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Proceedings of the 16th

Italian Association of Equine Veterinarians
Congress

Carrara, Italy
January 29-31, 2010

Next SIVE Meeting:

Feb. 4-6, 2011 – Montesilvano, Pescara, Italy

Reprinted in the IVIS website with the permission of the

Italian Association of Equine Veterinarians – SIVE

http://www.ivis.org
Published in IVIS with the permission of SIVE
Morphologic abnormalities
Abnormalities of
of sperm
Sperm
Claire Card
DVM PhD diplomate ACT, Dept LACS, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, Sk S7N 5B4 Canada
Claire.Card@usask.ca
Introduction: Van Leeuwenhoek in 1677 was
the first scientist to observe sperm. His suc-
cess was due to improvements in the quality
of lenses that were available for microscopy.
He observed what he described as “animalac-
ula” in seminal fluid in a variety of species.
He noted and recorded the heterogeneity in
sperm head shapes in men and other animal
species. Over time it was shown that beyond
normal species specific differences in sperm,
there were other morphologic changes present
that were associated with abnormal sperm.
Many of these morphologic abnormalities of
sperm were associated with either intrinsic or
transient male sub- or infertility. Many inves-
tigators usd systematic studies of sperm mor-
phology to devise a number of classification
systems with the goal of finding an efficient
and accurate way to evaluate the potential fer-
tility of a male.
Reason for Morphologic Assessment: Many
stallions are investigated for their fertility po-
tential before retirement to stud, as part of a
pre-purchase examination for a sire prospect,
or prior to semen shipment or cryopreserva-
tion. Stallions may also present with a history
of low fertility, or declining fertility. Semen
parameters are monitored during a breeding
season to allow management adjustments if
the quality of the sperm produced declines, or
the pregnancy rate suffers. It is essential that
all health, fertility and semen characteristics
are used in context when interpreting a mor-
phologic assessment of the semen.
It is customary when working with equine
semen to only evaluate the concentration and
motility of the sperm. Progressive motility is
correlated with fertility, as is the dose of the
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semen1,2. The variation in the percentage of
normal spermatozoa among stallion seasons
accounted for 15% of the variation in stallion
fertility, and the combination of sperm mor-
phology and motility accounted for 37% of
the variation1. A fuller picture of a stallion’s
fertility is achieved through a morphologic
assessment of the sperm. A morphologic as-
sessment of the sperm from 2 ejaculates is a
requirement for a Breeding Soundness Ex-
amination of a stallion. In addition in combi-
nation with motility measurements and preg-
nancy rates, morphologic assessment allows
the detection of negative or positive trends in
spermatogenesis.
The advent of cooled transported and frozen
semen has increased the number of semen
samples handled by veterinarians. There are
currently few guidelines about semen quali-
ty, and many samples arrive with no infor-
mation.
A quick assessment of the sample allows for
a determination of the number of progres-
sively motile and morphologically normal
sperm available in the inseminate. A clini-
cian is then in a position to determine if 1 or
2 insemination dosages were received, and
will gain insight into the mare’s relative
chance of conceiving based on the number
of morphologically normal and progressive-
ly motile sperm.
Anatomy of a Sperm: Sperm have a head
covered (2/3 of the head) by an acrosome to
the equatorial region, a midpiece, a principle
piece and an end piece. The supporting 9 +2
arrangement of microtubule doublets running
through the mid, principle and end piece com-
prise the axoneme.
Published in IVIS with the permission of SIVE
Choice of Morphologic Classification Sys-
tem: If we look to history to understand the
methods of morphologic classification of
sperm we find that Lagerlof, Milovanov, and
Blom wrote about methods of classifying
sperm in bulls. Blom described 2 systems of
classification where sperm defects were either
termed as “Primary or Secondary” or “Ma-
jor or Minor”3, 4. In the first system 2 mor-
phological categories for sperm defects were
used. Primary defects were those that oc-
curred during spermatogenesis, thus repre-
senting a failure of spermatogenesis; and Sec-
ondary defects were those that occurred in the
passage of the sperm through the excurrent
ducts, which represented a failure of matura-
tion. A Primary defect was therefore deemed
to be of testicular origin (examples include
nuclear vacuoles or acrosome defects), and a
Secondary defect would be a defect of matu-
ration such as a proximal droplet5. Some au-
thors referred to Tertiary defects, which are
defects that occur from improper handling of
the sample or faculty preparation of the slide.
Blom’s classification system is one of the
most widely used system of classification of
sperm defects. Another classification system
by Blom used “Major or Minor” to describe
defects4. Major defects included problems
with heads, midpieces, and proximal droplets,
which are defects that are thought to have a
greater impact on fertility. Minor defects were
deemed those defects which have an unknown
role or inconsequential role in fertility, such as
distal droplets.
These are binary classification systems. There
are problems with using a binary process of
classifying sperm. Using Blom’s systems
sperm are first classified as normal or defec-
tive, followed by another binary classification
of the defects as Primary or Secondary, or in
the alternative as Major or Minor defects. The
binary system means that each sperm must be
assigned one category. The reality is that dur-
ing spermatogenesis billions of sperm are pro-
duced, and inevitably some abnormal sperm
are produced. Testis tissue produces sperm
with both Major and Minor defects, and sperm
with a Primary defect(s) may gain a Second-
ary defects as they undergo maturation. There-
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fore both Major and Minor, Primary or Sec-
ondary defects may be present on the same
sperm. The testes also produce sperm with
more than one type of Major defect on a sin-
gle sperm. An example of this would be a
sperm with a large (macrocephalic) head and
broken midpiece. There are many situations
