energy stores)
than species with more monogamous mating systems, suggesting that
increased resources were devoted to locomotion (Johnson and Briskie 1999).
Birkhead et al. (2005), however, found no relation between sperm length and
sperm velocity among a sample of 105 Zebra finch males with flagellum
lengths varying from about 37 to 68 µm, and midpieces varying from 15 to
48 µm. Measures of sperm swimming speed across a wide range of species are
needed to resolve this issue but it seems unlikely that increased sperm length
is simply a function of the benefits accrued by greater swimming speed.
The storage of sperm in a female’s SSTs for days or even weeks before
fertilization (Birkhead and Møller 1992b) suggests that variation in sperm
length might be related to variation in sperm storage. Both the number and
size of SSTs vary widely across bird species, and sperm length is positively
correlated with SST length (Fig. 9.11A; Briskie and Montgomerie 1992, 1993;
Briskie et al. 1997). In most species studied to date, the length of a single SST is
about twice that of the sperm that they store (Fig. 9.11A). A few species have
sperm only slightly shorter than an SST (e.g., Red-eyed vireo, Vireo olivaceus),
while others have SSTs that are more than 3 times as long as the sperm (e.g.,
American robin; Briskie and Montgomerie 1993). A similar correspondence
between the size of sperm and the structures that females use to store sperm
has also been observed in other taxa (e.g., Dybas and Dybas 1981; Presgraves
et al. 1999).
Variation among species in the ratio of SST length to sperm length, and the
fact that SSTs are usually about twice as long as a single sperm, suggests that
SSTs have not simply evolved to store sperm (Briskie and Montgomerie 1992).
In some species, sperm appear to form layers within each SST, and it has been
suggested this might be a mechanism by which females could control
paternity through the differential storage of sperm from different males
Testis Size, Sperm Size and Sperm Competition #!%
Fig. 9.11 Sperm length in relation to A length and B number of sperm storage
tubules (SSTs) in females of 20 passerine bird species. Solid lines are model II
regressions; dashed line in (A) indicates SSTs that are twice as long as a single
sperm. Both relations are significant with and without controlling for phylogeny.
Modified after Briskie, J. V. and Montgomerie, R. 1992. Proceedings of the Royal
Society of London B 247: 89-95, Fig. 4a, b.
(Compton et al. 1978; Briskie and Montgomerie 1993). More recent work
suggests that such sperm stratification is unlikely to play a role in control of
paternity (Birkhead 1998a,c). Nonetheless, differences between species in the
lengths of their sperm and the lengths of SSTs (Fig. 9.11A) suggest that the
way in which sperm are used by females may vary interspecifically. Using
species in which genetic profiling techniques were used to estimate levels of
promiscuity, Briskie et al. (1997) found that sperm length was correlated
#!& Reproductive Biology and Phylogeny of Birds
positively with levels of extrapair paternity (Fig. 9.10). However, a path

analysis revealed that the relation between sperm length and extrapair
paternity arose only indirectly through the positive relation between the
length of the SSTs and extrapair paternity (Briskie et al. 1997). In other words,
SST length may evolve in response to mating with multiple males, and males
in turn, respond by evolving longer sperm.
Why increased length of SSTs would be advantageous to females with
promiscuous mating systems is not clear. One possibility is that increased SST
size incites competition between the sperm of rival males, and thus selects for
males that produce sperm that are successful competitors (Keller and Reeve
1995). This might then select for males that have longer sperm, if such sperm
are better are competing for access to SSTs, or can displace rival sperm from
SSTs.
Like SST size, SST number also varies across species, ranging from 325 per
female in the Blackpoll warbler (Dendroica striata; Briskie 1996) to over 24,000
per female in the Domestic turkey (Meleagris gallopavo; Goodrich-Smith and
Marquez 1978). In an early study of SST variation, Briskie and Montgomerie
(1992) found that sperm length was inversely correlated with SST number (Fig.
9.11B). Such a pattern was explained by long sperm being advantageous,
perhaps via increased swimming speed, in those species in which a lower
number of SSTs increased competition for access to limited storage sites
(Briskie and Montgomerie 1992). However, subsequent work failed to confirm
a link between sperm length and SST number, and it is now thought this
relation is due to an inverse relation between SST number and SST size, which
instead reflects the necessity of decreasing SST number to accommodate larger
SSTs in the limited size of the uterovaginal junction where SSTs reside (Briskie
et al. 1997).
An alternative view to long sperm evolving as an adaptation to sperm
competition was suggested by Eberhard (1996), who proposed that
postcopulatory female choice (or ‘cryptic female choice’) could explain the
elaborate structures associated with mating, including sperm morphology.
Under this scenario, females may prefer males with long sperm for many of
the same reasons they might choose males with bright plumage or long tails
feathers during the precopulatory phases of mate choice (Keller and Reeve
1995). Whether such cryptic mate choice occurs, and whether it might lead to
directional selection on sperm length in birds is still unresolved, although
there is evidence that female domestic fowl can preferentially eject sperm from
subordinate sires (Pizzari and Birkhead 2000). Although there is no evidence
at present that female birds selectively favor longer sperm, Briskie et al. (1997)
suggested that by increasing the length of their SSTs, females might increase
the probability that the sperm from two or more males mix in the SSTs. This
would incite greater sperm competition and indirectly favor longer sperm if
increased sperm length is advantageous in situations of sperm competition
(see above).
Studying the processes of differential sperm use in birds poses technical
problems that will not be easy to overcome, nor will it be easy to separate the
Testis Size, Sperm Size and Sperm Competition #!'
effects of mate choice from sperm competition (Birkhead 1998b). At present it

is not possible to determine whether the correlation observed between
increased sperm length and high levels of promiscuity among different
species of birds is the result of sperm competition or cryptic female choice (or
both). However, the patterns observed to date do suggest that sexual selection
plays a key role in shaping sperm morphology in birds. The challenge is now
to determine how changes in sperm morphology, such as increased length,
have evolved to overcome both the barriers they encounter in the female
reproductive tract and the competition they face from sperm of rival males.
9.3.4 Costs of Large Sperm Size

Compared to the production of a single egg, the costs (energetic or otherwise)
of producing a single spermatozoon would appear trivial. This has fostered
the view that sperm are ‘cheap’ relative to the costs of producing eggs. Of
course, males do not produce a single sperm for each ovum produced by a
female, and it is now clear that sperm production can limit ejaculate size and,
ultimately, male reproductive success (Nakatsuru and Kramer 1982; Birkhead
1991). If sperm are sometimes limiting for males, then long sperm may be more
costly to produce than short sperm. Thus the differences between costs and
benefits of long sperm should vary within and among species in ways that
might explain the extensive variation in the sperm length of birds.
One obvious cost of increased sperm length is the greater energetic and
material costs of producing a larger structure. The energetic costs to birds of
producing sperm are unknown, but it is logical that larger sperm must be
more costly to produce. Birkhead et al. (1998) found that male Zebra finches on
experimentally reduced diets developed significantly smaller testes and
produced fewer sperm, but did not produce smaller sperm than control males.
However, spermatozoa in this species are relatively short, and further tests of
this type are needed to determine if sperm size is limited by resource
availability in species with long sperm. Indeed, sperm size might not be
influenced much by environment, requiring selection experiments to examine
the influence of resource availability on the evolution of sperm size (e.g.,
Morrow and Gage 2001b).
Long sperm may also be costly if they have reduced longevity due the
increased demands on energy stores when propelling a long flagellum
(Stockley et al. 1997). Fish species with longer sperm have reduced periods of
swimming activity of the sperm, suggesting just such a trade-off between
sperm size and sperm longevity (Stockley et al. 1997), and therefore a potential
cost to production of long sperm. Although we do know that sperm can be
stored in a female’s SSTs for periods up to several weeks (Birkhead and Møller
1992b), the factors that influence the duration of sperm storage (and thus
sperm longevity) in birds have not yet been studied. If increased sperm length
in birds reduces their longevity then an inverse relation between the duration
of sperm storage and sperm size would be expected.
9.3.5 Other Explanations for Diversity in Sperm Morphology

At present, variation in sperm size in birds seems likely to be a product of
sexual selection. However, few studies of sperm size evolution in birds have
been undertaken, and it is possible that other factors may explain some of the
variation in sperm morphology. For example, sperm size in butterflies
increases with body size across species (Gage 1994) and it is possible that
some variation in sperm length in birds could be explained by a similar
allometric effect. No significant relation between sperm length and body size
was found among waders (Johnson and Briskie 1999), but sperm length is
negatively related to body size among passerines (Fig. 9.12; Briskie and
Montgomerie 1992). Both of these analyses were based on small sample sizes
so further work is needed before the influence of allometry on sperm length
can be determined.
Sperm length might respond to selection on female gametes. If gamete size
is determined by a similar proximate process in both sexes, then selection for
increased ovum size might result in a correlated response in sperm size
(Halliday and Arnold 1987). Johnson and Briskie (1999) examined this
linkage disequilibrium hypothesis in a comparative study of sperm size in
waders, but found no significant relation between egg volume and sperm
length. There was also no relation between sperm length and egg size in a
comparative study of passerines (Briskie and Montgomerie 1992).
Sperm size might also be influenced by genome size. As genome size varies
across species, those species with larger (and presumably heavier) genomes
Fig. 9.12 Relation between sperm length (SL) and body mass (BM) in 20
passerine bird species (both variables log-transformed). Model II regression is
shown (logSL = 2.4 –1.1 logBM, r2 = 0.19, P = 0.05). Modified after Briskie, J. V. and
Montgomerie, R. 1992. Proceedings of the Royal Society of London B 247: 89-95,
Fig. 4a, b.
Testis Size, Sperm Size and Sperm Competition #"
might require longer tails and midpieces to carry the greater load. For
example, the size of the enucleated red blood cells in birds is known to be
positively related to genome size (Gregory 2001), suggesting that a similar
pattern might be found with respect to sperm size. Gage (1998) tested this idea
across a wide range of mammal species but found no significant relation
between sperm length and nucleus size. Though genome size has not been
compared to sperm size in birds, genome size varies only two-fold among bird
species (Gregory 2001) and thus seems unlikely to explain the more than
6-fold variation in sperm length (Briskie and Montgomerie 1992).
Most modern comparative studies have used statistical methods to control
for phylogenetic effects (Harvey and Pagel 1991) on the assumption that at
least some of the observed variation in sperm morphology may be due to
phylogeny and may thus not be an adaptation to current selective pressures.
In other words, two species may have similar sperm length as a consequence
of inheriting this trait from a common ancestor. Although phylogeny clearly
plays a major role in determining some of the variation in sperm structure
(e.g., compare passerine and non-passerine sperm; McFarlane 1963; Birkhead
et al. 2006; Chapter 8), sperm length varies so much between even closely
related species that phylogeny does not appear to have much influence on
sperm size. Artificial selection experiments in insects confirm the evolvability
of sperm length (Morrow and Gage 2001b) and sperm length in birds has been
shown to be heritable (Birkhead et al. 2005), so there is clearly potential for
selection to favor changes in sperm size in response to some of the factors
described above. However, the degree to which phylogeny limits adaptive
changes in sperm length are poorly understood and there is a need for a
broad scale comparative study of sperm morphology to assess the degree to
which sperm size in birds depends on their phylogenetic history.
9.4 SCOPE FOR FUTURE RESEARCH

Despite a long and productive tradition among anatomists, physiologists and
histologists, the study of avian reproductive morphology has only recently
attracted the attention of evolutionary biologists and field-based
ornithologists. Much of the work done to date has been highly descriptive and
based on correlation analyses, rather than focussing on experiments explicitly
designed to distinguish among potential causes and control confounding
variables. Comparative analyses of testis and sperm morphology, in
particular, have mainly been limited to the analysis of variation in size. Little
of the intricate detail of sperm morphology and variation among species
revealed by histologists has been examined from an evolutionary perspective
(but see Chapter 8). Much could be learnt from the application of evolutionary
theory to the ultrastructure of the reproductive systems of birds. Mitochondrial
size and number, and tail structure, for example, are known to differ among
bird species (e.g., Henley et al. 1978; Soley 1993) and it would be interesting to
determine whether variation in the intensity of sperm competition can explain
this variation. Intense sperm competition appears to have led to convergent

evolution among unrelated species in relative testis size and absolute sperm
length, but whether similar patterns will be found at a finer structural level
remains to be seen.
The patterns of testis size variation and sperm morphology reviewed here
might give the impression that there is little new to learn by examining and
studying the reproductive anatomy of additional species. However, sperm
size in birds has been measured in only about 120 species worldwide, and the
sperm of relatively few species has been examined in finer detail at the
microscopic level (Chapter 8). Moreover, most of the species whose sperm size
is known are North American and European birds, and almost nothing is
known about variation in sperm morphology in the majority of avian families
(Chapter 8).
Even among common bird species, we lack basic information. Recently, for
example, Birkhead et al. (2006) discovered that the structure of Eurasian
bullfinch sperm is unlike that of any other passerine, though the reasons for
this remain obscure. One can only wonder what sort of variation might be
found in the >9000 other species of birds whose sperm have yet to be
examined. The collection of sperm from birds is easy and non-destructive, and
can be mastered with a little practise (Wolfson 1952; Immler and Birkhead
2005). Thus, systematic surveys of sperm morphology can be undertaken
during field studies of birds and we encourage field ornithologists to consider
semen collection as one of their routine procedures.
More data on interspecific variation in testis size is available than on sperm
size, but there is still a strong bias to species in temperate areas of the northern
hemisphere. For a small set of species, testis mass has been measured directly,
but most of the available data on testis mass has been taken from labels of
museum skins, in which length (and sometimes width) of one or both testes
were measured by the collector (Pitcher et al. 2005). These measurements are
then used to estimate testes mass, assuming that testis shape, density, and
asymmetry is the same for all species (Pitcher et al. 2005; but see Kempenaers
et al. 2002). The collection of birds from the wild has long gone out of favor,
but non-destructive techniques are available to measure testis dimensions
(e.g., laparotomy; Risser 1971), and recent developments in the use of
ultrasound and magnetic resonance imaging (Dietz et al. 1999; Czisch et al.
2001) may provide less invasive ways of estimating testis size. Where
permitted by law, birds salvaged from road kills, building collisions, and
extreme weather events can also provide considerable information on testis
mass and asymmetry without the need for destructive sampling (e.g., Brown
and Brown 2003).
The development of sperm competition theory in the 1970s provided a
sound framework (Parker 1970) that later workers used to guide their studies.
There is no doubt that this approach has been fruitful and that sperm
competition is likely the major factor (aside from body size) that explains
much of the variation in testis size and sperm size in birds (Birkhead 1998a, c).
Testis Size, Sperm Size and Sperm Competition #"!
However, it is too early to rule out other potential explanations for this
variation, and it is perhaps simplistic to conclude that variation in the
intensity of sperm competition alone can account for all of the variation in
testis and sperm size. Recent studies that have found high levels of
promiscuity in species with both small testes (e.g., Moustached warbler,
Acrocephalus melanopogon; Schulze-Hagen et al. 1995; Blomqvist et al. 2005) and
short sperm (e.g., Malurus fairy-wrens; Tuttle and Pruett-Jones 2004), provide
striking anomalies that do not fit the patterns expected from broad-scale
comparisons with respect to sperm competition. These examples suggest that
we are missing the full picture. Likewise, the reasons for testicular asymmetry,
and why it varies with age, are almost a complete mystery. More data, not only
on male reproductive morphology in a greater variety of species, but also on
factors that are likely to be important (e.g., mating frequency, levels of
infidelity, breeding season duration, patterns of sperm storage by females,
sperm longevity, etc.), are now needed to separate the causal factors and
determine how they interact. Such data will also provide a firm basis for
future work as the field develops from one of description and correlation, to
one in which controlled experiments are used to test the robustness of
proposed causal pathways and elucidate the mechanisms responsible for the
observed patterns.
9.5 ACKNOWLEDGMENTS
We are particularly grateful to Meghan Goodchild, Kristen Scott, Jason Clarke
and Christina Cliffe for help with library work; to Trevor Pitcher for supplying
us with his data on testis size; to Tim Birkhead and Barrie Jamieson for useful
insights; and to the University of Canterbury (to J. V. B.) and the Natural
Sciences and Engineering Research Council of Canada (to R. M.) for
supporting our research on avian reproduction. During manuscript
preparation R. M. was supported by a Killam Research Fellowship, and the
Queen’s University Research Chairs program.

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n n
CHAPTER
10
Fertilization
Urszula Stepinska1 and Murray R. Bakst2
10.1 INTRODUCTION
Until recently, the cellular and molecular events comprising fertilization in
birds were rather poorly understood. However, several recent studies in
mammals as well as in birds have considerably contributed to our knowledge
of the fertilization process. Mammals exhibit physiological monospermy, that
is, only a single sperm enters the ovum. Monospermy is ensured by a variety
of mechanisms, which is collectively known as the block to polyspermy.
Unlike mammals, the principal feature of fertilization in birds, as well as in
many fishes, amphibians and reptiles, is physiological polyspermy, that is,
penetration of the ovum by many sperm. It has been suggested that
polyspermy occurs in animals that produce large yolky (megalecithal) ova.
This review is not a comprehensive account of all aspects of avian
fertilization but rather concentrates on the following: oviductal sperm
selection, storage and transport; the interaction of gametes; the role of
envelope of the ovum; and formation and further fate of pronuclei. The main
focus is on the complexities of polyspermic fertilization and the mechanisms
preventing excessive pathological polyspermy that ensure normal embryonic
development. This chapter also includes some data on assisted reproductive
technologies used in birds, such as artificial insemination (AI), in vitro
fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Although the
latter two methods have yet to find practical application, they can shed some
light on at least some mechanisms of avian fertilization in vivo. Most of the
available information on avian fertilization relates to the chicken (Gallus
gallus), since it is commonly used as an experimental bird. In this chapter, all
observations will be derived from research with the chicken unless otherwise
noted.
1
Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec n.
Warsaw, 05-552 Poland. E-mail: ustepinska@yahoo.com
2
Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Center, ARS/
USDA, Beltsville, MD 20705 USA. E-mail: murray@anri.barc.usda.gov
##" Reproductive Biology and Phylogeny of Birds
10.2 MORPHOLOGY OF THE OVULATED OVUM

As in most other vertebrates, the avian ovum is ovulated with its nucleus at
metaphase of the second meiotic division. The voluminous 3-4 cm diameter
ovum is primarily comprised of yellow yolk. On its surface is a 3-5 mm
diameter germinal disc (GD), composed of white yolk that forms the animal
pole. The GD contains the female complement of genetic material as well as
the majority of cytoplasmic organelles (Bakst and Howarth 1977a; Perry et al.
1978). At the GD the plasma membrane, the oolemma forms an array of
microvillar projections (Fig. 10.1), which are longer and more abundant than
that of the vegetal hemisphere where the oolemma is discontinuous and forms
few elaborations (Bakst and Howarth 1977a; Bakst 1978). Given the role of
physiological polyspermy in the fertilization process, it should not be
surprising that cortical granules have not been observed in the ooplasm of
avian oocytes (Guraya 1982).
10.2.1 The Inner Perivitelline Layer

At the time of ovulation the ovum is enveloped by an acellular glycoprotein
coat known as the inner perivitelline layer (IPVL), sometimes erroneously
referred to as the inner layer of vitelline membrane in the laid egg (Bellairs et
al. 1963; Bakst and Howarth 1977a; Okamura and Nishiyama 1978a; for
Fig. 10.1 Gallus gallus. A scanning electron micrograph (SEM) showing the
microvilli-like extensions of the oolemma at the germinal disc (GD). White yolk
spheres are observed in the GD. Original.
Fertilization ###
nomenclature see Baumel et al. 1979) (Fig. 10.2A, B). The IPVL is considered to
be analogous to the egg envelope of other animal species such as the zona
pellucida (ZP) of mammals, vitelline envelope of frogs, and the chorion of
fishes (see Dumont and Brummett 1985). The IPVL of birds is a three-
dimensional network of fibers (Bellairs et al. 1963), where spaces between the
fibers are filled with a ground substance (Bakst and Howarth 1977a) (Fig.
10.2A, B). Bakst and Howarth (1977a) observed that the part of the IPVL
overlying the GD and that covering yolk had an identical structure and
exhibited similar variation in thickness (2-4 mm). On the other hand, it has
been claimed that the IPVL from the GD region appears thinner, and has
smaller and less numerous fibers than that from other regions of the ovum
(Bellairs et al. 1963; Perry et al. 1978).
In mammals, the ZP is composed of three glycoproteins designated as ZP1,
ZP2 and ZP3 (Wassarman 1988), also known as ZPB, ZPA and ZPC,
respectively (Harris et al. 1994). Two glycoproteins have been identified in the
IPVL of the avian ovum. One displays significant homology to members of the
ZP3/ZPC family of mammals and therefore has been named either chicken
ZP3 (chkZP3, 32 kDa) (Kido and Doi 1988; Waclawek et al. 1998; Takeuchi et
al. 1999) or quail ZP3 (qZP3, 33 kDa) (Mori and Masuda 1993; Kuroki and
Mori 1995; Pan et al. 2000). The other IPVL glycoprotein is a homologue of the
ZP1/ZPB family, about 95 kDa, and designated chkZP1 (Bausek et al. 2000) or
qZP1 (Sasanami et al. 2003). The presence of immunological homologues of
chkZP1 and chkZP3, identified by Western blotting with anti-chicken ZP1
and ZP3 antibodies, respectively, has been demonstrated in the IPVL from laid
eggs of several avian species including Japanese quail, duck, goose, pheasant
and turkey (Stewart et al. 2004). Both glycoproteins showed some differences
in molecular mass among the avian species studied: ZP1 from 91 to 106 kDa
and ZP3 from 34 to 42 kDa. There are no data indicating the presence of a
third glycoprotein in avian IPVL, a homologue of ZP2 found in the
mammalian ovum envelope. It is possible that the putative ZP2 homologue is
much less abundant in avian IPVL than ZP1 and ZP3, and/or that it is lost
during IPVL isolation and solubilization (Bausek et al. 2000).
In chicken and quail, glycoprotein ZP3 is synthesized by the granulosa
cells surrounding the oocyte during the rapid growth phase of the ovarian
follicle (Kuroki and Mori 1995; Waclawek et al. 1998; Takeuchi et al. 1999; Pan
et al. 2001; Sasanami et al. 2002). It has been suggested that testosterone
stimulates ZP3 production at the gene transcription level in the granulosa
cells (Pan et al. 2001). In contrast, avian ZP1 glycoprotein seems to be
synthesized under estrogen control in the liver and then transported through
the bloodstream to the ovarian follicle (Bausek et al. 2000; Sasanami et al.
2003). Thus, the site of biosynthesis and the hormonal regulation of avian
IPVL are different for ZP1 and ZP3. In fish, chorion proteins appear to be
synthesized in the liver of spawning females in response to estrogens and are
then transported to the ovary by blood circulation (Hamazaki et al. 1989). In
Xenopus, there is clear evidence that all egg envelope glycoproteins are
##$ Reproductive Biology and Phylogeny of Birds
Fig. 10.2 Gallus gallus. Initial step of interaction of sperm with inner perivitelline
layer (IPVL). A. A differential interference contrast (DIC) micrograph of the IPVL with
a single sperm (arrow) highlighted by a fluorescent nuclear stain. Note the fibrous
nature of the IPVL. Original. B. A SEM of a sperm with an intact acrosome on the
surface of the IPVL. The arrows indicate remnant cytoplasmic processes possibly
from the oolemma (top arrow) and the granulosa cells (bottom arrow). Original.
Fertilization ##%
synthesized by the oocytes (Yamaguchi et al. 1989). This is likewise true for
mice (Bleil and Wassarman 1980). However, in other mammals, such as
rabbits, humans and pigs both the oocyte and its accompanying granulosa
cells contribute to the synthesis of the ZP glycoproteins (Grootenhuis et al.
1996; Kolle et al. 1996).
Interestingly, Olszanska et al. (2001) have identified by RT-PCR the
presence of ZP3 transcripts in the GD and the ooplasm in quail oocytes. This
highlights the possibility of ZP3 synthesis in the avian oocyte in addition to
that in the granulosae cells.
10.3 OVIDUCTAL SPERM SELECTION, STORAGE AND TRANSPORT

This section will provide a brief overview of oviductal sperm selection, storage
and transport in birds with emphasis on poultry. More detail explanations of
the subjects can be found in reviews by Bakst et al. (1994), Wishart and
Horrocks (2000) and Birkhead and Moller (1998), and the proceedings of a
workshop edited by Hamlett (2003).
The left oviduct in birds has three major functions: egg formation; sperm
selection, storage and transport; and is the site of fertilization and early
embryonic development. Egg formation begins when the ovulated ovum is
captured by the fimbriated region of the infundibulum. Whether sperm are
present or not, the ovum passes through infundibulum where albumen-like
proteins form the attenuated continuous layer and the more extensive outer
perivitelline layer (OPVL). This fibrous investment strengthens the PVL
complex supporting the megalecithal ovum in its transit through the oviduct.
The oviductal ovum progresses through the magnum where the albumen is
secreted, the isthmus where the shell membrane is formed, and then the egg
resides in the shell gland (uterus) for shell formation prior to its brief transit
through the vagina at oviposition. This “ovulatory cycle”, that is, ovulation,
egg formation, and oviposition is about 24-25 h in duration and is repeated
with each egg in a clutch. If the ovum was fertilized in the infundibulum, the
initial cleavage furrows would appear in about 5-7 h after sperm penetrates
the ovum, about when the egg was in the isthmus or uterus. Depending on the
species, the fertilized, fresh laid egg posseses a blastoderm consisting of
30,000 to 60,000 cells.
That being said about egg formation and the site of fertilization and early
embryonic development, the main objective of this section is to present our
current understanding of oviductal sperm selection, storage and transport.
Briefly, in hens that have mated naturally or subjected to AI, a relatively small
number of sperm are transported to storage sites at the anterior end of the
vagina, a region referred to as the utero-vaginal junction (UVJ). Here sperm
enter the sperm storage tubules (SST) which are discrete tubular structures
originating as invaginations of the UVJ surface epithelium (Fig. 10.3A, B).
Sperm that successfully traverse the vagina and populate the SST are distinct
in both mobility characteristics and surface membrane composition.
##& Reproductive Biology and Phylogeny of Birds
Fig. 10.3 Gallus gallus. Sperm in sperm storage tubule (SST). A. Sperm are
observed at the orifice of a SST. The arrow indicates the sharp transition between
the ciliated epithelium of the uterovaginal junction and the SST epithelium. Original.
B. Fluorescing sperm are observed in the basal lumen of a SST viewed by DIC.
The outside diameter of the SST is about 40 mm. Original.
Fertilization ##'
Depending on the species, sperm reside for variable lengths of time in the SST
before they are released and ascend to the infundibulum, the site of
fertilization and presumptive secondary sperm storage site.
Sperm transport is probably a combination of sperm mobility and activity
of the ciliated epithelium lining the oviductal mucosa. There has been no
significant research done in the factors controlling oviductal sperm transport
in birds for many years (see Bakst et al. 1994 and Wishart and Horrocks 2000
for reviews). What we do know that the vast majority of sperm initially
transferred into vagina at copulation or AI fail to populate the SST. The
known exception to this is when a commercial turkey is inseminated 10-
14 days after photostimulation but prior to the actual onset of egg production.
For reasons that we can only speculate, sperm numbers in the SST are nearly
twice as great in the SST when inseminated just prior to the onset of egg
production as when the hen was AI initially after the onset of egg production
(Brillard and Bakst 1990; Bakst 1994). But even under these circumstances, the
maximum number of sperm in the SST remains below 10 million even though
the hen was inseminated with 150-350 million sperm. Obviously, the vagina
orchestrates an intense sperm selection process. The mechanisms behind such
selection remain to be elucidated. Interestingly, if a hen is inseminated within
0.5 h following oviposition, upon manual eversion of the vagina, the
inseminator may actually be able to deposit the semen in the uterus, by-
passing the vagina and UVJ. While large numbers of sperm are transported to
the infundibulum, this results in increased embryo mortality, possibly by
pathological polyspermy.
The sperm which do reach the UVJ and enter the SST can be viewed as
“selected” sperm (see Bakst et al. 1994). Yet, what favors the transport of some
sperm and not other sperm to the SST remains unknown. As pointed out by
Wishart and Horrocks (2000), sperm transport from the site of deposition in
the vagina to the UVJ requires that the sperm be motile and that the sperm
plasmalemma possesses certain glycoproteins. Differences in sperm mobility,
as measured by the ability of sperm to move progressively through a viscous
medium, contribute to the likelihood of sperm success in traversing the vagina
(Froman et al. 1999; Birkhead et al. 1999). Likewise there appears to be control
by the hen as to which sperm she will accept in the SST or to interact with the
hen’s ovum (Pizzari et al. 2002). Elucidating the cellular and molecular basis
for successful sperm selection will no doubt lead to improvements in AI
technology, specifically, the insemination of lower numbers of sperm at longer
intervals between successive inseminations.
One must assume that sperm residing in UVJ SST undergo a reversible
suppression of certain activities. They may include: 1) reversible suppression
of sperm metabolism and motility; 2) stabilization of membrane systems; 3)
stabilization of the acrosomal enzymes; and 4) suppression of sperm
immunogenicity (Bakst et al. 1994). One needs also to assume that the sperm
exiting the SST undergo some type of activation, possible comparable to
mammalian capacitation (see 10.4.2 and Chapter 4).
A long contentious issue is whether sperm release from the SST is

associated with a particular stage of the ovulatory cycle, such as immediately
following oviposition, or whether they are released slowly over the course of
the ovulatory cycle. Recently Froman (2003) suggested that sperm residing in
the SST swim against a fluid current generated by the SST epithelium that
flows in the direction of the UVJ lumen. Once the velocity of this current
exceeds the velocity of the sperm, sperm are swept out of the SST. The
localization of aquaporins in the SST epithelium (Zaniboni and Bakst 2004)
would appear to add support to Froman’s hypothesis. Interestingly, extensive
innervation of the UVJ mucosa surrounding the SST as well as an actin-
filament network in apical cytoplasm of the SST epithelial cells suggests that
individual SST may be capable of contracting, thereby facilitating the release
of resident sperm (Freedman et al. 2001). Table 10.1 contains a hypothetical
model describing the fate of sperm after transfer to the vagina.
Table 10.1 Hypothetical model for oviductal sperm transport and storage in birds. Based
on works by Bakst et al. (1994), Poultry Science Reviews 5: 117-143 and Froman (2003)
• A small percentage of the sperm transferred to the vagina reach the SST
• SST resident sperm are subjected to a reversible suppression of metabolism and motility,
inhibition of enzyme systems, and stabilization of the plasmalemma
• Resident sperm metabolize lipids and carbohydrates released from SST epithelium
• As resident sperm mobility decreases, increasing numbers of sperm are swept out of
the SST
• Liberated sperm are transported to the site of fertilization at the infundibulum
It has long been assumed that the infundibulum serves as a secondary

sperm storage site in the chicken and turkey oviduct (see Bakst et al. 1994 and
Wishart and Horrocks 2000 for reviews). However, upon extensive
examination of the infundibular mucosa within 48 h of insemination no more
than 1 to 3 sperm were ever located (Bakst 2003). When one considers the
benefits of the UVJ SST to reproductive success (Table 10.2) and given the
paucity of a sperm in the infundibulum after a normal insemination or
copulation, one is led to the conclusion that the infundibulum is not a true
sperm storage site analogous to the UVJ SST.
Table 10.2 Benefits of sperm storage in the UVJ SST
• Elimination of synchronization of copulation with ovulation

• Sperm transfer to hen is not necessary for production of fertile eggs over one or more
clutches
• Provides resevoir for “selected” sperm
• Affords protection to sperm during the daily ovulatory cycle
We have no knowledge of what cross-signaling may exist between resident

sperm and the SST or what genes are turned on and off in the presence or
Fertilization #$
absence of resident sperm. Long et al. (2003) constructed 2 SAGE (separate

serial analysis of gene expression) libraries with SST RNA obtained from
turkey hens inseminated with diluted semen or semen extender only. They
found that only 1 percent (214) of the putative genes was differentially
expressed between SST with or without resident sperm. These data support
the idea that differential gene expression occurs in the SST mucosal
epithelium within 48 h after AI with semen. This raises the possibility that
sperm themselves induce specific gene expression events required for
prolonged sperm storage and/or release from the SST.
10.4 INTERACTION OF SPERM WITH THE OVUM

10.4.1 Site of Fertilization
In birds, fertilization takes place in the infundibulum, the most anterior
segment of the oviduct, within about 15 minutes of ovulation (Olsen and
Neher 1948). Sperm must contact the surface of the ovum, specifically the IPVL
overlying the GD, before appreciable amounts of infundibular secretory
material is deposited around the IPVL. This tertiary investment derived from
the infundibular mucosa forms the OPVL, which sperm cannot penetrate. It is
the OPVL that prevents pathological polyspermy (Fig. 10.4). The mechanisms
responsible for drawing the sperm to the GD remain unknown.
Fig. 10.4 Gallus gallus. The perivitelline layer complex composed of the inner
(IPVL) and outer perivitelline layers (OPVL) are observed. The OPVL is formed by
infundibular secretory activity and functions as a block to pathological polyspermy.
Numerous sperm are seen embedded in the OPVL. After Bakst, M. R. and Howarth,
B. H. 1977b. Biology of Reproduction 17: 370-379, Fig.12.
10.4.2 Capacitation
In mammals, ejaculated sperm are progressively motile yet do not have an
immediate capacity for fertilization. In the case of in vivo fertilization they
gain this ability after residing in the female genital tract or, in the case of IVF,
after incubation for some time in an appropriate medium. The physiological
changes that render the sperm competent to fertilize is referred to as
capacitation (see Austin 1952; Yanagimachi 1994).
It is generally believed that capacitation of sperm is not essential for
fertilization in birds (see Howarth 1984; also Chapter 4). This was primarily
based on observations that avian ova could be fertilized in vitro by fresh
ejaculated sperm which had not been previously treated with a special
capacitation medium (Howarth 1971; Nakanishi et al. 1990; Olszanska et al.
2002). It was demonstrated that the duration of gamete interaction needed for
successful IVF in birds is short and does not exceed 10-15 min (Nakanishi et
al. 1990; Olszanska et al. 2002). Likewise, initiation of in vitro hydrolysis of
IPVL fragments by ejaculated sperm was found to occur within 2.5 min
(Robertson et al. 1997). Moreover, it was shown in vivo that avian sperm do
not need to reside long in the female reproductive tract to be competent for
fertilization, since fertilized eggs could be observed within 15 min after AI into
the vagina (Bobr et al. 1964) or infundibulum (Olsen and Neher 1948).
While fresh ejaculated sperm can fertilize an ovum in vitro, the sperm that
fertilize a daily succession of ova have resided days or weeks in the oviductal
SST. Bakst et al. (1994) suggested that sperm residing within the SST are
quiescent, with motility, metabolism, enzyme systems reversibly suppressed
(“decapacitated” in the mammalian sense). Upon release from the SST, the
oviductal milieu may activate sperm (capacitate?) prior to ascent to the site of
fertilization.
10.4.3 Sperm-Inner Perivitelline Layer Interaction

What can be considered the first step in the process of fertilization is binding
of sperm to the IPVL (Fig. 10.5A, B). Sperm binding properties have been
observed in chicken and quail IPVL even after its removal from oocytes
(Howarth 1990; Kuroki and Mori 1997).
Sperm-binding induces the acrosome reaction (AR) (Fig. 10.5B) thereby
releasing hydrolytic enzymes that digest a path through the IPVL (Fig.
10.6A,B) (Okamura and Nishiyama 1978a; see Howarth 1984). The ability of
IPVL to stimulate an AR was demonstrated in chicken in vitro by incubating
sperm with fragments of IPVL isolated from freshly ovulated ova (Koyanagi et
al. 1988) or with homogenized IPVL isolated from the PVL complex of laid
eggs (Horrocks et al. 2000). Okamura and Nishiyama (1978a) documented the
process of the AR and subsequent passage of sperm through the IPVL of the
hen. They have shown that when the acrosome region of a sperm contacts the
IPVL the outer acrosomal membrane and overlying plasmalemma of the sperm
fuse at different points, thereby releasing its complement of hydrolytic
enzymes. These hydrolytic enzymes digest small holes in the IPVL,
Fertilization #$!
Fig. 10.5 Gallus gallus. Interaction of sperm with IPVL. A. A SEM of a sperm
making initial contact with IPVL. The arrow highlights remnant cytoplasmic
processes on the IPVL surface. After Bakst, M. R. and Howarth, B. H. 1977b.
Biology of Reproduction 17: 370-379, Fig. 14. B. A transmission electron
micrograph (TEM) of a sperm head interaction with the surface of the IPVL. The
plasmalemma overlying the acrosome is highly ruffled and discontinuous in
places (arrow) suggesting the beginning of acrosome reaction. Original.
#$" Reproductive Biology and Phylogeny of Birds
Fig. 10.6 Gallus gallus. “Sperm holes” in the IPVL. A. A light micrographs of
“sperm holes” (arrows) in the IPVL overlying the GD. These holes are sites where
the sperm have hydrolyzed a path through the IPVL to reach the oolemma. Original.
B. A TEM shows the hydrolyzed fibers and ground substances composing a portion
of a sperm hole in the IPVL. Original.
Fertilization #$#
approximately 10 mm in diameter, through which the sperm penetrate to reach