where a sperm has more than one defect, these
Major Minor or Primary Secondary systems
suggest that the most proximal defect or the
most severe defect when classifying sperm
should be chosen. In the Primary and Second-
ary defect system if a sperm has 3 defects such
as nuclear vacuoles, a swollen midpiece and a
proximal droplet, only a primary defect would
be enumerated, even though there is another
primary defect (the swollen midpiece), and
another secondary defect (proximal droplet).
Using the Major Minor system where a sperm
has a microcephalic head, a distal midpiece
reflex, and a coiled principle piece with a dis-
tal droplet, only one major head defect would
be enumerated. The process of choosing a de-
fect on a sperm, even when multiple defects
are present, results in a percentage distribution
that will total to 100%. The examiner will not
however have a record of the distribution of
the sperm defects in the ejaculate. This fact
makes it much harder to determine if there are
any changes in the types of defects present in
sperm over time. This is relevant when we
consider the process of spermatogenesis and
determine that multiple developmental stages
of the sperm may be affected by an injury.
More recently a concept of classifying sperm
defects as Compensable and Non-compen-
sable System was also proposed6. A Compen-
sable defect is a defect where either the sperm
are not able to reach the site of fertilization, or
the sperm is unable to: bind to the oocyte, in-
duce the cortical reaction, and initiate fertil-
ization. There is no effect on fertility if a nu-
merically adequate population of normal
sperm are present for fertilization, because the
oocyte may still be fertilized by other fertile
sperm. The fertility of the individual is not af-
fected by defects that are effectively overcome
by having adequate levels of normal sperm.
The normal sperm compensate for the abnor-
mal sperm. Similarly a non-compensable de-
Published in IVIS with the permission of SIVE
fect is a defect that does not interfere with the
sperm reaching the site of fertilization, and the
cortical reaction is induced by the sperm as it
binds to the oocyte, and may cause syngamy,
but the sperm is defective in that it is capable
of supporting embryonic development. These
non-compensable sperm defects interfere with
fertility. An example of a non-compensable
defect is sperm DNA condensation or DNA
compaction problems.
Introduction to the Differential Spermiogram:
Another popular system of morphologic evalu-
ation is called the Differential Spermiogram7.
The Differential Spermiogram System is per-
formed in an analogous fashion to a differential
white count in a leukogram. A total white count
and a differential cell count is performed on a
blood sample. The differential white blood cell
count gives us a variety of information such as
the percentage of lymphocytes, neutrophils,
monocytes, eosinophils, and basophils in a sam-
ple. Changes in the absolute number and per-
centages of neutrophils and lymphocytes are
evaluated because they provide us with valu-
able clinical information. Neutrophils are also
scored for signs of activation, such as toxic
changes, which indicate impending cell death.
Serial daily and weekly samples are used to
compare changes in terms of the absolute cell
numbers, percentages of cells such as neu-
trophils, and the condition of the cells in terms
of toxic changes in the leukogram. This infor-
mation helps us to determine if our patient is
improving, staying the same or deteriorating.
This same principle applies to use of the Differ-
ential Spermiogram in the stallion. The slide is
examined for the different cell types in the
ejaculate (sperm, neutrophils, lymphocytes,
red cells, squames, germinal epithelial cells).
The sperm cells are differentially classified
by enumerating all the defects on the sperm.
Using the supravital stain Eosin Nigrosin, live
and dead sperm are evaluated in a separate
count. Cells which stain pink are considered
dead or devitalized, while the cells that stain
white are considered alive. The Live / Dead
sperm ratios are determined, to indicate the
vitality of the sperm, similar to the identification
of toxic changes in neutrophils.
4
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Preparation of a sample: In a complete se-
men analysis the total sperm number is deter-
mined (concentration x volume), the motility,
and the morphology of the semen is exam-
ined. The stallion’s sperm sample must be fil-
tered to remove the gel fraction because gel
makes the sample impossible to interpret.
Sperm are mixed with Eosin Nigrosin for the
morphologic assessment A 60:40 stain to
sperm ratio is recommended to provide good
contrast and density of sperm for evaluation.
The stain is hyposmotic to the sperm so the
slide should be dried quickly, such as on a
warming tray, to prevent any hyposmotic arte-
facts from forming (usually curved mid and
end pieces). Wet mount preparations viewed
under phase contrast do not usually allow the
same detailed examination of the defects pres-
ent. The examination is performed using 100x
objective (oil immersion) which yields 1000x
magnification7.
Laboratory Errors in Preparation: Labora-
tory errors causing the appearance of sperm
defects are sometimes referred to as tertiary
defects. There are a few defects that may be
produced through errors in sample handling
such as failure to filter the semen, excessive
stain, cold shock and hyposmotic shock. Gel
in the sample makes the background appear
white and makes the sperm difficult to see.
Excessive stain will create stain cracks along
the sperm. Artefacts associated with cold
shock of sperm include distal midpiece reflex-
es, while hyposmotic shock may result in
curved midpieces or curled endpieces. In gen-
eral it is very important to emphasize that the
vast majority of defects present in a sample
come out of the stallion. Errors or rough han-
dling of the sample will not produce defects
such as detached heads, or nuclear vacuoles.
Most sperm defects are either developmental
or maturational in nature.
Differential spermiogram: A spermiogram,
which is a frequency distribution of all de-
fects is performed on a minmum of 100
sperm cells, using a cell counter, and a sepa-
rate live dead count of at least 100 sperm are
performed8. All the sperm in a representative
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microscopic field are categorized. The pres-