the oolemma (Figs. 10.6A, B, 10.7A, B) (Bakst and Howarth 1977b; Okamura
and Nishiyama 1978a). Interestingly, sperm penetration of the IPVL is seen
preferentially at the GD region of the ovum (see 10.4.3.4). Sites of sperm
penetration through the IPVL can be observed in uterine eggs (Okamura and
Nishiyama 1978a) or after oviposition (Bramwell and Howarth 1992a;
Birkhead et al. 1994; Bramwell et al. 1995; Wishart 1997). Similar holes were
found after the coincubation of sperm with fragments of the IPVL isolated
from the largest preovulatory oocytes (Steele et al. 1994; Kuroki and Mori 1997;
Takeuchi et al. 2001), from recently ovulated ova (Howarth 1990; Bramwell
and Howarth 1992b; Steele et al. 1994) or from oviposited eggs (Steele et al.
1994; Robertson et al. 1997).
It is assumed that a trypsin-like acrosomal enzyme, acrosin, is involved in
the hydrolysis of the IPVL. Acrosin has been isolated from chicken (Ho and
Meizel 1970; Froman 1990) and turkey (Richardson et al. 1988) sperm. The
acrosin activity of turkey sperm is much higher (about 100 times) than that of
human sperm and is similar to that of boar sperm (Glogowski et al. 2001). Ho
and Meizel (1975) observed in vitro hydrolysis of IPVL of chicken ova by
partially purified acrosin. It has been reported that the presence of trypsin
inhibitors in the proteins forming the OPVL reduced sperm fertility in vivo
(Palmer and Howarth 1973) and inhibited sperm hydrolysis of chicken and
quail IPVL in vitro (Howarth and Digby 1973; Kuroki and Mori 1997;
Takeuchi et al. 2001).
10.4.3.1 Molecular aspects of sperm-inner perivitelline layer
interaction
Our understanding of the role of the ZP glycoproteins and the mechanism of
fertilization is quite advanced for the mouse. All ZP proteins (ZP1-3) are
structural glycoproteins necessary for the formation of the ZP during mouse
oocyte growth (see Wassarman et al. 2004 and Yanagimachi 1994 for reviews).
ZP2 and ZP3 form long filaments which are cross linked by ZP1 (see
Wassarman et al. 2004). In addition to their structural properties, ZP2 and ZP3
also play roles in gamete interaction. Mouse ZP3 glycoprotein has been
identified both as the acrosome-intact sperm receptor (primary sperm receptor)
as well as the inducer of AR in the bound sperm (Litscher et al. 1995; see
Wassarman et al. 2004 and Yanagimachi 1994 for reviews). Considerable
evidence shows that O-linked oligosaccharides of mouse ZP3 account for the
sperm receptor activity (Florman and Wassarman 1985; Bleil and Wassarman
1988), whereas the induction of AR reaction in the sperm is mainly dependent
on its polypeptide chain (Leyton and Saling 1989). In turn, mouse ZP2
glycoprotein seems to be mainly responsible for the binding of acrosome-
reacted sperm to the ZP (secondary sperm receptors) (Bleil et al. 1988).
Initially, ZP3 glycoprotein was postulated to be the most likely candidate
for the sperm receptor also in avian IPVL (Waclawek et al. 1998; Pan et al.
2000). However, the above suggestion was based only on the extensive amino
#$$ Reproductive Biology and Phylogeny of Birds
Fig. 10.7 A. Gallus gallus. After staining the perivitelline layer (PVL) complex
overlying the GD, seven sperm holes (arrows show 2 pairs of holes) can be viewed.
Fertilization #$%
acid homology of avian and mammalian ZP3, and not on any experimental
data. It has recently been suggested that another major glycoprotein of the
IPVL, chkZP1, is responsible for the interaction of sperm with the IPVL. In
vitro sperm penetration of the IPVL from the largest ovarian follicles was
completely inhibited after pretreatment of the IPVL with monoclonal
antibodies directed against chkZP1 (Takeuchi et al. 2001; Bausek et al. 2004). It
has also been shown that in vitro sperm interaction with the IPVL leads to
proteolytic activity that only affects chkZP1, degrading it into discrete
fragments, while chkZP3 remains intact (Bausek et al. 2004). Alternatively,
Takeuchi et al. (2001) have reported that sperm degraded IPVL chkZP1 and
chkZP3 in vitro. Bausek et al. (2004) showed that chkZP1 and chkZP3 bind
specifically to the acrosome of sperm in vitro. Thus, it appears that in birds
the interaction between sperm and the IPVL is mediated by both chkZP1 and
chkZP3.
There is evidence that N-linked oligosaccharides of the IPVL with terminal
N-acetylglucosamine residues may play a crucial role in the interaction of
avian sperm with IPVL (Robertson et al. 2000). In their study, the in vitro
interaction of sperm and IPVL from laid chicken eggs, measured as the
number of holes produced in the IPVL, was significantly decreased after
treatment of the IPVL with either: 1) N-glycanase (removing N-linked sugars
from IPVL); 2) lectins with affinity for N-acetylglucosamine (masking the
terminal N-acetylglucosamine in IPVL); or 3) when the sperm:IPVL interaction
was carried out in the presence of N-acetylglucosamine (competing with
endogenous N-acetylglucosamine). One should realize that the interaction of
sperm with IPVL involves both the binding of sperm to the IPVL, the induction
of the AR, and then the hydrolysis of the IPVL. In the above study, the overall
sperm-IPVL interaction was measured by the number of holes produced in the
IPVL, thus it is not possible to determine which stage of the interaction was
affected by the experimental manipulation. However, using a stain specific for
acrosome, it was revealed that N-linked oligosaccharides of the IPVL
glycoproteins can induce the AR in chicken sperm (Horrocks et al. 2000). Of
significance, their study revealed that the oligosaccharides need to have
terminal N-acetylglucosamine groups and that they are capable of inducing
the AR even when detached from their protein anchor. At present, it is not
known to which IPVL protein the N-linked oligosaccharides are attached.
That the IPVL N-linked oligosaccharides appear to be responsible for the
induction of the AR does not exclude the possibility that they may influence
the binding of sperm to IPVL and its hydrolysis after the AR.

Note the texture, lighter color, and doughnut-shape of the PVL overlying the GD.
The diameter of the GD is about 3mm. Original. B. A SEM of the underside of the
IPVL shows two sperm exiting a sperm hole. The sperm on the right appears to
have lost its acrosome but not the one on the left. Complements of B. Howarth, Jr.
#$& Reproductive Biology and Phylogeny of Birds
10.4.3.2 Changes in the inner perivitelline layer between

ovulation and oviposition
Steele et al. (1994) found that the number of holes in IPVL produced in vitro by
chicken sperm was several times less in IPVL from laid eggs compared with
that isolated from the largest follicular and freshly ovulated oocytes. However,
it should be mentioned that in case of laid eggs whole PVL, not isolated IPVL,
was used for the experiment. Likewise, the number of sperm that could bind
in vitro to the IPVL at the surface of the GD was lower in laid eggs than in
follicular oocytes (Kuroki and Mori 1997). A simple explanation may be that
the IPVL sperm binding site are masked by the OPVL or the sperm are
sterically hindered from reaching the IPVL by the components of the OPVL. In
addition, the IPVL from laid eggs differs, from that of both follicular oocytes
and ovulated ova. Chicken and quail ZP3 glycoproteins from laid eggs had
lower molecular weights than the ZP3 glycoproteins isolated from follicular
oocytes and ovulated ova (Kuroki and Mori 1995; Waclawek et al. 1998; Pan et
al. 2000). Alterations of the quail ZP3 glycoproteins on the oviductal ovum
includes removal of N-terminal amino acids (probably due to the action of a
protease secreted by the infundibulum) and modifications of N-linked and O-
linked oligosaccharide chains (Pan et al. 2000). Even though these changes
were observed in virgin hens, the modifications of the ZP3 glycoproteins were
suggested to be a block to excessive sperm IPVL penetration before the OPVL
formed (Waclawek et al. 1998; Pan et al. 2000).
In contrast to the observations of Steele et al. (1994), Robertson et al. (1997)
observed that the IPVL from laid chicken eggs, from follicular oocytes and
ovulated ova gave similar responses to hydrolysis by sperm in vitro. However,
it should be noted that in the above experiment IPVL was used not only in
case of follicular oocytes and ovulated ova but also in case of laid eggs, as
IPVL was isolated from PVL, whereas Steele et al. (1994) used whole PVL from
laid eggs. Robertson et al. (1997) did observe that one of the IPVL
glycoproteins, probably ZP3, appeared smaller in laid eggs than from
follicular oocytes. Notwithstanding, these authors proposed the use of IPVL
from laid eggs instead of IPVL from follicular oocytes or ovulated ova. The
former are considerably easier to obtain when performing in vitro assays of
sperm-IPVL interaction for predicting the fertilizing ability of semen from
different males.
10.4.3.3 Species specificity in sperm-inner perivitelline layer
interaction
In mammals, sperm interaction with the ZP is usually described as a species-
specific event, although cross fertilization may occur between closely related
species (see Yanagimachi 1994). The interaction between sperm and IPVL
from guinea fowl, turkey and chicken was demonstrated by IPVL hole
formation following homologous and heterologous coincubation in vitro
(Steele et al. 1994). However, sperm showed a slightly higher specificity
towards the homologous IPVL. Stewart et al. (2004) coincubated chicken sperm
Fertilization #$'
with IPVLs from laid, unfertilized eggs from closely and distantly related
avian species and demonstrated that chicken sperm were able to hydrolyze
the IPVL of all species examined. The functional cross reactivity between
chicken sperm and heterologous IPVL seemed to correlate with the
phylogenetic distance between the species. Within the order Galliformes, the
interaction of chicken sperm with the IPVL from turkey, quail, peafowl,
pheasant and guinea fowl was not significantly different from that with the
homologous chicken IPVL. The hydrolytic activity of chicken sperm towards
IPVL was strongest within Galliformes, intermediate in Anseriformes (goose
and duck) and the weakest in Passeriformes (zebra finch) and Columbiformes
(dove). These findings show that, unlike the mammalian ZP, the IPVL does
not represent such strong species specific barrier to heterologous sperm
penetration. This may be a factor facilitating hybrid production both in wild
and domesticated birds after AI.
10.4.3.4 Preferential attraction of sperm to the inner perivitelline
layer over the germinal disc
During fertilization in birds sperm bind to and penetrate the IPVL over the
entire surface of the ovum, although with a much greater frequency on the
IPVL overlying the GD. This is advantageous in ensuring syngamy of the male
and female nuclei. Preferential sperm penetration of IPVL overlying GD has
been demonstrated in laid eggs of several avian species after natural mating
(Bramwell and Howarth 1992a; Birkhead et al. 1994; Steele et al. 1994;
Bramwell et al. 1995; Wishart 1997; Wishart and Staines 1999) and in freshly
ovulated and then in vitro fertilized chicken ova (Bakst and Howarth 1977b).
The number of sperm penetrating the IPVL (number of holes produced by
sperm in IPVL observed in fertilized laid eggs) over GD is approximately
20 times greater than in other regions of the egg in both chicken and turkey
(Bramwell et al. 1995; Wishart 1997) (Fig. 10.7A). The total number of holes in
the IPVL region over the GD, which is about 3 mm in diameter, can reach
several hundred. However, because this represents a minor fraction of the
area of the whole IPVL, there are approximately 50-fold more holes found
evenly distributed in the IPVL outside this region (Wishart 1997; Wishart and
Staines 1999).
The mechanisms responsible for the preferential hydrolysis of the IPVL
overlying the GD remain unknown, despite considerable interest and
speculation (Ho and Meizel 1975; Bramwell and Howarth 1992b; Steele et al.
1994; Kuroki and Mori 1997). This may be attributed to different densities of
sperm receptors, differential induction of the AR or different susceptibility of
various regions of the IPVL to hydrolysis. Kuroki and Mori (1997) have found
that sperm receptors are concentrated in the IPVL over the GD disc region of
quail oocytes from preovulatory follicles. This was demonstrated in an assay
in which hydrolysis of the IPVL by sperm in vitro was inhibited with trypsin
inhibitors and the binding of sperm to the IPVL could be observed. An
alternative explanation is that the IPVL overlying the GD region may be more
#% Reproductive Biology and Phylogeny of Birds
susceptible to hydrolysis since that region of the IPVL of chicken ova is

preferentially hydrolyzed in vitro by partially purified cock sperm acrosin (Ho
and Meizel 1975). Steele et al. (1994) observed a higher density of holes in the
IPVL over GD in fertile laid eggs, but after in vitro coincubation of fragments
of the IPVL from ovulated ova with sperm, holes appeared with similar
frequency throughout the whole IPVL surface. These data do not support the
conclusions of Bramwell and Howarth (1992b) that in vitro sperm
preferentially hydrolyze the GD region of IPVL (isolated from ova) and that
this region is particularly rich in sperm receptors. However, it should be noted
that the above conclusions were based on a single result only in which the
concentration of holes in the IPVL over the GD region was only 1.33 times that
found in the IPVL from other ovum areas.
Although some authors reported that the IPVL appeared thinner in the GD
region, Bakst and Howarth (1977a) found the IPVL over the GD and remaining
regions of the ovum structurally identical. It was also demonstrated that the
electrophoretic protein patterns of IPVL over the GD and non-GD regions
were indistinguishable (Steele et al. 1994). It is therefore possible that the
preferential hole formation in the GD region is not associated with the IPVL
but originates from the GD itself. Bakst (1978) found that the microvillar
projections of the oolemma were longer and more abundant at the GD region
when compared to the remainder of the oolemma (Figs. 10.1, 10.2B, 10.5A). He
suggested that the oolemma projections may protrude through the IPVL and
that they were possibly the site of sperm receptors. He later indicated that the
remnant processes seen on the surface of the IPVL by scanning electron
microscopy could also have originated from the granulosa cells (Bakst 1979)
(Fig. 10.2B). Wishart (personal communication) has taken the view that some
components in the yellow yolk act to suppress sperm hydrolysis of the IPVL.
His suggestion supports the observation of few IPVL sperm holes in intact ova
are observed overlying the yellow yolk whereas, if the IPVL is washed free of
the yellow yolk, sperm holes are more abundant.
It is interesting to note that sperm penetrate the IPVL over GD in such a
manner as to form a doughnut-shaped configuration with the center of the GD
remaining largely unpenetrated (Fig. 10.7A). This was demonstrated both in
vivo, in the IPVL isolated from fertile laid eggs of several avian species
(Birkhead et al. 1994; Wishart 1997), as well as in vitro in chicken after
incubation of IPVL fragments (isolated from ovum) with sperm (Bramwell and
Howarth 1992b). The hypothetical physiological advantage of the central area
of IPVL over the GD being less attractive to sperm is unclear at present but it
is likely that it may restrict excessive sperm entry into the GD in the vicinity of
the female nucleus (see 10.5).
10.4.4 The Outer Perivitelline Layer and its Role in

Blocking Excessive Polyspermy
In birds there is no block to polyspermy at the level of IPVL. However, a block
to pathological polyspermy is in the form of tertiary investment, the OPVL,
Fertilization #%
deposited by the distal infundibulum and proximal magnum around the IPVL
(Bellairs et al. 1963; Bakst and Howarth, 1977b; Okamura and Nishiyama
1978a). Ultrastructurally, the OPVL is composed of irregularly deposited
concentric arrays of what are assumed to be fibrous proteins. These fibers are
densely arranged near the IPVL, then the space between the concretions
gradually widens. A single “continuous layer” is observed separating the
IPVL from the OPVL. Biochemically, the OPVL is composed of enzymatically
active lysozyme (about 60% of dry weight), an insoluble ovomucin complex
(Back et al. 1982), and two basic proteins, the 17.5-kDa VMO I (vitelline
membrane outer I) (Back et al. 1982) and the 6-kDa VMO II (Kido et al. 1992).
Ovomucin appears to form the skeleton of OPVL, but some other proteins,
especially lysozyme, are responsible for its integrity (Back et al. 1982). The
functions of VMO I and VMO II are not known.
Luminal sperm in the upper region of the oviduct become passively
trapped between and within the fibers of the OPVL and are incapable of
penetrating this investment (Fig. 10.4). Sperm embedded in the OPVL can be
observed in fertilized eggs recovered from distal infundibulum and proximal
magnum (Bakst and Howarth 1977b) or from laid eggs (Wishart 1987; Wishart
1997). The OPVL is thought to act as a block to excessive pathological
polyspermy and to ensure the integrity of the ovum by preventing the rupture
of IPVL, weakened by the sperm hydrolytic activity (Bakst and Howarth
1977b; Okamura and Nishiyama 1978a). Under in vitro conditions,
preparations of OPVL can inhibit the hydrolysis of IPVL by sperm as well as
interfere with the induction of AR by IPVL (see Wishart and Horrocks 2000).
Sperm trapped in the OPVL of chicken eggs do not appear to have undergone
an AR (Okamura and Nishiyama 1977b) and if acrosin were released, the
OPVL contains trypsin inhibitors (see Howarth 1984) that would inhibit
hydrolytic activity. Therefore, it is possible that OPVL presents not only a
mechanical barrier preventing further sperm penetration into the IPVL but can
also inhibit the AR and acrosin activity.
Fertile eggs possess about ten times more sperm trapped in the OPVL than
there are holes in the IPVL (Wishart 1997). In chicken and turkey eggs sperm
trapped in OPVL were evenly distributed over the ovum (Wishart 1987;
Wishart 1997). However, in several other avian species the density of sperm
in OPVL appeared higher over the GD area than elsewhere (Birkhead et al.
1994). In fertile eggs of the emu and ostrich the number of sperm trapped in
OPVL over the GD was very low in the center of GD and then increased
rapidly in a band around the center of GD. Here the concentration of sperm
was the highest and then gradually declined with increasing distance from
the center (Malecki and Martin 2003). Thus, the pattern of distribution of
sperm trapped in the GD region of the OPVL of emu and ostrich eggs
corresponds with the pattern of holes produced by sperm in the same region
of the IPVL that has been observed by some authors in laid eggs of several
avian species (Birkhead et al. 1994; Wishart 1997).
#% Reproductive Biology and Phylogeny of Birds
In laid eggs of chickens and turkeys there is nearly a linear relationship

between the number of holes in the IPVL over the GD, the number of holes in
the IPVL over non-GD regions, and the number of sperm trapped in the OPVL
(Bramwell et al. 1995; Wishart 1997; Wishart and Staines 1999). The above
authors found a logarithmic relationship between these three parameters and
the egg fertility status in chicken and turkey and concluded that estimation of
any of these parameters could be applied for assessing the mating efficiency
of commercial turkey and broiler breeders.
10.4.5 Sperm Penetration into the Ovum

Following their passage through the IPVL, one or more sperm interact with
the oolemma and are incorporated into the ooplasm (Fovanova 1965; Bekhtina
1966; Okamura and Nishiyama 1978b; Perry 1987; Nakanishi et al. 1990;
Waddington et al. 1998). As the studies on the entry of sperm into the avian
ovum have been mainly limited to the GD region, the fate of sperm that
penetrated the non-GD region remains unknown. Although sperm holes are
seen throughout the IPVL, the non-GD oolemma is discontinuous and there is
an absence of ooplasm containing organelles. It is assumed that these sperm
do not contribute to the fertilization process. It has been suggested that holes
in the IPVL in the non-GD region were artifactual and the result of acrosin
release from sperm trapped in the OPVL adjacent to the IPVL (Birkhead et al.
1994; Steele et al. 1994). Likewise, Okamura and Nishiyama (1978b) claimed
that although sperm may penetrate any part of the IPVL, the entry of sperm
into the ovum occurs only within the GD region.
Following penetration of IPVL over the GD, the apex of the sperm head
which has undergone the AR comes in close contact with the oolemma
(Okamura and Nishiyama 1978b). After the surface of the inner acrosomal
membrane has come in contact with the oolemma, the two membranes fuse
and the sperm passes into ooplasm. Sperm penetration into the ovum by
means of a phagocytic process has also been observed in chickens (Okamura
and Nishiyama 1978b). However, as the sperm plasmalemma is intact, it is
not clear if male pronuclei are formed.
10.4.6 Formation of Pronuclei and Syngamy

Sperm nuclei decondense within the GD to form male pronuclei (Okamura
and Nishiyama 1978b; Perry 1987). During this process there is a
disintegration of the nuclear envelope and a subsequently swelling of the
nucleus as it transforms from an elongated to spherical organelle. This is
accompanied by a decondensation of the chromatin and, at later stages, a
reconstitution of the degraded nuclear envelope. This transformation was
observed in chicken ova removed from the upper part of magnum after in vivo
fertilization (Okamura and Nishiyama 1978b; Perry 1987) as well as after IVF
(Nakanishi et al. 1990). As a consequence of polyspermy many male pronuclei
are formed in the GD. The number of pronuclei observed by different authors
in the GD of naturally fertilized chicken ovum was as follows: 1-60, average
Fertilization #%!
10 (Fofanova 1965); 20-60 (Bekhtina 1966); 5-15 (Perry 1987); 6-16

(Waddington et al. 1998); and 1-30 in the case of IVF (Nakanishi et al. 1990).
Prior to syngamy, the association of the female and male pronuclei, the
supernumerary male pronuclei are evenly scattered in the GD and are
indistinguishable from the pronuclei destined to form the zygote nucleus. At
the onset of male pronuclei formation, the female nucleus was arrested in
metaphase of the second meiotic division. Formation of the female pronucleus
commences with the completion of its second meiotic division at the time
when male pronuclei formation is nearly completed (Bekhtina 1966; Perry
1987). Syngamy takes place when a fertilized ovum has progressed to the
posterior part of the magnum, i.e. about 3-5 h after ovulation (Perry 1987;
Waddington et al. 1998) or about 4 h following IVF (Nakanishi et al. 1990).
10.4.7 The Fate of Supernumerary Male Pronuclei

The supernumerary male pronuclei, which do not take part in syngamy,
gradually disperse to the periphery of the GD where they can undergo one or
even two rounds of mitotic division, but all of them degenerate and disappear
at the early cleavage stage (Fofanova 1965; Bekhtina 1966; Perry 1987; Gupta
and Bakst 1993; Waddington et al. 1998). The mechanisms responsible for the
degradation and disappearance of supernumerary male pronuclei in early
avian embryos remain unknown. In the polyspermic egg of the newt Cynops
pyrrhogaster displacement of the supernumerary male pronuclei from their
points of entry in the animal hemisphere into the vegetal hemisphere has been
observed (Iwao et al. 1993). However, unlike birds, the accessory pronuclei
never undergo mitotic divisions and degenerate before the first cleavage
division.
10.5 POSSIBLE INVOLVEMENT OF DNASES IN LATE

CYTOPLASMIC BLOCK TO POLYSPERMY
In mammals, transgenic animals can be produced by the microinjection of
exogenous DNA directly into a pronucleus of the fertilized ovum (Brinster et
al. 1985). In birds, direct injection of DNA into zygote pronuclei is not possible
because: 1) they are masked by the presence of yolk spheres and 2) they
cannot be distinguished from the supernumerary male pronuclei. So in birds
the injections of DNA are done into the central part of the GD, where the
zygote pronuclei are usually located (Perry 1987; Waddington et al. 1998).
DNA injection into the ooplasm of a fertilized avian ovum rarely, if at all,
leads to chromosomal integration of the foreign DNA. Most of it gradually
disappears during early embryonic development (Sang and Perry 1989; Perry
et al. 1991), probably contributing to the low efficiency of the method for
obtaining transgenic chickens (Love et al. 1994) and quails (Naito et al. 1994).
The fate of supernumerary sperm and foreign DNA in the GD led us to
suspect that avian oocyte DNases are responsible for the degradation of
excess DNA in early embryos. In Japanese quail oocytes DNase I and DNase
#%" Reproductive Biology and Phylogeny of Birds
II activities were high and capable of digesting, under in vitro conditions, both
naked lDNA/HindIII substrate as well as DNA contained in quail sperm
(Stepinska and Olszanska 2001, 2003). It was also found that the activities of
both DNases increased during oogenesis and reached maximum in mature
preovulatory oocytes and ovulated ova. Stepinska and Olszanska (2001, 2003)
speculated that these enzymes degraded the DNA of supernumerary male
pronuclei resulting from polyspermic fertilization. Interestingly, the presence
of high DNase activities in the GD would explain the low efficiency of
production of transgenic birds by DNA injection into GD since the exogenous
DNA would be targeted by the DNases.
DNase I and II activity is not limited to the GD of ovulated quail ova but is
found outside the GD in the thin layer of cytoplasm surrounding the yolk
subjacent to the IPVL (Stepinska and Olszanska 2003). No DNase activities
were found in the yellow yolk of ovulated quail ova (Stepinska and Olszanska
2003), which is consistent with the observation of visually unchanged sperm
in the yolk of fertilized ova removed from the uterus (Bekhtina 1966).
In contrast to avian ova, DNase I and II activities could not be observed in
ovulated mouse ova under in vitro conditions (Stepinska and Olszanska
2003). Boone and Tsang (1997) also showed the absence of DNase I in rat
oocytes from antral follicles, although they found the presence of the enzyme
in the younger oocytes from preantral follicles. Monospermic fertilization of
mammalian ova is ensured by an early block to polyspermy activated
immediately after penetration of a single sperm. This block depends on
modifications of the ZP (cortical granule-mediated zona reaction) and/or the
oolemma (cortical granule-independent plasma membrane block) (see
Yanagimachi 1994). Thus, from a functional perspective, the DNase activity
in mature mammalian oocytes would be needless or even harmful for the
fertilization process, since the enzymes could destroy the single sperm.
However, polyspermic fertilization of mammalian ova has often been observed
under a variety of experimental conditions, such as IVF, or excess of sperm in
the case of AI. This usually results in abnormal embryo development,
polyploidy, mosaics etc. (see Yanagimachi 1994). Thus, in mammals, when
several sperm do overcome the block to polyspermy, these sperm may remain
functional and they may participate in cleavage divisions causing
developmental abnormalities.
It seems that in birds the lack of an early block preventing multiple sperm
penetration into the ovum might be compensated by the high DNase activities
in the GD. While not a true block to polyspermy, the enzymes participating in
the degradation of the supernumerary male pronuclei would certainly help
minimize polyploidy and subsequent embryo mortality.
Given the high activity of the GD-DNases around the time of pronuclei
formation, why are the male and female pronuclei destined for syngamy not
destroyed? The germinal vesicle (nucleus) of F1 follicle (largest oocyte
destined to ovulate) is quite large and visible by eye at the center of the GD
prior to germinal vesicle breakdown (GVBD). According to the studies of
Fertilization #%#
Bekhtina (1966) and Yoshimura et al (1993), the germinal vesicle enters the
maturation phase by resuming meiosis and is transformed from a spherical to
a discoidal form as it moves towards the surface of the GD. With extrusion of
the first polar body, the second metaphase plate is formed and GVBD has
commenced (Bekhtina 1966; Yoshimura et al. 1993). Given its vast volume, it is
assumed that GVBD and the subsequent liberation of the nucleoplasm dilutes
the impact of DNases on the pronuclei involved in syngamy and activation of
the ovum (Stepinska and Olszanska 2003).
Studies on natural polyspermic fertilization in the newt ovum indicated a
possible influence of a gradient of the maturation/M phase-promoting factor
(MPF) activity on the behavior of accessory male pronuclei (Iwao and Elinson
1990; Iwao et al. 1993; Sakamoto et al. 1998). A higher level of MPF activity
was observed in the center of the animal hemisphere of the newt ovum versus
a lower level in the vegetal hemisphere where the accessory male pronuclei
degenerate. An injection of cytoplasm from unfertilized ova or germinal
vesicle material from full-grown immature oocytes into fertilized newt vegetal
hemisphere rescue accessory male pronuclei that subsequently form extra
bipolar spindle (Iwao and Elinson 1990). These results suggested cytoplasmic
factors present in the animal but not in the vegetal hemisphere control entry of
the pronuclei into the M phase and that the nucleus is involved in the control
of MPF activation. It has also been demonstrated that nuclear material is
indispensable for the increase of MPF activity during oocyte maturation in
several amphibian species (Gautier 1987; Iwashita et al. 1998; Sakamoto et al.
1998).
Our knowledge concerning the role of MPF in oocyte maturation and its
presence in fertilized ova of birds is rather scarce. It has been reported that the
largest vitellogenic quail oocytes contain MPF in the GD and that the MPF
activity is markedly increased during oocyte maturation (Mori et al. 1991).
Injection of a GD extract from the mature oocytes causes maturation of full-
grown immature Xenopus oocytes. No MPF activity was found outside the GD
in either immature or mature quail oocytes. It has been suggested that the
gradient of MPF activity may play a role in the behavior of pronuclei in avian
ova after polyspermic fertilization (Stepinska and Olszanska 2003). DNase I
and II might be responsible for the degradation of all but one of the numerous
sperm entering the avian ovum. The choice of the single sperm predestined to
form the zygote nucleus might depend on its point of entry into the avian
ovum (Fig. 10.8). The one sperm that does enter the center of GD is presumably
drenched with nucleoplasm and therefore might be saved from the degrading
activity of DNases and would be subject to the action of MPF and nuclear
MPF-stimulating factor(s). Other sperm entering periphery of the GD and non-
GD region, would lack the joint effect of MPF and MPF stimulating nuclear
component(s) and would be degraded by DNases present there. It should be
noted here that the probability of many sperm entering at the central region of
a GD is greatly reduced since, as mentioned before, penetration of IPVL over
GD is much less frequent in the center (corresponding to the nuclear territory
#%$ Reproductive Biology and Phylogeny of Birds
Fig. 10. 8 Hypothetical behavior of sperm depending on their point of entry into the
avian egg during polyspermic fertilization. Regions of sperm entry: A. Center of GD;
the single sperm takes part in the formation of the zygote nucleus. B. Periphery of
GD; the sperm form pronuclei but finally are degraded by DNases. C. Non-GD
region; the sperm are degraded by the DNases. After Stepinska, U. and Olszanska,
B. 2003. Zygote 11: 35-42, Fig. 5.
of GD) than in the surrounding ring (corresponding to the cytoplasmic region

of GD) (see 10.4.3.4).
10.6 IS POLYSPERMIC FERTILIZATION IN BIRDS OBLIGATORY?

The findings that a greater proportion of inseminated sperm reach the site of
fertilization in the female reproductive tract in birds than in mammals
(Birkhead et al. 1993) and that the number of sperm reaching the ovum at
fertilization is positively correlated with ovum size in a variety of avian
species (Birkhead et al. 1994) are consistent with the idea that a larger ovum
requires more sperm in order to be fertilized.
In the past, it was generally believed that polyspermic fertilization in birds
is necessary to ensure activation of their megalecithal ova. The conclusion that
a minimum six accessory spermatozoa were required for normal development
of chicken ovum supported the above idea (Fofanova 1965). However, other
data provide evidence against the hypothesis of obligatory polyspermy in
birds. First, it was observed that avian ovum may be fertilized even when few
sperm are found in the OPVL of the GD region (Wishart 1987; Malecki and
Martin 2002) or even when only one sperm penetrated the IPVL over that
region (Wishart 1997). Second, if polyspermy were obligatory, we would
expect a large number of sperm to be concentrated in the center of the GD, the
most likely location of the female pronucleus (Perry 1987; Waddington et al.
1998). However, as mentioned earlier, the sperm penetration of IPVL, although
higher in the GD region than outside it, is much lower in the very center of the
GD than in the ring immediately around the center (Bramwell and Howarth
1992b; Birkhead et al. 1994). Third, excessive sperm penetration into the GD
has been observed to cause abnormal development of avian embryos (Fofanova
1965; Bramwell et al. 1997). Finally, conclusive evidence against the necessity
of polyspermy in birds was supplied by the recent study of Hrabia et al. (2003)
Fertilization #%%
which demonstrated that a single sperm introduced by ICSI into the GD of

quail ovum was capable of activating the ovum for embryonic development.
Thus, it seems that the large avian ovum, with a relatively small GD, requires
physiological polyspermy in order to increase the chance of a sperm entering
the right place in the GD, but not for fertilization process to commence.
Otherwise, in the case of monospermy, a single sperm entering a non-GD
region would prevent the ovum being fertilized.
10.7 ASSISTED REPRODUCTIVE TECHNOLOGIES

10.7.1 Artificial Insemination
Included in AI technology are aspects of semen collection, semen evaluation,
semen dilution and storage, insemination of the female, and evaluation of hen
fertility. The first comprehensive manual on the subject is that from Lake and
Stewart (1978). Somewhat more comprehensive is a text by Bakst and Wishart
(1995) and laboratory guide book by Bakst and Cecil (1997) addressing AI
technology. For captive bird propagation, there is excellent review by Gee et al.
(2004).
Fundamentally, AI is a 2-step procedure. First, the semen is collected and
second, the semen is inseminated. What transpires between these two steps is
a function of management objectives, the skill level of the personnel involved,
and the degree to which purposeful selection of breeder stock and their
progeny drives the breeding program. Manual semen collection in birds was
first described by Burrows and Quinn (1937) and the same basic “abdominal
massage” technique is still in use today. A gentle massaging and stroking of
the cloacal region and abdomen elicits an ejaculatory response from the male.
The semen can be collected and left undiluted, or “neat” prior to AI. However,
unless inseminated within 30-45 min of semen collection, the sperm will lose
their viability. An alternative approach is to collect and dilute the semen with
a semen extender that has been formulated to prolong the survival and
viability of sperm. Semen dilution and storage has been reviewed several times
over the years. Unfortunately, the technology here has not advanced
significantly since the 1980s (Christensen 1995). While some have added
antibiotics and anti-oxidants to commercially available extenders, conclusive
prove of their efficacies remains equivocal (Surai 1999; Breque et al. 2003).
The benefits of using semen extender are numerous, with more possibilities
as the duration to store sperm in vitro increases. By diluting the semen with
extender, one increases the ratio of male:female for breeding purposes. For
example, when using neat semen, or when birds are allowed to copulate, the
ratio of male: female can be 1:8 or 1:10. After dilution of semen with an
appropriate extender, the ratio of male: female can be 1:20 to 1:30. The
potential ramifications of using extenders for dilution and the storage of
semen include: increased selection pressure on male breeders, an opportunity
to evaluate semen quality, an opportunity to inseminate a known number of
sperm, use of fewer sperm per insemination, and better male management.
#%& Reproductive Biology and Phylogeny of Birds
That being said, no significant changes have been made in semen collection,
dilution, and insemination over the past few decades. What has improved are
the means by which semen is evaluated and fertility estimated (see Bakst and
Cecil 1997).
In this millennium, some poultry breeders are selecting males based on
sperm mobility through a viscous medium, referred to as the sperm mobility
assay (Froman et al. 1999). They found a strong positive correlation in
reproductive success (paternity) and the ability of sperm from a given male to
progressively move through a viscous medium. This observation appears to
be applicable to both domestic and feral birds (Pizzari et al. 2002). Devices that
integrate progressive motility and sperm concentration into a sperm index are
available on the market and have proven to be useful to some.
Careful dissection of fresh laid eggs can provide considerable information
about the fertility status of a flock. By examining the morphology of the GD, a
trainer observer can determine if the ovum was fertilized or left unfertilized.
At the time of lay, a fertilized GD will be at the blastoderm stage of
development (Wilson 1995; Wishart 1995; Bakst et al. 1998). This blastoderm
will be symmetrical, uniformly opaque, and about 3 mm in diameter. An
unfertilized GD is small, asymmetrical and be characterized by variable
number of vacuoles sometimes visible by eye. If one is unsure if the GD was
fertilized, two additional procedures can be quickly performed. With a fine
pipette, puncture the ovum just outside the GD area and bring the pipette tip
to the center of the GD. Gently aspirate some of the white yolk from the GD
region and mix with a nuclear fluorescent stain such as bis-benzimide. If cells
are present, whether from an early dead or a viable embryo, their nuclei will
intensely fluoresce. If done carefully the PVL overlying the GD is still intact.
This can be removed, washed clean of adhering yolk material and either stain
with the same fluorescent nuclear stain to reveal sperm embedded in the
OPVL, or the PVL could be examined using the PVL hole assay (see 10.4.3.4
and 10.4.4). The PVL hole assay has provided a means to estimate flock
fertility as well as the duration of fertility for individuals hens. While time
consuming to perform, the procedure is simple and provides objective results.
10.7.2 In vitro Fertilization

The development of IVF and embryo manipulation techniques in mammals
has significantly improved our understanding of the mechanisms of
fertilization, activation of the ovum, and early embryonic development. In
birds, IVF and associated technologies are not extensively developed, possibly
due to the difficulties in both obtaining and manipulating mature yolk-laden
ova.
Howarth (1971) reported the first successful production of chicken embryos
by IVF. Recently ovulated chicken ova retrieved from the body cavity or
infundibulum were co-cultured with fresh sperm. The fertilization status of
the ova was examined after 24 h of in vitro culture by hematoxylin staining of
nuclei, which confirmed the presence of multicellular embryos. More recently,
Nakanishi et al. (1990) provided direct evidence of IVF of chicken oocytes by
Fertilization #%'
demonstrating formation of male and female pronuclei after staining sections

of a GD with 4,6-diamino-2-phenylindole (DAPI). Nakanishi et al. (1990) also
concluded that early nuclear events, such as pronuclear formation,
association of the male and female pronuclei, and elimination of
supernumerary pronuclei, between in vivo and IVF during 1-4 h of in vitro
culture were essentially the same. Tanaka et al. (1994) obtained viable chicks
by transplanting in vitro fertilized ova into the oviduct of recipient hens
followed by incubation of the eggs. However, the method used by those
authors is complicated and requires surgical intervention. The oocytes used
for IVF by all above authors were ovulated and recovered immediately from
the body cavity or oviduct. Olszanska et al. (2002) successfully showed that
quail embryos would develop normally after IVF of oocytes which had been
ovulated in vitro. After IVF with fresh semen, and 20-22 h incubation, 14% of
the blastoderms were between Stages IV-VI (see Eyal-Giladi and Kochav 1976)
and contained DAPI-stained nuclei. Yet, under identical conditions, 25% of
the “embryos” appeared normal but did not contain nuclei. Olszanska et al.
(2002) suggested that the GD underwent segmentation without accompanying
divisions of the nuclei. This phenomenon underlines the importance of
monitoring the presence/absence of nuclei in early avian embryos, especially
ones derived by IVF, to distinguish true cleavage from segmentation.
10.7.3 Intracytoplasmic Sperm Injection

Methods of direct injection of sperm into an ovum, referred to as
intracytoplasmic sperm injection (ICSI) have not only resulted in normal
offspring and transgenic animals, but increased our knowledge of some
aspects of mammalian fertilization. For the first time an ICSI method in birds
was reported by Hrabia et al. (2003). An intracytoplasmic injection of a single
sperm into a quail ovulated ovum activated the GD and led to fertilization
and the onset of development. The efficiency of fertilization after ICSI of ova
was similar to that reported by Olszanska et al. (2002) for conventional IVF
using 1-2 ¥ 104 sperm per ovum. The blastoderms after ICSI and 24 h culture
reached Stages II-VII (see Eyal-Giladi and Kochav 1976), which is also
comparable to the results of Olszanska et al. (2002). It was also shown that
injection of a single testicular sperm or elongated spermatid into a quail ovum
resulted in fertilization and embryo development to Stages VI-VII. These
results indicated that both testicular sperm and elongated spermatids were
capable of activating the ovum, form pronuclei and begin early embryonic
development when introduced into the ovum. In contrast, injection of a round
spermatid into quail ovum did not result in successful fertilization. The
findings of Hrabia et al. (2003) provide direct evidence against polyspermy
being obligatory in birds.
We are very grateful to B. Olszanska and G. Wishart for their helpful
comments on the manuscript.

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n n
Index
A Acrosome Reaction (AR) 295, 376, 377,

A. renalis medius 41 379, 508, 562, 563, 583, 584
Abdominal Aperture 153 Acrosome Vesicle 293, 295, 299, 350, 360,
Abdominal Ostium of the Oviduct 153 367-369, 371, 373, 374, 376, 377, 379,
Acanthizidae 20 383, 385, 386, 390, 392, 397, 399, 400,
Accessory Ducts 155 403, 405-407, 410, 411, 414-417, 419,
Accessory Sex Organs or Glands 37 420, 425, 426, 428, 429, 433, 434, 436,
Accipitres 11 438-440, 442, 443, 450, 454, 458, 488
Accipitridae 11, 13, 35, 149, 358 Actin 44, 72, 85, 376, 377, 379
Achaetops 23 Actions of FSH on Follicular
Acid Phosphatase Activity 100 Development 202
Acrocephalidae 23 Actitus 441
Acrocephaline Warblers 23 Activin 51, 53, 110, 158, 202, 232, 257-
Acrocephalus 147, 448, 451, 457, 478, 259, 261, 264, 273, 275
517, 543, 549 Additional Apertures 155
Acrosin 565, 570, 571, 572, 582, 583, 585 Adenohypophysis 190, 192, 194, 233,
Acrosome 282, 283, 285, 287, 289, 291, 234, 239, 327
293, 295, 297, 299, 335, 339, 350, 360, Adrenal Gland 40, 96, 97, 103, 165
365-369, 371-374, 376, 377, 379, 381, Adsorptive 72, 83
383-385, 388-392, 394-397, 399-403, Aedon 357, 463, 467
405-407, 410, 411, 414-420, 422, 423, Aegithalidae 23
425, 426, 428, 429, 433, 434, 436, 438- Aegithinidae 20, 21
450, 452, 454, 455, 458-461, 463, 467- Aegothelidae 8, 10, 12
469, 471-473, 475, 476, 478-488, 490, African Stonechats 207
491, 493, 494, 496, 497, 499-502, 506, Agapornis 362, 428, 434
508, 532-534, 556, 562, 563, 565, 567, Agelaius 141, 262, 304, 364, 449, 481, 486,
581 583, 584 487, 520
Acrosome granule 283, 285, 287, 288, 295 Alauda 357, 463, 465
*
Some taxa given in tables (e.g. Table 8.3) are not listed in this index.
#' Reproductive Biology and Phylogeny of Birds
Alaudidae 21, 358, 463 Anseranas 7, 131

Albumen 149, 166, 167, 171, 201, 557 Anseranatidae 7, 131
Alcae 12 Anseriformes 4, 7, 28, 31, 34, 116, 117,
Alcedinidae 9, 12, 14 123, 125, 126, 140, 143, 157, 297, 355,
Alcidae 14, 29, 358, 440 356, 358, 359, 361, 383, 405, 410, 427,
Alectoris 209, 226 497, 502, 527, 569
Alkaline Phosphatase Activity 100 Anseriforms 99, 353, 410, 426, 429, 436,
Allometric 540 443
Allometry 540 Antbirds 19, 30, 207
Alpine Accentors 513 Anthus 357, 465, 467
Ant-pittas 19
Amazonetta 8
Ant-thrushes 19, 445
Ambient Temperature 319
Apicolateral Junctional Complexes 93
American Avocet 519, 550
Apodiformes 8, 9, 10, 12, 339, 357, 358,
American Robin 363, 449, 450, 452, 454,
362, 410, 411, 418, 448, 497, 498, 502,
456, 467-470, 534, 536
506
AMH Synthesis 161 Apomorphy 2, 354, 355, 450, 496, 501
AMH Transcription 161 Apoptosis 158, 159, 178, 179, 221, 244,
Amino Acids 184, 199, 568
249, 252, 255, 265, 267-270, 272-276
Ammodramus 363
Apostlebird 21
Amniote Spermatozoon 350
Appendix Epididymidis 96-98
Amniote Symplesiomorphies 360
Appendix Paradidymidis 40
Amniote Synapomorphy 354, 360, 364,
Apterygidae 6, 129, 200, 360
365, 436
Apus 289, 297, 339, 357, 362, 411, 413,
Amphibians 157, 236, 247, 350, 354, 553
414, 418, 448, 498, 499, 500, 506, 510
Ampulla 37, 164, 165, 168
Aramidae 9, 11
Anas 3, 7, 8, 31, 35, 44, 46, 55, 57, 59, 68,
Arcanator 24
79, 88, 91, 92, 101, 115, 131, 199, 208,
Ardeidae 13, 358
251, 280, 305, 322, 324, 325, 326, 330,
Ardeotis 15
331, 332, 343, 356, 359, 361, 382, 405,
Artamidae 20
406, 409, 410, 425
Arteria cranialis renalis 41
Anatidae 7, 32, 126, 128, 129, 131, 137,
Arteria pudenta 41
139, 142, 143, 281, 241, 358
Arteria renalis caudalis 41
Anatomical Nomenclature 168
Androgen 38, 51, 82, 94, 105, 106, 113, Arteria testicularis 41
166, 199, 226, 233, 236, 241, 256, 257, Arteriae testiculares accessoria 41
263, 314, 315, 317, 319, 329, 333, 342, Artificial Insemination 113, 505, 509,
346, 531 553, 577, 580-582, 584, 586
Androgen Binding 51, 106, 113, 342 Asymmetrical Testes 517
Androgenic Hormones 166 Atrichornithidae 20
Androstenedione 51, 200, 256, 257, 263, Auks 27, 440
274 Australasian Treecreepers 20
Anhingidae 8, 11 Australian Babblers 20
Annulus 283, 291, 293, 294, 300, 350, 352, Australian Magpie 20
360, 365, 369, 370-373, 379, 380, 383, Australian Robins 21
387, 389, 391, 399, 400, 403, 404, 407- Autoantibodies 53
410, 413, 414, 418, 421, 425, 428, 433, Autoimmunocytes 53
434, 439, 442, 443, 447, 451, 478, 491, Aves 3, 26, 28-35, 113, 115, 145, 147, 178,
494, 497, 500, 533 181, 339, 346, 352, 355, 357, 360, 421,
Anser 109, 157, 250, 328, 342, 272 438, 497, 499, 504-506, 508
Index #'
Avian Egg 150, 176, 576 Block to Polyspermy 553, 570, 573, 574
Avocets 13, 440, 540 Blood-epididymal Barrier 71, 108
Axoneme 61, 297, 299, 350-352, 360, 364, Blood-testis Barrier 49, 51, 53, 102, 104,
365, 370, 371, 379-371, 374, 376, 379, 105, 110, 111, 279, 337, 338
383, 385-387, 389, 391-393, 396, 397, Blue Tit 486
399, 400, 401, 403, 404, 408, 409-411, Blue-flycatchers 21
413, 415, 417, 418, 420, 421, 423, 425, Body Temperatures 516
426, 428, 431-434, 436, 438, 439, 440, Bombycillidae 23, 358, 467
442, 443, 447, 451, 452, 454-456, 458, Bonasa 7
460, 461, 463, 469, 470, 472-480, 482- Boobies 8, 11
486, 488, 493, 496, 497, 500, 532 Boundary Tissue 53-59, 61, 87, 89, 95, 112
Aythya 356, 382, 405 Bowerbirds 20, 504
Brachypteracidae 9, 12, 14, 31
B Bradypterus 23
Babblers 20, 23 Brain 182, 184, 188, 189, 194, 197, 198,
Balaenicipitidae 8, 440 200, 202, 205-207, 210, 216, 218-222,
Ballowitz 349, 355, 371, 405, 415, 417-419, 224, 225, 228, 231, 233-240, 262, 272,
426, 441, 443, 456, 461, 463, 467, 477, 327
501 Brambling 521
Bank Swallows 520 Branta 128, 259, 361, 405, 410
Barbets 8, 13, 26, 32, 34 Breeding 40, 109, 124, 126, 128, 133-135,
Barn Swallows 517 138, 141, 147, 148, 158, 162, 166, 181-
Basal Cells 87, 89, 90, 95, 321 186, 188, 199, 200, 203-209, 211, 213,
Basal Lamina 46, 50, 53, 54, 56-58, 61, 63, 215, 221, 224, 226, 228-231, 233, 235-
80, 96, 153, 155, 157, 158, 162-164, 200, 237, 242, 246, 248, 250, 251, 253, 258,
254, 255, 271, 307, 309, 323-325, 335, 266, 267, 271, 272, 274, 275, 312, 315-
367, 431 319, 335, 339-347, 505, 513, 515, 521-
Basal Plate or Capitulum 291 528, 530-532, 543-548, 550, 551, 553,
577, 586
Batis 21
Breeding Strategies 182
Batises 21
Bristleheads 20
Baya Weaver 203
Broadbilled Sapayoa 17
Bayesian Analysis 16, 23, 24
Bucerotidae 9, 12, 15
Bearded Tit 21, 135, 144
Budgerigars 509, 522, 551
Beavan’s Bullfinch 451, 490, 533
Bulbuls 23
Bee-eaters 9
Bullfinch 448, 451, 457, 490, 492, 501, 502,
Bengalese Finch 299, 300, 493, 544 504, 533, 542, 544
Berrypeckers 20 Buntings 25
b-catenin 159, 175 Buphagus 24
Biological Clock 181-183, 229 Bush Warblers 23
Blackbirds 25, 147, 503, 520, 533, 543, Bush-shrikes 21
549, 550 Bustards 11, 26, 33
Blackcap 356, 457, 478 Butcherbirds 20
Black-headed Grosbeaks 523, 547 Butterflies 540, 546
Black-necked Stilt 441, 519 Buttonquails 9
Blackpoll Warbler 538 Ballowitz 349, 355, 371, 405, 415, 417-419,
Black-throated Blue Warblers 518 421, 423, 425, 426, 440, 443, 456, 461,
Blastoderm 166, 262, 557, 578, 580 463, 467, 477, 501, 504, 532, 543
#' Reproductive Biology and Phylogeny of Birds
C Certhiidae 23, 24, 363, 455, 478

Cacatuidae 13-15, 26 Certhioidea 19, 23, 363, 455, 478
Caiman 352, 354, 355, 509 Cettiidae 23
Cairina 280 Chachalacas 7
Calcareous Shell 166 Chalaziferous Region 168
Calcarius 25, 527 Charadrii 12, 34, 440
Calidris 357, 382, 441, 443 Charadriidae 12, 15, 358, 440, 441, 533
Callaeatidae 20 Charadriiformes 9, 11, 12, 15, 28, 29, 33,
Caluromys 352, 508 353, 355, 356, 358, 362, 405, 418, 426,
Canary 21, 208, 236, 364, 410, 455 427, 440, 443, 448, 498, 499, 501, 533
Canary-flycatchers 21 Charadrius 383, 441, 533
Capacitation 85, 315, 339, 559, 562, 580, Chatshrike 21
583 Chelidon 356, 463
Cape Sparrow 457 Chelonia 350, 352-354, 371, 374, 381, 436,
Cape Weaver 364, 450, 484, 485 494, 495, 496, 505
Capitonidae 2, 12-15, 34 Chick Embryos 150, 153, 159, 161, 164,
Capitulum 291, 292, 385 176, 242
Caprimulgiformes 8, 9, 12, 32, 355, 358, Chick-quail Chimeras 165
362, 410, 411, 415, 497, 498 Chiffchaff 457, 478
Caprimulgus 289, 291, 297, 355, 362, 415- Chionidae 13
418, 426, 443, 498, 499 Chloris 356, 357, 364, 455, 463, 464, 467,
Carbendazim 83, 334 526, 548
Cardinalidae 25 Chordae Retis 64, 66, 70, 72
Cardinalis 364, 449, 481, 488 Chromatoid Body 291, 292, 335, 337,
Carduelis 218, 233, 356, 357, 364, 451, 338, 345, 346
455, 457, 463, 464, 526, 548 Chrysomitris 357, 463
Cariamidae 11 Ciconiidae 3, 9, 10, 13, 34
Carpodacus 211, 355, 356, 526 Ciconiiformes 8, 10, 149, 358, 362, 440,
Carrion Crow 448, 451, 453, 456-458 443, 498, 500
Caspase-3 161, 268, 269 Ciliated Cells 66, 68, 73, 74, 77, 80, 83, 84,
Casuariidae 3, 6, 124, 360 89, 94, 99, 163, 168, 171, 172, 328-323
Casuarius 124, 360 Cinclidae 23, 24
Cathartidae 3, 9, 13, 35, 149 Circadian Rhythm 203, 211, 212, 226
Catoptrophorus 441 Circadian Rhythmicity 229, 240, 270
cDNA 198, 199, 218, 219, 223, 235, 236, Circannual Rhythms 230, 272, 316
238, 586 Circus 40
Cellular Associations 280, 301, 302, 305, Cirl Bunting 364, 455, 456, 488
306, 347, Cisticolas 23
Centriole 283, 285, 289, 291, 300, 338, Cisticolidae 23
345, 350, 351-353, 355, 360, 367, 369, Classification and Phylogeny 1, 37
371-374, 376, 397, 377, 379-381, 383, Cliff Swallows 523, 532, 545
385-387, 389, 391, 393, 395-397, 399, Climacteridae 20
400, 403-405, 407, 408, 410, 411, 413- Cloaca 37, 41, 85, 86, 95, 120-124, 127-
415, 417-423, 425, 428, 431 434, 436, 129, 131, 133-137, 139, 142, 146, 158,
439, 442, 443, 446, 451, 455, 463, 472, 162, 166, 172, 175, 201, 434, 519
473, 475, 478, 480, 482, 483, 486, 494, Cloacal Protuberance 85-87, 112, 134-
496-498, 500, 502, 509 136, 145-148, 199, 319, 516, 548, 551
Centropus 516 Clutch Size 245, 246, 248, 250, 258, 266,
Certhia 363, 455, 478, 488 272, 530, 531
Index #'!
CNS 182-184, 188, 193 Cost of Flight Hypothesis 518

Coerebidae 358, 467 Cotingas 20, 445, 446
Colies 8 Cotingidae 17, 20, 345, 358, 445, 446
Coliiformes 8, 361, 499 Coturnix 39, 44, 47, 48, 50, 54, 55, 57, 60,
Columba 15, 31, 38, 102, 112, 232, 287, 65, 67, 68, 83, 84, 98, 100, 103, 108, 110,
289, 293, 318, 355, 357, 362, 412, 418, 157, 176, 198, 204, 224, 226, 230, 232-
419, 421, 423, 425, 448 234, 236, 240, 242, 254, 259, 264, 277,
Columbidae 9, 14, 15, 358, 410 281, 287, 302, 309, 310, 313, 334, 336,
Columbiformes 8, 31, 297, 355, 358, 359, 340, 342, 344, 347, 361, 385, 387, 388,
362, 392, 411, 418, 427, 448, 494, 497- 390-394, 405, 421, 423, 448, 451, 457,
500, 502, 569 497, 502, 507, 508, 511, 534, 551, 584,
Commericial Turkey 166 585, 587
Common Magpie 521 Coucals 516, 517, 548
Common Tern 85, 252, 274 Coursers 13
Comparative Methods 137, 514 Cracidae 7, 33, 126, 131, 132, 136, 143
Compensation Hypothesis 518, 547 Cranes 9, 11, 31
Connecting Duct 61, 72, 73, 83, 85 Crescent-chests 19
Connecting Piece 292, 300, 385, 397, Crex 356, 382, 438, 440
399, 400, 404 Cristae 59, 60, 289, 295, 323, 325, 326, 352,
354, 365, 369, 370, 373, 387, 397, 404,
Conopophagidae 19
407, 408, 413, 428, 433, 436, 470, 480
Continuous Layer 166, 168, 557, 571
Crocodile + Avian Stock 355
Contopus 362, 444, 445, 447
Control of Avian Reproduction 216 Crocodiles 116, 117, 120, 121, 350-354,
371, 436, 443, 498
Coprodeum 122, 172, 173, 175
Copulation 87, 98, 102, 103, 115, 116, Crocodiloid Spermatozoon 371
120, 122-124, 129, 131, 133-138, 140, Crocodylia 116, 350, 352, 354, 355, 381,
144-147, 185, 198, 222, 312, 318, 335, 436, 496
504, 514, 518-521, 525, 530, 535, 544, Crocodylus 352-355, 494, 495, 506, 509
549, 559, 560 Crossbill 207, 231, 251, 271
Copulatory Structures 116, 120, 136, Crotophaga 289, 293, 344, 362, 424-426,
137, 144 500, 508, 509
Coraciidae 9, 12 Cryptic Female Choice 535, 538, 539,
Coraciiformes 9, 12, 15, 361 544, 546
Corcorax 21 Cuckoos 3, 9, 10, 25, 27, 30, 32
Cormorants 8, 11 Cuculidae 9, 10, 14, 30, 32, 358
Corn Bunting 457, 490 Cuculiformes 3, 9, 10, 344, 355, 358, 361,
Coronaves 4, 5, 9, 10, 410, 415, 423, 426, 426, 498-500, 508, 509
498, 499, 500, 501 Cuculus 355, 425, 426
Corticosterone 211, 235, 262, 272, 532, Culicicapa 21
545 Curassows 7
Corvida 19, 355, 357-359, 363, 444, 446, Currawongs 20, 21
448, 449, 452, 455-459, 461, 501, 502 Cyanistes 485, 486
Corvidae 17, 21, 113, 346, 358, 363, 427, Cyanocitta 363, 447, 454, 456
445, 451, 455-459, 461 Cycle of the Seminiferous Epithelium
Corvoidea 17, 19, 20 281, 301-303, 305-307, 311, 312, 336,
Corvus 85, 208, 233, 271, 328, 355, 357, 340, 343
363, 427, 446, 448, 449, 451-460, 494, Cynops 573, 585
501, 504, 521 Cypselus 357, 362, 411, 413
#'" Reproductive Biology and Phylogeny of Birds
D 446, 451, 463, 472, 473, 475, 478, 483,

Dapple-throat 24 486, 494, 496-498, 500
Darters 8, 11 Divers 8
Dasyornis 20 Diving-petrels 11, 13
Deconychura 427, 445 DNA Hybridization 3, 7, 26, 27, 30, 33,
Dehydrogenases 94, 99, 113 34, 117, 118, 120, 441, 498, 500, 504,
Dendrocitta 203, 226 509
Dendrocolaptidae 17, 19, 20, 358, 445 DNA Sequences 1, 25, 26-31, 33, 34, 494,
505, 583
Dendrocalaptinae 446
DNase 573-575, 585
Dendrocopos 355, 357, 412, 423, 425, 448
Domestic Fowl 38, 40, 63, 95, 98, 101,
Dendrocygninae 7
102, 104, 106, 108-113, 150, 175, 177,
Dendroica 363, 391, 427, 513, 518, 538,
193, 198, 229, 230, 232, 238, 239, 241,
546
275, 280, 321, 338, 342, 343, 345, 347,
Dense Fibers 299, 336, 339, 353, 369, 371,
392, 503, 505, 507, 508, 510, 521, 538,
372, 376, 379, 381, 385, 387, 393, 396,
543, 547, 580-586
399, 407-409, 413, 417, 421, 423, 425,
Domestic Turkey 133, 538
428, 433, 436, 439, 440, 442, 446, 447,
Donacobius 23
451, 456, 461, 463, 467, 469, 471-476,
Double Oviducts 149
478, 480, 482, 483, 486, 488, 494, 496,
Doublets 353, 354, 365, 369-372, 385, 387,
497, 501, 502
389, 391, 393, 399, 400, 405, 408, 410,
Desmin 72
417, 418, 420, 421, 423, 433, 434, 436,
Development 49, 95, 102, 103, 105, 107,
442, 456, 461, 470, 473, 475, 482, 483
109, 110, 112, 113, 137, 141, 142, 149,
Dromaius 38, 109, 318, 341, 360, 361, 364,
150, 157-159, 161, 162, 166, 175-179,
365, 374, 448, 451, 496, 584
182-186, 188, 189, 194, 199, 201, 202,
Drongos 21
204, 208, 221, 227, 230, 232, 235, 239-
Dryoscopus 21
245, 247, 249-251, 253-257, 259, 262-
267, 270-276, 279, 283, 287, 289, 292, Duck 7, 54, 73, 128-130, 146, 157, 162,
295, 300-302, 306, 312, 313, 317, 320, 210, 224, 280, 338, 343, 356, 382, 405,
321, 323, 336, 337, 340, 343-345, 347, 418, 425, 433, 555, 569
352, 391, 413, 418, 428, 440, 447, 450, Ductuli Aberrantes 97, 98, 103
453, 454, 478, 488, 506-509, 517, 542, Ductuli Efferentes Distalis 72
553, 557, 573, 574, 576-579, 581-585, Ductuli Efferentes Proximalis 72, 73
587 Ductus Aberrans 97, 98
Developmental Abnormalities 516, 574 Ductus Conjugens 61, 65, 72, 83, 98
Dicruridae 21 Ductus Deferens 37, 39, 41, 44, 61, 64, 71,
Didelphis 352, 510 82, 83, 85, 86, 89, 90, 94, 95, 100, 106,
Diethylstilbestrol 161, 179, 585 107, 110, 113, 135, 300, 328, 333, 430,
434
Differentiation of the Left Oviduct 150
Ductus Epididymidis 61, 64-66, 83, 84,
Diomedeidae 13, 251
91, 97, 98, 100, 101, 105-107, 110, 328
Dipnoi 352
Dunnock 144, 335, 527, 544
Dippers 23
Dynein Arms 370, 371, 387, 399, 408, 431,
Distal Centriole 283, 285, 289, 291, 300,
476
338, 351-353, 355, 360, 367, 369, 371-
374, 376, 379, 381, 386, 387, 389, 391,
E
393, 396, 399, 400, 403-405, 407, 408,
410, 411, 413-415, 417, 418, 420, 421, Early Embryonic Development 150, 166,
422, 425, 428, 431-434, 436, 439, 443, 557, 573, 578, 579
Index #'#
Ectoparasite 532 328-330, 333-345, 557-561

Efferent Ducts/Ductuli Efferentes 61, 63, Epithelium of the Müllerian Duct 157
65, 72-75, 77, 80-83, 85, 93, 95, 98, 99- Epoophoron 165, 176
103, 105-109, 111, 113, 314, 328, 334, Eptatretus 377, 508
344 Estrildidae 230, 364, 493
Egg Formation; Sperm Selection 166, Estrogen Receptors 82, 99, 100, 108, 161,
557 247
Egg Production 143, 162, 166, 171, 252, Estrogenic Hormones 161
276, 559 Ethynyloestradiol 162, 176
Egg-mass 166 Eudromia 287, 293, 361, 377, 378, 496
Ejaculate Size 520, 535, 539, 549 Euplectes 364, 450, 484, 485
Elminia 21 Eupodotis 15
Emberiza 356, 357, 364, 455-457, 465, 467, Eurasian Bullfinch 448, 451, 457, 490, 492,
488, 490, 533 501, 502, 504, 533, 544
Emberizidae 25, 134, 449, 455, 457, 488 Eurasian Tree Sparrow 521
Empidonax 534 European Quail 204, 230
Emx-2 157 European Starlings 194, 206, 212-214,
Endonuclear canal 285, 287, 289, 293, 224, 226, 227, 229, 230, 232, 521, 545
295, 355, 360, 367, 369, 371, 372, 374, Eurylaimidae 17, 19, 33
376, 377, 379, 381, 383-385, 392, 399, Eurylaimides 19
403, 407, 410, 415-419, 431-434, 436, Eurypygidae 9, 11, 410
438, 439, 494, 496, 497, 500 Excurrent Ducts of the Testis 61, 82, 87,
Endpiece 350, 360, 365, 366, 369, 371, 89, 101, 107, 113, 313, 314, 327, 329,
374, 376, 381, 383, 385, 387-389, 393, 333
396, 400, 401, 405, 407-410, 413, 415, Presecretion Phase 320, 333
471, 473, 475, 477, 483, 532 Reproduction Phase 320, 328, 333
Environmental Clues, Stimuli or Effects Regressive Phase 320, 329, 521
183, 184, 188, 216, 243, 250, 270, 316, Refractory Phase 320, 321, 329
319, 321, 327 External (Coelomic) Glomeruli 150
Environmental Signals 181-184, 186-188, Extracellular Matrix 157
205, 315, 318 Extra-Pair Copulations 145, 313, 335,
Epididymal Duct/Ductus Epididymidis 514, 518, 525, 530, 544, 545
61, 71, 83, 85, 87-92, 94-97, 100, 110, Extremitates Caudales 37
112, 315, 328-330, 333, 515 Extremitates Craniales 37
Epididymis 37, 39, 40, 41, 44, 46, 61, 63-
65, 71, 72, 75, 83, 85, 86, 89, 94-98, 100- F
104, 107-112, 177, 199, 314, 315, 327, Fairy Bluebirds 24
329, 334, 335, 339, 340, 342-344, 346, Fairy Warbler 21
386, 434, 510 Fairy Wrens 20, 110, 136, 137, 146, 147,
Epithelio-mesenchymal Transformation 543, 548, 550
157 Falcipennis 7
Epithelium 45-49, 51, 54, 57, 58, 61, 64, Falco 149, 362, 426, 444, 448, 499, 501
66-68, 70-73, 75, 80-82, 84, 85, 87-90, Falconidae 13, 29, 149
95-100, 102, 104-107, 110, 112, 113, 141, Falconiformes 9, 11, 29, 149, 355, 358,
150, 153, 155, 157-159, 162, 163, 164, 362, 443, 448, 499, 501
166, 168, 171, 172, 174, 175, 244, 253, Fantails 21
256, 271, 279, 280, 281, 282, 283, 301- Female Choice 141-145, 525, 535, 538,
303, 305-308, 311, 312, 314, 321-324, 539, 544, 546, 548
#'$ Reproductive Biology and Phylogeny of Birds
FEPC 142 Fulica 356, 359, 382, 438, 440

Fertilization 115, 141, 142, 150, 166, 168, Furnarii 19
171, 201, 252, 261, 262, 344, 376, 442, Furnariidae 17, 19, 358, 445, 446
503, 504, 507, 508, 514, 530, 535, 536,
545, 547, 550, 553, 554, 557, 559-562, G
565, 568, 569, 572, 574-579, 583-587 Galbuliformes 8, 10, 15, 361
Fibrolymphatic Bodies 122, 123, 125, 127 Galliformes 4, 7, 26, 28, 32, 33, 75, 94,
Fibrous Sheath 285, 291, 294, 336, 339, 116, 117, 126, 131, 140, 143, 157, 218,
350, 352, 354, 360, 365, 369-372, 374, 252, 297, 300, 301, 355, 356, 358, 361,
376, 379, 380, 381, 383, 385, 387, 389, 383, 405, 410, 427, 448, 497, 502, 569
393, 400, 401, 404, 405, 410, 423, 428, Galliforms 353, 389, 391, 400, 405, 410,
433, 434, 436, 442, 494, 496, 497 426, 428, 436, 443, 451
Ficedula 357, 449, 463, 465, 467 Galloanserae 4, 6, 28, 120, 126, 131, 132,
Fimbria 168 134, 136, 140, 143, 371, 383, 400, 410,
Fimbriated Region of the Infundibulum 413, 418, 419, 423, 428, 431, 497, 498,
166, 167, 557 500, 502
Finches 25, 207, 225, 236, 242, 251, 262, Gallus 38, 42, 43, 64, 67, 71, 74, 76, 78, 79,
451, 518, 520, 534, 539, 549, 551 81, 96, 97, 101-104, 109, 111-113, 116,
Finfoots 9, 11 150, 152, 154, 156, 157, 159, 160, 165,
Fitch Trees 3 167, 176, 193, 232, 245, 247, 254, 256,
Fitch-margoliash 3 264, 271-276, 280, 312, 343, 345, 346,
Flagellum 282, 283, 289, 291, 299, 300, 355, 356, 361, 382-386, 391-393, 396,
341, 345, 372, 374, 380, 388, 389, 391, 398, 401, 405, 410, 425, 427, 497, 504,
392, 396, 399, 400, 401, 404, 413, 417, 505, 508, 510, 511, 521, 543, 547, 553,
418, 423, 425, 426, 429, 433, 438-440, 554, 556, 558, 561, 563, 564, 566, 582,
451, 454, 457, 458, 466, 469-476, 480, 584-586
486, 492, 493, 509, 532, 534, 536, 539 Gartner’s Ducts 165
Flamingos 8, 10, 32 GATA-3 157
Flight Costs 138, 518, 532 Gaviidae 10
Fluid-phase Endocytosis 83, 109 Gaviiformes 8, 362
Follicle Atresia 265-267 Genetic Distances 3
Genetic Paternity 529
Follicle Selection 246, 252, 255, 258, 260,
Genome Size 540, 541, 546
264, 266, 270, 276
Geopelia 352, 362, 418, 419, 421, 422, 423
Follicle Stimulating Hormone (FSH) 51,
Germ Cells 38, 40, 46, 47, 49, 53, 72, 83,
194, 197-199, 201, 202, 205, 208, 220,
99, 108, 247, 249, 273, 274, 276, 279,
227, 232, 237, 242, 249, 250, 256-259,
292, 300-302, 304, 306-308, 311, 312,
261, 263, 264, 266, 269, 270, 317, 327
321-323, 328, 329, 342, 344, 347, 517,
Food Availability 204, 243, 250, 251, 316,
584
318, 321
Germinal Disc (GD) 554
Forced Extrapair Copulations 129, 138,
Glandular Grooves 168
139, 142, 143, 528
Glareolidae 13
Formicariidae 17, 19, 358, 445, 446
Glycoprotein Hormones 197
Francolinus 7
Gnatcatchers 23
Fregatidae 8, 11, 13, 31
Gnateaters 19
Frigatebirds 8, 11, 31 GnIH 189, 203, 210, 217-223, 225, 239
Fringilla 356, 357, 363, 455, 463, 464, 505, GnIH Peptide 218
521 GnRH 183, 189, 197, 198, 201, 205-207,
Fringillidae 25, 35, 358, 363, 427, 455, 457, 212, 214-216, 219-223, 231-238, 243,
463, 490 250, 251
Index #'%
Goblet Cells 171 Gulls 27, 34, 247, 252, 440

Goldfinch 364, 457 Gymnorhina 20
Golgi Complex 49, 50, 56, 59, 63, 68, 70,
77, 80, 91, 94, 283, 285, 286, 292, 299, H
307, 328 Haematopodidae 13, 15
Gonad 38, 157, 159, 161, 162, 165, 179, Hamerkop 8, 440
181, 184, 194, 197, 198, 200, 205, 207- Hawks 11, 112, 149, 150
209, 212, 213, 247, 317, 320, 516, 517, Head 155, 176, 210, 300, 301, 335, 337,
545, 547 341, 347, 360, 365-367, 369, 372, 374,
Gonadal Recrudescence 319, 332 376, 377, 379, 385, 392, 405-408, 413,
Gonadotropin Releasing Hormone 417, 421, 429, 438, 444, 445, 448, 451,
(GnRH) 189, 206, 235, 343 454, 457, 475-477, 479, 480, 486, 487,
Gonadotropin Synthesis 220, 221, 240 490, 492, 493, 504, 505, 507, 532, 533,
Grallina 21, 359, 363, 456, 461, 462 563, 572
Grallinidae 363, 456 Helical Membrane 413, 418, 425, 428,
Granular Body 455, 478, 479, 486, 488 444, 446, 447, 449, 450, 452, 454, 461,
Granular Helix 455, 478-480, 482, 483, 463, 467, 472, 473, 475, 477, 478, 480,
488, 502 481, 485, 486, 488, 492-494, 497, 500,
Granulosa 159, 200, 201, 202, 253-261, 501, 502
263-276, 555-557, 570, 585, 586 Heliornithidae 9, 11, 30
Granulosa Cells 159, 200, 201, 202, 254- Helmet Shrikes 21
261, 263, 264, 267-276, 555-557, 570, Hemiprocnidae 12, 498
585, 586 Himantopus 441, 519
Great Crested Flycatcher 444, 447, 449, Hippolais 355, 356
455, 534 Hirundinidae 23, 358, 363, 449, 450, 455,
Great Tit 232, 252, 342, 346, 457, 486 463, 477
Grebes 8, 10, 32 Hirundo 85, 113, 251, 252, 274, 356, 449,
Greenfinch 356, 357, 455, 464, 467, 526, 463, 477, 517
548 Hoatzin 3, 8, 10, 27, 30, 34
Grosbeaks 25, 523, 547 Homo 64, 280, 305
Ground-rollers 9 Honeyeaters 20, 28
Grouse 7, 28, 116, 132, 238, 525, 549, 550 Honeyguides 8
Growth 112, 158, 162, 164, 179, 193, 197, Hoopoe 9
199, 202-205, 210, 221, 224, 228, 229, Hormonal Cascade 182, 189
232, 234, 240, 242-245, 250-259, 261, Hormone Control 181, 275
262, 266, 268-276, 319, 327, 346, 417, Hormones of the Pars Distalis 197
508, 521, 523, 545, 555, 565, 583, 585, Hornbills 9, 28
587 House Crow 521
Gruidae 9, 11, 13 House Finch 211, 231, 526
Gruiformes 9-11, 15, 30, 32, 33, 356, 358, House Martin 356, 455, 463
359, 362, 418, 438, 498-501 House Sparrows 147, 210, 212, 218, 225,
Gruiforms 2, 9, 10, 11, 15, 353, 440, 443 231, 237, 239, 345, 517, 519, 547, 548
Grus 362, 418, 431, 438-440, 443, 499, 501 Hume’s Ground Jay 16
Guans 7, 132 Hummingbirds 8, 26, 27
Guineafowl 7, 27, 54, 59, 61, 64, 73, 132, Hydatids of Morgagni 165
281, 287, 291-293, 295, 302, 315, 328, Hydrobatidae 11, 13, 15, 358
329, 352, 361, 392, 396-399, 401, 402, Hydroxysteroid 94, 99, 109, 113, 341
404, 405, 421, 433, 448, 451, 497 Hylia 23
#'& Reproductive Biology and Phylogeny of Birds
Hyliota 21, 29 Irenidae 24

Hylophylax 207 Iridoprocne 463, 477
Hypothalamic Nuclei and Tracts 190 Isthmus 162, 166-168, 171, 172, 201, 557
Hypothalamo-hypophysial Portal
System 192 J
Hypothalamo-pituitary Unit 181, 183, Jacamars 8, 28, 34
187, 188 Jacana 362, 418, 442, 448, 500, 501, 509
Hypothalamus 181, 182, 187-190, 192, Jacanidae 14, 441, 442, 533
197, 198, 201, 206, 210, 212, 217, 218, Jackdaw 85, 328, 329, 357, 452, 453, 456
221, 233-235, 270, 276 Japanese Quail 44, 56, 71, 82, 95, 103,
108, 176, 234, 262, 287, 297, 302, 305,
I 307, 311, 340, 511, 534, 551, 555
Ibidorhynchidae 12 Junctional Complexes 68, 70, 77, 93, 110,
Ibisbill 12 279
Ichthyophis 153
Icteridae 25, 358, 364, 449, 455, 467, 486, K
487, 503, 533, 543 Kagu 11
Icterus 364, 486, 487 Kinetics of spermatogenesis 301, 305,
Immune Responses 532 311, 336, 344
Implantation Fossa 287, 291, 300, 350, Kingfishers 9
367, 385, 393, 397, 400, 403, 404, 405, Kinglets 21
407, 411
In Vitro Fertilization (IVF) 553, 562, 572- L
574, 579, 584, 586
Lagorchestes 352, 507
Indian Tree Pie 203
Lampetra 376, 505, 508, 510
Indicatoridae 12
Laniidae 21, 358, 455
Indifferent Gonad 38, 161
Lanioturdus 21
Infundibulum 153, 162, 165-168, 171, 192,
Lanius 355, 357, 451-453, 455, 456, 458,
201, 536, 557, 559-562, 568, 571, 578,
461, 513
580
Lapwings 440
Inhibin 51, 53, 107, 108, 110, 197, 202,
Lari 12
232, 264, 273
Laridae 14, 15, 33, 358, 440, 441
Inner Perivitelline Layer (IPVL) 166, 554
Larus 15, 355, 356, 382, 426, 427, 440, 443
Internal Coincidence Model 211
Leaf Warblers 23
Interphase 281
Interrenal (Adrenal Cortical-like) 165 Least Flycatcher 534
Interstitial Nodules 165 Left Oviduct 122, 150, 162-165, 167, 173,
Interstitial Tissue of the Testis 53, 105, 248, 519, 557
113 Lek 525, 528
Interstitium 53-56, 59, 61, 321, 323, 326, Leydig Cell 53, 55, 59, 60, 62, 102, 103,
341 105, 112, 319, 326
Intracellular Lipid 175 Life History Stage 182, 184-187
Intracytoplasmic Sperm Injection 553, Limpkin 9, 11
579, 583 Linkage Disequilibrium Hypothesis 540
Intraepithelial Lymphocytes 68, 70, 80, Linnet 356, 451, 457
87, 95, 101, 104 Lissamphibia 350, 352, 354
Intravascular Perfusion Fixation 93 Logrunners 20
Intromittent Organ 115, 139, 145, 545 Lonchura 299, 300, 340, 364, 450, 452, 490,
Ioras 20 492, 493, 507, 510, 544
Index #''
Longitudinal Columns 354, 365, 369, 370, Matrix Metalloprotease MMP2 159
371 Maturation/M Phase-promoting Factor
Long-tailed Tits 23 (MPF) 575
Loons 8, 10 Mature Follicular Oocyte 149
Lophonetta 8 Median Eminence 188-194, 198, 205, 215,
Lovebird 295, 299, 300, 347, 362, 428, 217, 218, 226, 228, 232
434, 490, 492, 511 Megaluridae 23
Loxia 207, 218, 231, 251, 271 Megapodes 7, 26, 116, 131, 146
Luscinia 357, 463, 465, 467 Megapodiidae 7, 26, 118-120, 126, 131,
Luteinizing (Leutenising) Hormone (LH) 136, 137
109, 193, 194, 196-202, 204, 205, 208, Melampitta 21
211, 215, 220-222, 227, 235, 238, 239, Melanerpes 361, 423-425, 447, 454, 500
241, 242, 256-259, 261, 263, 264, 266, Melanocharitidae 20
269, 270, 277, 316-318, 327, 341, 343, Melatonin 202-204, 210, 215, 222-227,
346 230, 232, 233, 236, 237, 240
Lymphoid Enhancer Factor 1 159 Meleagrididae 7, 15
Meleagris 38, 54, 88, 101, 106, 107, 116,
M 193, 226, 254, 264, 281, 283-286, 288,
Macgregor’s Bird of Paradise 17 290, 294, 296, 330, 334, 346, 355, 361,
Macgregoria 17, 27 391, 393, 396, 398, 401, 402, 448, 510,
Macropus 107, 285, 340 538, 582
Macrosphenus 23 Meliphagidae 17, 20, 27, 28, 145
Magellanic Plover 13 Meliphagoidea 17, 19, 20
Magnum 162, 163, 166-168, 171, 172, 201, Melopsittacus 49, 85, 285, 339, 362, 428-
557, 571-573 430, 432, 434, 448, 499, 506, 509, 522,
Magpie Goose 7, 131 551
Malaconotinae 20, 21 Melospiza 198, 225, 238, 241, 251
Male Germ Cells 40 Menuroidea 19, 20
Mallard 115, 127, 128, 131, 132, 199, 208, Meropidae 9, 12, 24
280, 293, 302, 306, 323, 328, 329, 341, Mesenchymal Sheath 157, 158, 164
359, 361, 405, 406, 409, 410, 433 Mesites 10, 11, 32
Maluridae 20 Mesitornithidae 9-11, 32, 410
Malurus 87, 136, 147, 519, 527, 543, 550 Mesonephric Tubules 150, 159, 165
Mammals 37, 40, 41, 44, 46, 49, 51, 53, Mesonephros 97, 104, 150, 152, 153, 157,
54, 56, 59, 61, 63, 64, 66, 68, 70-73, 77, 161, 162, 165, 166, 178
81-83, 85, 86, 94, 98-100, 109, 158, 159, Mesorchium 37
161, 165, 198-200, 203, 209, 210, 212, Mesotocin 192, 193
223, 228, 235, 236, 244, 247, 249, 255, Metanephros 161, 162
256, 265, 267, 280-282, 285, 289, 291, Metaves 4, 9, 10, 410, 411, 423, 497-500
292, 297, 300, 301, 302, 303, 305, 306, Microrecanalization 83, 101, 105
312-315, 327, 336, 341, 342, 344, 350, Midpiece 282, 291, 293, 296, 297, 335,
352, 353, 372, 436, 508, 514, 516, 536, 351, 352, 360, 364-367, 369-374, 376,
546, 547, 548, 550, 551, 553, 555, 557, 377, 379-381, 383-389, 391-393, 395-
562, 568, 573, 574, 576, 578, 586 397, 399-405, 407-409, 411, 413-415,
Manakins 20, 445 417-421, 423, 425, 426, 428, 429, 431-
Manchette 287, 289-293, 297, 300, 344, 434, 436, 438-440, 442, 443, 445-447,
345, 415, 417, 418, 428, 492, 509 451, 452, 454-461, 463, 467-469, 472-
Mating Systems 126, 135, 141, 142, 346, 478, 480, 483, 484, 486-488, 490-494,
527, 528, 536, 538, 549, 550 496-502, 504, 532-534
Mimic Thrushes 23 Myofibroblast Cells 56, 57

Mimidae 3, 23, 24, 358, 467 Myrmecocichla 359, 363, 447, 449-452,
Mitochondrion/Mitochondria 50, 56, 59, 456, 467, 468, 472-474, 477, 479, 483,
60, 68, 70, 77-80, 91, 94, 95, 217, 263, 502
287, 289, 293, 295-297, 299, 307, 309,
310, 323, 325-329, 332, 351, 352, 354, N
365, 367, 369-372, 374, 376, 378-381, Neck 167, 168, 170, 171, 292, 300, 301,
383, 385-387, 389, 391-393, 396, 397, 335, 354, 367, 369, 372, 373, 377, 379,
399-401, 403, 404, 407, 408, 410, 411, 381, 389, 391, 393, 396, 397, 400, 407,
413, 414, 417, 418, 420, 421, 423, 425, 446, 451, 475, 476, 478, 504
426, 428, 431-434, 436, 438-440, 442, Neoaves 4, 8, 9, 120, 133-136, 383, 410,
443, 446, 447, 451, 452, 454, 455, 458- 423, 497-500
461, 463, 469, 470, 472, 473, 475, 477, Neognathae 4, 29, 117, 132, 358, 383,
478, 479, 480, 483, 486, 488, 493, 494, 494, 497, 498
497, 500, 501, 502, 532 Neornithes 3, 27, 116, 145, 358, 496
Mitochondrial Genomes 4, 32, 507 Nephrostome-like Depressions 150, 153
Mitochondrial Helix 450-452, 454-456, Neural Pathways 182-184
463, 472-480, 482-485, 488, 490, 493, Neuroendocrine Secretions 183, 184,
502 188
Mitochondrial Sheath 297, 299, 389, 407, Neurohormones 189, 192
408, 454, 455, 466, 478, 488, 490, 491 Neurohypophysis 194
Mockingbirds 3 Neuromodulator Mechanisms 184
Molecular Data 1, 10, 32, 497 Neurosecretions 182, 190
Molluscs 354 New World Blackbirds 25, 533
Molothrus 364, 449, 486 New World Sparrows 25
Momotidae 9, 12 New World Vultures 3, 9, 27, 35
Monarch Flycatchers 21 New Zealand Wattlebirds 20
Monarchidae 21 Nine-primaried Oscines 24, 25, 35
Monogamy 129, 138, 142, 312, 318, 340, Noduli Epididymidis 98, 103
519, 527, 528, 530, 536, 546 Non-ciliated Cell Types 72
Monotremes 350, 352 Non-ciliated Cells 66, 73, 74, 77, 83, 89,
Motacillidae 24 99, 163, 328, 331-333
Motmots 9 Non-monophyly 2, 6, 14
Moustached Warbler 451, 478, 543, 544 Non-passerine Birds 2, 15, 16, 17, 85, 87,
Mucin 171, 175 89, 135, 138, 140, 295, 299, 300, 352,
Mucous Part of the Magnum 171 355, 358, 365, 371, 374, 381, 383, 391,
Müllerian (Paramesonephric) Duct 150, 392, 399, 412, 418-420, 423, 428, 431,
153-156, 158, 159, 161, 162, 164, 175- 433, 435, 436, 440, 444, 445, 446, 448,
179 451, 456, 475, 486, 500, 516, 533, 534
Müllerian Duct Aplasia 157 Nuclear Fossa 360, 367, 383-385, 392,
Müllerian Ridge 150, 157 394, 400, 403, 405, 407, 411, 414, 438,
Multicellular Glands 168, 171 446, 447, 463, 467, 469, 478, 480, 482,
Mus 82, 311 486
Muscicapa 356, 357, 463, 467 Nucleus 45-48, 60, 63, 70, 75, 77, 79, 80,
Muscicapidae 23, 24, 358, 363, 449, 463, 91, 93, 95, 96, 119, 191, 203, 210, 217,
467, 472 218, 261, 276, 280-287, 289, 291-293,
Muscicapoidea 19, 23 295, 297, 299, 300, 307, 309, 310, 323,
Musophagidae 9, 10, 14, 15 324, 326, 328, 329, 332, 342, 343, 350,
Myiarchus 363, 444, 445, 447, 449, 534 355, 360, 365-369, 372-374, 376, 377,
Index $
379, 381, 383-385, 388-397, 399-405, Overcrowding 319

407, 410, 411, 414, 415, 417-423, 425, Oviduct 66, 101, 122, 146, 149, 150, 153,
426, 428, 429, 431-434, 436, 438-440, 155, 158, 161-168, 172, 173, 175, 176,
442-450, 452, 454, 455, 458, 459, 460, 178, 179, 193, 201, 233, 237, 239, 248,
461, 463, 467-469, 471, 472-476, 478, 265, 293, 516, 518, 519, 536, 546, 550,
480, 482-486, 488, 490, 491, 493, 494, 557, 560, 561, 571, 579, 581, 582, 587
497, 500, 501, 502, 532-534, 541, 554, Oviposition 166, 183, 188, 193, 201, 262,
570, 572-576, 583, 584 265, 270, 557, 559, 560, 565, 580
Numida 54, 101, 281, 330, 334, 346, 361, Ovulation 149, 166, 171, 184, 197, 201,
391, 393, 397, 398, 401, 402, 448 238, 243, 244, 253, 254, 259, 261-265,
Numididae 7, 120, 126, 132, 136, 143, 401 267, 270, 272, 554, 557, 560, 561, 573,
Nuthatch 23, 356, 455 580
Nyctibiidae 14, 32 Ovulatory Cycle 166, 167, 238, 255, 264,
Nymphicus 362, 428, 434, 436, 448, 499 271, 557, 560
Ovum 149, 166-168, 171, 201, 536, 539,
O 540, 553-555, 557, 559, 561, 562, 565,
Ocyphaps 354, 359, 362, 418-421, 423 568-581, 583-586
Odontophoridae 7, 120, 126, 132, 136, Owlet-nightjars 8, 10, 415
143 Owls 8, 27, 202, 272
Old World Flycatchers 23, 24 Oxpeckers 24
Old World Sparrows 24 Oxyruncus 20
Opisthocomidae 3, 9, 10, 410 Oystercatchers 13
Opsin 210, 238
P
Oriolus 355, 451, 456, 461
Orthonychidae 20 Palaeognathae 4, 282, 358
Oscines 17, 19, 20, 24, 25, 28, 35, 358, 363, Pandionidae 11
423, 425, 445-447, 449, 451, 454, 457, Pangolin 350, 560
458, 467, 494, 500, 501 Panurus 21, 135, 144, 147
Papilla Ductus Deferentis 41, 85, 328
Osprey 11
Papillae 134
Ostium 153, 155, 157, 161, 162, 166-168,
Paradidymidis 40, 97
173, 201
Paradiseidae 17, 20, 21
Ostrich 6, 27, 40, 41, 44-46, 54, 56, 59, 61,
Paramythia 20
63, 64, 66, 68, 70-73, 75, 80, 87, 89, 93,
Paraphyly 6, 14, 32, 383, 415, 496
96, 98, 101, 103, 109, 112, 116, 117, 124,
Parasitemia 531
125, 193, 199, 225, 235, 285, 287, 289,
Pardalotes 20
292, 293, 295, 297, 301, 311, 314, 334,
Pardalotidae 20
341, 344, 345, 354-356, 360, 361, 365,
Paridae 17, 21, 29, 30, 358, 363, 449, 450,
366, 368, 371-374, 376, 377, 381, 448,
457, 467, 485
497, 509, 510, 550, 571, 584
Parrots 8, 13, 133, 136, 138, 147, 339, 506
Otididae 11, 13, 15, 26, 33 Pars Cranialis Uteri 172, 173
Outer Perivitelline Layer (OPVL) 166, Pars Distalis 190, 192, 194, 197, 201, 202
557 Pars Nervosa 183, 188, 190, 192-194
Ovarian Dynamics 244 Pars Recta Ductus Deferentis 39, 41, 85
Ovarian Growth Factors 202 Pars Translucens 167, 171
Ovary 111, 149, 159, 161, 162, 165, 167, Parulidae 25, 32, 358, 363, 467
181, 200, 201, 229, 241, 243-250, 252- Parus 17, 232, 252, 363, 447, 449, 450,
256, 258, 259, 266-268, 270-277, 516, 457, 485, 486
519, 555, 581, 583 Passer 86, 103, 203, 219, 224, 225, 228,
Ovenbirds 19, 445 231, 236, 237, 239, 283, 298-300, 335,
338, 356, 357, 364, 449, 450, 452, 455, Persistent Right Oviduct 164, 165, 178
457, 464, 467, 471, 479-481, 484, 494, Persisting Right Genital Organs 149
504, 505, 517, 521, 544, 547, 548, 550 Petrels 11, 13
Passerculus 523, 549 Petrochelidon 463, 477, 523
Passeri (Oscines) 494, 501 Petroicidae 21
Passerida 18-22, 24, 28, 29, 356-359, 363, Phaethontidae 8-11, 13, 31, 410
392, 425, 447, 448, 451, 458, 461, 463- Phalacrocoracidae 8, 11
465, 481, 494, 500-502, 532 Phalaropes 137, 441
Passeridae 24, 364, 449, 450, 455, 457, Phalloid Organ 136, 144, 145, 147, 148
463, 467, 479, 484 Phallus 41, 98, 99, 108, 115-128, 130-134,
Passeridan Sperm Type 463, 467 136, 142, 143, 146, 147
Passeriformes 3, 8, 15, 16, 28, 30, 35, 218, Phasianidae 7, 15, 120, 132, 136, 143, 358,
236, 355-359, 362, 427, 444, 458, 494, 383, 387, 396, 400
498-502, 506, 507, 532, 547, 569 Pheasants 7, 31, 116, 132, 237
Passerimorphae 500 Pheucticus 523
Passerines 1-4, 15-17, 26, 27, 28, 33, 37, Philetairus 364, 447, 449-452, 455, 456,
61, 85-87, 89, 113, 135, 138, 140, 145, 473, 479, 482, 483, 493, 502
147, 148, 238, 261, 282, 283, 287, 289,
Philomachus 357, 412, 441, 448, 533
291, 292, 295, 297, 299, 300, 313, 314,
Phoenicopteridae 9, 10, 32, 410
328, 335, 337, 347, 355, 358, 374, 377,
Phoenicopteriformes 8
381, 382, 391, 399, 413, 418, 423, 428,
Phoeniculidae 9, 12, 15
431, 433, 436, 441, 444-448, 451, 456,
Photoperiodism 203, 205, 224, 231, 236,
458, 461, 475, 480, 486, 490, 492-494,
237, 250, 270, 276, 316, 317, 337, 341,
496, 499-501, 510, 516, 519, 522, 532-
344
535, 540, 544, 547, 549, 551
Photosensitivity 203, 205, 207, 209, 210,
Passeroidea 19, 24, 363, 455, 478, 479
214, 224, 225, 227, 235
Pathological Polyspermy 168, 553, 559,
Phylloscopidae 23
561, 570, 571
Phylloscopus 355-357, 457, 463, 465, 478
Pavo 228, 520, 544
Phylogenetic Analyses 2, 120
Peafowl 520, 544, 569
Pedionomidae 9 Phylogeny of Spermatozoa 493
Pelecanidae 8, 11, 440 Physiological Monospermy 553
Pelecaniformes 8, 10, 11, 15, 31, 34, 362, Physiological Polyspermy 553, 554, 577
440 Pica 357, 452, 453, 455, 456, 521
Pelecanoididae 11, 13, 15 Picathartidae 21
Pelicans 8, 11, 15, 440 Picidae 12, 14, 15, 35, 358
Penduline Tits 21 Piciformes 8, 10, 12, 15, 26, 30, 32, 34,
Penguins 8, 10, 26, 32, 246, 250, 259, 276 355, 357, 358, 361, 423, 448, 498-500
Peptides 181, 189, 190, 192, 193, 197, 216, Picoides 15, 34, 412
218, 220, 222, 223, 235, 236, 240 Piculus 15
Perforatorium 287, 289, 293, 295, 299, Picus 15, 355, 423
334, 350, 354, 360, 367, 369, 371-374, Pigeons 8, 31, 38, 111, 193, 247, 377
376, 377, 379, 381, 383-386, 389-392, Pine Grosbeak 493
394, 397, 399-401, 403, 405-407, 410, Pineal Gland (Epiphysis) 202
411, 413, 415-419, 423, 426, 428, 429, Pinicola 493
431-434, 436, 438-440, 443, 450, 478, Pipilo 364, 447
480, 494, 496-498, 500, 503, 508, 533 Pipits 24
Perisoreus 357, 449, 452, 453, 456, 459 Pipridae 17, 20, 358, 445, 446
Peritoneal Funnels 150 Piranga 364, 447, 449, 450, 478
Index $!
Pittas 19 Procellariiformes 8, 10, 11

Pittidae 19 Proctodeum 98, 120, 122, 124, 127, 132,
Pituitary Gonadotropins 243 133
Plains Wanderer 9 productive demands on testis 312
Platycercus 359, 362, 428, 434 Progesterone 51, 200, 201, 229, 257-259,
Plectrophenax 25 261-264, 273, 274, 276
Pleomorphic Sperm 440 Prolactin 194, 197, 204, 205, 208, 211, 213,
Plesiomorphy 2, 365 214, 220, 227-230, 234, 251, 545
Plethornithes 8 Prolactin Releasing Hormone 213
Ploceidae 358, 449, 450, 467, 479, 482-485 Promerops 24
Ploceus 203, 224, 238, 364, 450, 484, 485 Promiscuity 135, 142, 147, 303, 313, 315,
Plovers 12, 13, 440 517, 519, 521, 527, 528, 536-539, 543
Plumage Ornaments 518 546, 550
Pluvianellus 13 Pronephric Tubules 153
Pluvianus 13 Pronephros 150, 153, 155, 156
Podicipediformes 8 Pronephrostomes 153
Poephila 251, 275, 299, 452, 453, 493 Pronuclei 261, 553, 572-576, 579
Polioptilidae 23 Pro-opiomelanocortin Derived Peptides
Polyandry 313, 514, 527, 528, 549 197
Polygynandrous 145, 148, 504, 527, 544 Protein 40, 51, 75, 77, 82, 83, 94, 99, 100,
Polygyny 142, 312, 313, 318, 346, 525, 106, 108, 109, 111, 113, 158, 166, 171,
528, 530, 550 176, 178, 179, 197, 200, 210, 220, 224,
Polypeptides 110, 197 232-234, 242, 254-259, 261-263, 267-
Pomatostomidae 20 270, 272, 273, 275, 292, 315, 336, 342,
Postcopulatory Female Choice 538 343, 377, 554, 555, 557, 559, 565, 567,
Pratincoles 13 568, 570, 571, 580-587
Premeiotic Protonataria 363
Interphase 281 Proximal Centriole 360, 367, 369, 372,
Preleptotene 281 373, 377, 379, 380, 385-387, 389, 391,
Leptotene 281, 303, 308 393, 395, 397, 399, 400, 404, 407, 408,
Zygotene 281, 308 411, 415, 419,421, 423, 431-434, 436,
Pachytene 281, 303, 308 439, 442, 472, 480, 483, 494, 502
Diplotene 249, 281, 308 Prunella 134, 144-146, 335, 364, 493, 504,
Diakinesis 281 513, 527, 544, 548
Preovulatory Follicle 246, 254, 263, 264, Prunellidae 24, 364, 493
276, 277 Pseudophallus 116
Primary Folds 162, 163, 175 Pseudostratified Columnar Epithelium
Primary Follicle 252, 254, 255 168, 171, 172, 175
Primary Spermatocyte 46, 49, 281, 303, Psittacidae 8, 13-15
306, 308, 311, 323, 344 Psittacidae/ Cacatuidae 14
Principal Piece 295, 350, 354, 360, 365, Psittaciformes 8, 26, 27, 120, 339, 357,
366, 369, 370-374, 379, 381, 383-385, 359, 362, 418, 428, 448, 494, 498, 499,
387-389, 391, 393, 396, 400, 405, 407- 502, 506
409, 413, 414, 418, 423, 425, 428, 433, Psittacus 357, 412, 428, 448
442, 445, 446, 451, 452, 487, 488, 500, Psophiidae 9, 11
532 Pteroclidae 9, 410
Prionopidae 21 Pterocliformes 8
Proacrosomal Granule 283, 285 Pteroicnemia 360
Procellariidae 11, 13, 15 Ptilonorhynchidae 20
Puffbacks 21 Remizidae 21
Puffbirds 8 Reproductive Cycle (see also Testicular
Puffins 137, 440, 441 Cycles) 40, 51, 86, 101, 214, 265, 270,
Pycnonotidae 23 271, 315-321, 327, 328, 329, 333, 334,
Pycnonotus 522 338, 551
Pyrrhula 451, 457, 490, 493, 504, 533, 544 Rete Testis (RT) 44, 54, 61, 63-68, 71, 84,
101-109, 111-113, 305, 328, 329, 330,
Q 333, 335
Quelea 328, 340, 341, 364, 450, 484, 522 Retronuclear Body 354
Quiscalus 364, 481, 486, 487 Retzius 349, 355, 356, 371, 382, 405, 411,
413, 417, 419, 421, 423, 425, 426, 428,
R 438, 440, 441, 443, 448, 452, 453, 456,
Rails 9, 11, 32 458, 459, 461, 463-465, 467, 475, 480,
Rallidae 9, 11, 13, 32, 356, 358, 438 501, 503, 508, 532, 549
Ramphastidae 2, 12-15, 33, 358 Rhabdornis 24
Ramus ureterodeferentialis cranialis 41 Rhea 6, 103, 125, 126, 283, 287, 292, 293,
Ratites 4, 6, 66, 99, 115, 117, 146, 193, 297, 343, 351, 360, 361, 364, 365, 371-
297, 352, 354, 355, 371, 374, 381, 383, 374, 376, 377, 381, 496, 508
387, 393, 405, 418, 428, 431, 436, 443, Rheidae 6, 125, 360
494, 496, 509 Rhinocryptidae 17, 19
Rattus 64, 280, 301, 305, 311, 313 Rhipiduridae 21
Receptaculum Ductus Deferentis 41, 85, Rhodopsin 202, 203, 210, 229
89, 90, 328 Rhynochetidae 9, 11, 410
Recessus Uterinus 172, 173 Ring Dove 210
Recrudescence 68, 231, 243-245, 251, 254, Riparia 449, 463, 477, 520
317, 319, 320, 327, 330, 332, 521-523, Rockfowl 21
532 Rollers 9, 31
Recurvirostra 519 Rooster 38, 41, 44, 46, 49, 51, 54, 56, 61,
Recurvirostridae 12, 15, 358, 440 64, 66, 70, 73, 75, 80, 82, 83, 87, 90, 93-
Red Bishop 364, 450, 484, 485 96, 98, 99, 102, 104, 107-109, 111, 134,
Red-backed Shrike 355, 357, 452, 453, 280-283, 285, 287, 293, 295, 300-302,
455, 456, 461, 513, 533 312, 314, 315, 328, 329, 337, 341, 342,
Red-billed Quelea 364, 450, 484, 522 347, 352, 355, 356, 361, 382, 383, 385,
Red-eyed Vireo 449, 457, 461, 526, 536 396-401, 403-405, 418, 421, 451, 508,
Red-legged Partridge 209, 226 511
Red-vented Bulbul 522, 547 Rough Endoplasmic Reticulum 58-60, 62,
Red-winged Blackbirds 147, 520, 550 70, 323
Reed Bunting 145, 533, 534, 545 Rudimentary Right Oviduct 164
Regressed Testes 38, 51, 63, 323 Rudiments of Mesonephros 165
Regression of the Male Duct 158 Rudiments of the Wolffian Ducts 162
Regression of the Right Müllerian Duct Ruff 357, 412, 441, 448, 533
150, 158, 159 Rufous-collared Sparrows 206, 207, 218
Regulation of GnIH Expression 222
Regulidae 21 S
Relationships Between Neoavian Orders Sagittariidae 11
9 Sandgrouse 8, 27
Relationships Within Neoavian Orders Sandpipers 441, 545
11 Sapayoa 17, 19, 29
Relationships Within Passerida 21 Sarcopterygian Fish 350
Index $#
Savannah Sparrows 523, 524, 526, 549 Sexually Transmitted Diseases 124, 139,
Saxicola 207, 228 146
Scolopaci 12 Sharpbill 20
Scolopacidae 14, 358, 441, 501, 530, 535 Sharp-tailed Grouse 525, 549
Scolopax 357, 382, 441, 443 Shearwaters 11, 13
Scopidae 8, 440 Sheathbills 13
Screamers 7, 116, 131 Shell 149, 162, 164, 166, 167, 172, 173,
Scrub-birds 20 178, 201, 557
Season 40, 124, 128, 133-135, 138, 141, Shell Gland 162, 164, 166, 167, 172, 173,
147, 158, 162, 166, 184-186, 199, 200, 201, 557
203, 205, 209, 221, 235, 239, 245, 248- Shell Membranes 149, 166, 172
251, 253, 256, 266, 267, 312, 316-318, Shoebill 8, 32, 440
327, 345, 346, 504, 515, 521, 523, 526, Shrikes 21
527, 532, 543 Silky Flycatchers 23
Seasonal Breeding 204, 209, 230, 237, Sitta 356, 363, 455, 456
315, 319, 344 Sittidae 23, 358, 363, 455, 467
Seasonal Dynamics 220 Skuas 26, 440
Seasonally Breeding Species 40 Smith’s Longspur 527
Secondary Folding 162 Smooth Endoplasmic Reticulum 49, 50,
Secondary Spermatocyte 46, 281, 303, 59, 60, 62, 285, 300, 327, 490, 491
308 Snow Buntings 25
Secretary Bird 11 Social Mating System 134, 514
Secretory Cells 89, 168, 170-172, 175, 194 Society Finch 493
Secretory Granules 168, 170, 172, 193, Song Sparrow 198, 207, 218, 238, 241,
194 251
Sedge Warbler 448, 457, 478, 517, 544 Song Thrush 357, 448, 449, 451, 457, 463,
Semen Extender 561, 577 467
Seminal Glomera 135, 314, 434, 519 Songbirds 26, 32, 198, 199, 216, 220, 223-
Seminal Glomus or Sac 85, 86, 89, 96, 225, 252, 549, 550
199, 313, 314, 320, 330, 333 SOX 161
Seminiferous Epithelium 45, 46, 48, 49, Specialized Receptors 182, 184
51, 54, 57, 61, 104, 113, 279-282, 301- Speculanas 8
303, 305-307, 308, 311, 312, 321-324, Sperm 40, 53, 70, 82, 83, 85-87, 90, 99,
334, 336, 337, 340, 342-344 100, 102, 103, 106-110, 113, 115, 116,
Seminiferous Tubule 46-48, 51, 53-57, 59- 122, 124, 131, 135-139, 141-148, 150,
61, 63, 105, 112, 279, 297, 301, 302, 305, 166, 168, 171-177, 225, 295, 300, 303,
306, 321, 323, 325, 335 313-315, 318, 328, 333-337, 339, 342,
Semipalmated Plover 533 343, 345, 347, 349-352, 354, 355, 357,
Seriemas 11, 208, 235, 239, 364 360, 365, 367, 369-372, 374, 376, 377,
Serinus 44, 45, 157, 208, 364 379, 381, 383, 385-387, 389-394, 396-
Serosa 44 405, 407, 408, 410-413, 415, 417-419,
Serotonin 202, 203 421, 423, 425-428, 431, 433, 434, 436,
Sertoli Cell 46-53, 61, 63, 104-107, 111, 438-447, 449-458, 461, 463, 464, 466-
112, 202, 279, 284, 285, 287, 289, 291, 468, 472, 473, 475-481, 483, 485-488,
297, 300, 301, 302, 311, 313, 321, 323- 490, 492, 493, 496-510, 513, 514, 516,
325, 327, 337, 339, 413, 415, 488, 490, 518-521, 525-551, 553, 556-587
Sexual Selection 116, 126, 137, 141, 145, Sperm Competition 85, 109, 110, 135,
176, 335, 539, 540, 544-546, 548, 580, 139, 141-148, 176, 225, 313, 314, 335,
585 342, 343, 451, 490, 504, 513, 514, 516,
520, 526, 527-532, 535, 536, 538, 539, 329, 334-336, 337, 339, 342-346, 349,
541-550, 580 350, 352-354, 360, 361, 365, 366, 369,
Sperm Depletion 109, 342, 520, 527, 530, 371-374, 376-383, 385, 387-389, 391-
544, 548 393, 396-398, 401, 403-405, 407, 408,
Sperm Head 337, 347, 377, 379, 407, 417, 410, 415, 417, 418, 420, 421, 424, 426-
421, 438, 451, 475, 476, 487, 492, 505, 431, 433, 434, 438-444, 447, 451, 452,
532, 563, 572 455, 456, 458, 460, 464, 465, 467-469,
Sperm Longevity 539, 543 470-474, 476-480, 484-487, 492-494,
Sperm Production 40, 82, 85, 87, 109, 496, 498, 500, 502-511, 513, 514, 532-
147, 303, 313, 314, 342, 514, 516, 518- 534, 539, 543, 544, 547, 548, 550, 551,
521, 525-527, 530, 539, 548, 550 576, 581-586.
Sperm Production Rate 40, 313 Spermiation 297, 300, 340, 507
Sperm Size 145, 504, 513, 514, 533-536, Spermiogenesis 111, 282, 283, 285, 289,
539-543, 545, 546 291, 292, 295, 297, 298, 300, 302, 334,
Sperm Storage 102, 113, 136, 141, 146, 336, 338-340, 343-347, 351, 362, 411,
148, 150, 171-177, 313, 335, 347, 392, 418, 426, 442, 450, 454, 455, 479, 488,
434, 493, 504, 514, 516, 536, 537, 539, 490, 503-505, 507-511
543-547, 550, 551, 557-561, 580, 582, Spheniscidae 10, 13, 32
584, 587 Sphenisciformes 8, 10, 26, 362
Sperm Storage Tubules (SST) 557 Sphenodon 350, 352, 353, 354, 381, 436,
Sperm Velocity 536 505, 506
Spermateliosis 111, 282 Sphenodontida 350, 352, 505, 506
Spermatid(s) 46-49, 53, 107, 108, 281- Sphenoeacus 23
302, 307, 308, 311, 334, 336-339, 341, Spinus 357, 427, 463, 464
342, 344-346, 351, 352, 411, 414, 417, Splanchnopleure 157
425, 429, 442, 454, 456, 459, 461-463, Spotted Antbirds 207
477, 490, 505, 507-509, 579 Spotted Sandpiper 441, 549
Spermatocytes 46, 49, 108, 280, 281, 292, Squamates 350, 352, 421
301, 302, 303, 306, 308, 311, 321, 323, SST 172, 175, 493, 536, 538, 557-562
337, 344 Starlings 3, 23, 194, 206, 209, 212-214,
Spermatocytogenesis 279-281, 301, 302, 224-230, 232, 235, 236, 241, 266, 274,
304, 306-313, 321, 323 276, 521, 545
Spermatogenesis 40, 46, 49, 51, 61, 104, Steamer Ducks 8
106, 108, 109, 112, 197, 198, 279, 280- Stem Cell 46, 255, 280, 301, 302, 304, 306,
282, 297, 302, 303, 306, 311, 312, 317, 307, 311, 312, 339, 343
327, 333, 336-338, 340-345, 347, 349, Type A (Ad, Ap1, Ap2) 280, 281, 307
365, 425, 447, 488, 506, 509, 510, 511, Type B 121, 127, 280, 281, 306-308,
521 310, 311, 326
Spermatogenesis, Duration of 311 Steps of Spermiogenesis 283, 298, 299,
Spermatogenic Tissue 531 302
Spermatogonium/ia 46, 47, 48, 49, 50, Stercorariidae 14, 15
51, 53, 108, 279-281, 301, 304, 306-309, Stercorarius 15
310, 311, 321, 323, 336, 337, 340, 341, Steroid Biosynthesis 200
344 Steroids 51, 99, 177, 182, 189, 201, 212,
Spermatozoon/Spermatozoa 38, 44, 46, 213, 224, 229, 231, 234, 236, 237, 239,
49, 61, 66, 70, 72, 75, 76, 82, 83, 85, 86, 256, 272, 341
89, 90, 94, 95, 98, 101-104, 107, 109, Stilts 12, 440, 550
112, 113, 199, 247, 279, 282, 289, 291, Storage 85, 90, 100, 102, 103, 113, 135,
292, 297, 299-301, 312-315, 318, 328, 141, 146, 148, 150, 166, 171-177, 234,
Index $%
313, 335, 336, 347, 392, 434, 493, 504, Sylvioidea 17, 19, 21, 23, 25, 363, 477
514, 516, 536-539, 543-547, 550, 551, Symmetrical Testes 517, 518
553, 557, 558, 559-561, 577, 580-582, Synapomorphies 3, 12, 350, 365, 371,
584, 587 495, 502
Storks 3, 9, 34 Synapomorphy of Aves 360
Storm Petrels 13 Syngamy 569, 573-575
Streptopelia 15, 31, 38, 210, 259, 293, 342,
T
362, 418, 423, 507
Striated Columns 354, 421 Tachycineta 134, 363, 449, 450, 455, 478,
Strigiformes 8, 357, 358, 415, 417, 426, 517, 534, 547
448 Tachyeres 8
Strix 357, 412, 426, 448 Tadorna 355, 405, 425
Struthidea 21 Taeniopygia 225, 230, 251, 262, 299, 313,
Struthio 40, 58, 62, 88, 90, 101, 109, 112, 364, 451-453, 455, 493, 513, 544, 549,
124, 193, 225, 285, 295, 322, 341, 345, 581
356, 357, 360, 361, 364-366, 368, 383, Tail 285, 289, 291, 300, 343, 365, 366, 369-
448, 451, 496, 509, 510, 550 371, 374, 376, 401, 403, 407-409, 419,
Struthionidae 360 425, 429, 433, 445, 451, 456, 461, 463,
Struthioniformes 6, 116, 297, 356, 360, 466, 469, 471, 477, 480, 493, 507, 508,
361, 379, 381, 448, 502 518, 532, 534, 541
Sturnella 487, 533 Tanagers 25
Sturnidae 3, 23, 24, 358, 363, 450, 455, Tapaculos 19
463, 473 Terminal Segment 62, 63, 105, 111
Sturnus 73, 102, 194, 206, 224-230, 235, Terns 252, 440
238, 241, 271, 328, 357, 363, 449, 450, Testicular Asymmetry 103, 105, 517-519,
452, 456, 463, 465, 473, 475, 476, 521, 523, 543, 546, 547, 549
545 Testicular Capsule 40, 44-46, 65, 104, 321,
Subacrosomal Cone 350, 360, 431 323
Subacrosomal Space 293, 295, 350, 360, Testicular Color 38
367, 376, 383-385, 392, 400, 403, 407, Testicular Cycles 241, 279
Acceleration (Accelerative) Phase
410, 415, 416, 419, 423, 429, 438, 496,
320, 327, 333
497
Active Secretory Phase 320
Suboscines 17, 19, 29, 30, 358, 362, 444,
Culmination Phase 320, 327
445, 447-449, 451, 458, 501, 502
Presecretion Phase 320, 333
Sugarbirds 24
Progressive Phase 320, 333
Sulidae 8, 11
Reconstruction (Reconstructive,
Sunbirds 24
Regeneration) Phase 320, 329
Sunbittern 11
Regressive Phase 320, 329, 526
Superb Fairy-wren 87, 527
Reproductive Phase 320, 327, 328, 333
Supernumerary Male Pronuclei 573, 574
Testicular Spermatozoa 44, 314, 315, 452
Supernumerary Testes 516, 547
Testiculares Accessoria 41
Surface Mucosa 168
Testis 37-41, 43-46, 51-54, 56, 61, 63-68,
Swallow(s) 20, 21, 23, 146, 517, 520, 523,
71, 82, 84-87, 89, 96, 97, 101-113, 145,
532, 545
159, 161, 177, 181, 197, 199, 206, 221,
Swifts 8, 12, 27, 38 225, 226, 229, 233, 234, 247, 279, 303,
Sylvia 23, 230, 356, 457, 478 305, 306, 311, 313, 314, 319-330, 333,
Sylvietta 23 334-342, 344-347, 452, 462, 513-527,
Sylviidae 23, 358, 457, 463, 477 529-532, 535, 536, 541-543, 545-550
Testis Size 38, 40, 109, 141, 145, 199, 212, Troglodytidae 23, 26, 358, 363, 455, 467,
338, 342, 513, 516-518, 521-523, 525- 478
527, 529-532, 535 , 542, 543, 545, 546, Trogon 426, 427
548, 549, 550 Trogonidae 10, 14, 30, 32, 358
Testosterone 51, 198-201, 221, 228, 239, Trogoniformes 8, 28, 33, 358, 405, 426,
262, 263, 317, 318, 327, 338, 342, 343, 427
346, 516, 529, 532, 546, 549, 550, 555, Trogons 8, 10, 30, 32
585 Tropicbirds 8, 11, 31
Tetrao 357, 360, 383 Trumpeters 9, 11
Tetraonidae 7, 15, 358 Tubal Ridge 153
Tetrapod(s) 350, 352, 354, 436, 506 Tubenoses 8
Thamnophilidae 17, 19, 30 Tubular Glands 162, 166, 168, 171, 172,
Thermotolerance 40 175
Thornbills 20 Tubular Neck Region 167, 168
Thraupidae 25, 358, 364, 449, 450, 467, Tubular Shell Gland 167, 172, 173
478 Tubuli 63, 97, 103, 105
Thrushes 19, 23, 445 Tubulus Rectus 63
Thryothorus 363, 380, 456 Tufted Titmouse 363, 449, 450, 485
Thyroid Hormones 204, 213, 215, 262, Tunica 44, 45, 175
276, 345 Tunica Albuginea 44
Thyroid-stimulating Hormone (TSH) Tunica Muscularis 175
197 Turacos 9, 10, 34
Timaliidae 23, 24 Turdidae 23, 24, 358, 363, 449, 450, 457,
Tinamidae 123, 357, 360, 377 467
Tinamiformes 6, 116, 147, 361, 377, 379, Turdus 165, 357, 363, 447-452, 454, 456,
381 457, 463, 464, 467-472, 477, 480, 493,
Tinamous 4, 6, 26, 116, 117, 122-124, 141, 522, 534, 549
142, 147, 377, 418, 494, 509 Turnicidae 9, 11, 15
Tip Granule 383, 385, 387, 391, 393, 401, Tympanuchus 525
405, 494, 497 Tyranni 19, 20, 358, 444, 445, 449, 501
Titmice 21, 29, 33, 229 Tyrannida 444
Tityras 20 Tyrannidae 17, 20, 358, 427, 444-446, 455
Tityridae 20 Tyrannus 362, 427, 444-447, 449, 456, 501,
Todidae 9, 12 502
Todies 9 Tyrant Flycatchers 20, 445, 446
Totanus 357, 441
Toucans 13, 26 U
Tragopan 361, 400, 510 Undulating Membrane 380, 425, 445-447,
Transferrin 53, 113 454, 488, 533
Transport 61, 68, 71, 80, 82, 85, 93, 103, Unilateral Development of Female
106, 111, 150, 166, 176, 192, 206, 247, Genital Organs 149
256, 257, 261, 313, 315, 335, 337, 503, Unit 61, 63, 64, 71, 72, 82, 83, 85, 87, 90-
553, 557, 559, 560, 580, 581, 585 92, 94, 95, 100, 181, 183, 187, 188, 315,
Tree Swallow 134, 463, 517, 518, 547 328-330, 333
Treecreeper(s) 20, 23, 455, 478 Upper Neck Region 168
Treeswifts 12 Upupidae 9, 12, 15
Tringa 357, 382, 441, 443 Ureterodeferentiales Caudales 41
Trochilidae 14, 26, 358, 498 Uria 356, 382, 440, 504
Troglodytes 363, 455, 478, 479, 521 Urodeles 354
Index $'
Urodeum 41, 85, 120, 175, 328 White-rumped Mannikin 493

Uterine Transudate 166 White-rumped Munia 493
Utero-vaginal Junction (UVJ) 172, 557 White-winged Chough 21
Uterus 162, 166, 167, 172, 173, 175, 201, White-winged Fairy-wren 519
557, 559, 574 Wild Birds 40, 68, 102, 115, 127, 150, 165,
244, 249, 318, 319, 335, 519
V Willow Warbler 457, 478
Vaginal Sphincter 175 Winter Wren 521
Vanellus 355, 357, 382, 440, 443 Wolffian Duct(s) 85, 94, 97, 99, 104, 109,
Vangas 21 150, 153, 155, 157, 162, 163, 165, 166,
Vangidae 21 176, 177, 315, 337, 342
Vasculosa 44, 45 Wolffian Duct Proteins 94, 99, 109, 315,
Vasectomy 83, 104, 105 342
Vasoactive Intestinal Polypeptide 213, Woodhewers 445, 446
227, 230 Woodhoopoes 9
Veniliornis 15 Woodpeckers 8, 28, 33, 34
Vimentin 72 Wood-swallows 20
Violet-green Swallow 363, 449, 450, 455, Wren 17, 20, 23, 87, 134, 363, 380, 381,
472, 534 455, 456, 478, 479, 519, 521, 527
Vireo 17, 363, 427, 447-449, 451, 454, 457,
458, 461, 463, 526, 536 X
Vireonidae 358, 363, 427, 457
Xenopus 555, 575, 583, 587
Vitellogenesis 200
Vitellogenin (VTG) 257
Y
VTG/VLDL Receptor 257
Vultur 358 Yellow Hammer 457, 490
Yellow Warbler 391, 513, 534
W Yuhina (Erpornis) 17
Waders 530, 533, 540
Warblers 17, 23, 25, 518, 546 Z
Waterfowl 31, 32, 34, 116, 131, 145, 261, Zebra Finch 193, 230, 251, 275, 299, 300,
527, 528, 545 313, 335, 364, 451-453, 493, 504, 513,
Wave of the Seminiferous Epithelium 519, 534, 536, 544, 569, 581
305, 343 Zona Pellucida (ZP) 555
Waxwings 23 Zonotrichia 187, 191, 195-199, 201, 206-
Weight of the Egg 149, 259 208, 210-212, 215, 217, 218, 225, 228,
Western Meadowlark 487, 533 229, 232-234, 236, 240-242, 250, 256,
White-bellied Yuhina 17 258, 274, 319, 337, 340, 342, 347, 364,
White-crowned Sparrow 187, 191, 194, 447
195, 217, 218, 221, 225, 228, 229, 233, Zosteropidae 23
234, 240-242, 274, 319, 342 Zosterops 23

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ISBN (Series) 978-1-57808-271-1

© 2007, Copyright reserved

All rights reserved. No part of this publication may be reproduced, stored in

Published by Science Publishers, Enfield, NH, USA

‘Natural History of Selborne’, and my good fortune in being joined by a most

Preface to this Volume

contribution of any magnitude by the editor. Former workers recognized the

Preface to the Series—Barrie G. M. Jamieson v

1. Classification and Phylogeny of Birds 1

About the Volume

About the Series and Volume Editor

4869 Pepperwood Way, San Jose, CA 95124; E-mail: jharshman@pacbell.net

those questions (Sheldon and Bledsoe 1993), leading another pair of

groups do have clear morphological synapomorphies, and these can be easy

1.2.1 Basal Relationships

Fig. 1.1 Relationships among non-passerine families, so far as they can be

Neognathae. Palaeognathae includes the ratites and tinamous. Neognathae is

Fig. 1.2 Relationships among non-passerine families, continued. Coronaves only.

sister groups to the remaining anatids), the remaining relationships differ

The monophyly of both Gruiformes and Charadriiformes is falsified by the

1.2.4.3 Relationships within neoavian orders

relationships of families within Charadriiformes. Paton et al. (2003) discuss

and avocets) and Haematopodidae (oystercatchers) (Ericson et al. 2003; Fain

Table 1.1 References to studies of phylogeny within individual neoavian families

Table 1.1 Contd. ...

1.2.4.6 Monophyly of neoavian genera

1.2.5 Classification of Non-passerines

1.3.1 Monophyly of Passeriformes

1.3.2 Monophyly of Passerine Families and Genera

member of Corvidae and thus of Corvoidea, is instead a member of Paridae (in

1.3.3 Basal Relationships within Passerines

1.3.4 Relationships within New World Suboscines

1.3.5 Relationships within the Corvidan Grade

shrikes), Dryoscopus (puffbacks), Batis (batises), Lanioturdus (chatshrike),

1.3.6 Relationships within Passerida

Fringillidae (finches), Emberizidae (buntings and New World sparrows),

1.6 LITERATURE CITED

Armstrong, M. H., Braun, E. L. and Kimball, R. T. 2001. Phylogenetic utility of avian

Livezey, B. C. 1998. A phylogenetic analysis of the Gruiformes (Aves) based on

Moyle, R. G. 2005. Phylogeny and biogeographical history of Trogoniformes, a

Short, L. L. and Horne, J. F. M. 2002. Family Capitonidae (barbets). Pp. 140-173. In J.

Whittingham, L. A., Sheldon, F. H. and Emlen, S. T. 2000. Molecular phylogeny of

2.1.1 Testis Shape, Asymmetry and Size

Anatomy of the Testis and Male Reproductive Tract !'

2.1.2 Body Temperature and Testis Function in Birds

2.1.3 Consistency and Structure of the Testis

2.1.4 The Excurrent Ducts of the Testis

2.1.5 Blood Supply to the Reproductive Organs

2.2 THE TESTIS

2.2.2 Seminiferous Tubules

2.2.3 Sertoli Cells

Fig. 2.6 Coturnix japonica. A. A survey photomicrograph of the seminiferous

and hence epithelium (Aire, T. A. personal observations). The cytoplasm

Fig. 2.9. A. A diagrammatic representation of Sertoli-germ cell and Sertoli-Sertoli

Fig. 2.9 Contd. ...

Pelletier 1990). It has an important protective role in safeguarding the germ

2.2.4 Interstitial Tissue of the Testis

Fig. 2.9 Contd. ...

Sg, spermatogonium; Sp, spermatocyte; Sd, elongating spermatids; B, basal

the testis has been described in a number of mammals (Christensen 1965;

Wilpe, E. and Aire, T. A. unpublished observations). The component tissue

! Reproductive Biology and Phylogeny of Birds