ence of other cell types is noted, and addi-
tional stained slides may be determined to
identify the other cell types. Therefore if a
sperm has a macrocephalic head and a mid-
piece defect both are enumerated. To enumer-
ate both defects simultaneously one presses
both keys down at the same time on a cell
counter device. This will only advance the
counter by one number in the total column.
For example using a cell counter if 100 sperm
are counted and all the abnormalities are enu-
merated (meaning one presses more than one
key at the same time), only one sperm is
added to the total count even when more than
one defect is present on a sperm. Mathemati-
cally the percentages for the categories of de-
fects when added to the percentage of normal Figure 1 - Normal sperm.
sperm will not add to 100% using the Differ-
ential Spermiogram System.
Subcategories of sperm: There are also sub-
Categories in the Differential Spermiogram: categories of the head and midpiece defects,
The categories used in the Differential and the relative frequency of occurrence with
Spermiogram include: head, midpiece, de- the most common subcategory being noted
tached normal head, detached abnormal first, followed by the others in a descending
head, principle piece, proximal droplet, fashion. This is done by memory. The subcat-
distal droplet, and acrosomes. Germinal egories of the main defects include:
epithelial cells are counted if more than just
an occasional cell is present. All the sperm in Head defects: microcephalic (too small),
a field are counted irrespective of whether macrocephalic (too large), pyriform (pear
they stain white or pink with the Eosin Ni- shaped), tapered (long and narrow sperm)
grosin stain. Figure 1 shows a normal sperm. teratoid (almost non-recognizable) and vac-
When detached heads are encountered the uolated. There are a number of reports on the
head but not the corresponding headless mid- head morphometry of sperm in a large number
pieces are counted7. of breeds10-12. The range of values for fertile
This will only advance the counter by one stallions was as follows: length, 4.9-5.7 µm;
number in the total column. For example us- width, 2.5-3.0 µm. Higher percentages of
ing a cell counter if 100 sperm are counted morphometrically normal sperm heads are
and all the abnormalities are enumerated found in fertile than in subfertile stallions (52
(meaning one presses more than one key at vs. 19%)11. Essentially sperm are twice as
the same time), only one sperm is added to long as they are wide, and variations in size
the total count even when more than one de- resulting in sperm heads that are >25% larger
fect is present on a sperm. Mathematically the or smaller should be considered abnormal.
percentages for the categories of defects Stallions however are noted as very having a
when added to the percentage of normal variety of head shapes and sizes in their ejac-
sperm will not add to 100% using the Differ- ulates. The exception is in the Warmblood
ential Spermiogram System. Certain abnor- breeds where selection for fertility has result-
malities when found together represent more ed in a much more uniform population of
severe disturbances in spermatogenesis and sperm with consistent head shapes and sizes.
influence the stallion’s prognosis8,9. Pyriform sperm (pear shaped with a narrow
5
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base, and a proportionately wider apex) may

be associated with chromatin instability. Ta-
pered cells are long and narrow and are may
not be significant if the whole population of
sperm are uniformly tapered. Teratoid sperm
have the mid and principle coiled around
head. Vacuoles in sperm are visible as dark
spots on the sperm head when using. Small
single apical vacuoles may not be highly sig-
nificant in stallions. Larger and more conflu-
ent vacuoles are of significance. Eosin Ni-
grosin. The overall frequency of the head de-
fects present in both the intact and detached
sperm are noted.
These are listed from most frequent to least
frequent. Regarding acrosomes, knobbed,
missing, or abnormal acrosomes are counted
separately. Figures 2 and 4 show sperm with Figure 2 - Sperm with pyriform head/segmen-
pyriform, vacuolated heads, and a knobbed tal aplasia of the mitochondrial sheath (upper)
acrosome. and vacuole/pyriform head/segmental aplasia/
abnormal principle piece (lower).
Midpiece defects: The subcategories of the
midpiece defects include: segmental apla-
sia of the mitochondrial sheath, swollen
mitochondrial sheath, pseudo-droplet,
fractured midpiece, and distal midpiece
reflexes. Abaxial insertion of the midpiece
is not considered an abnormality in the
stallion or the boar. Figure 3 shows sperm
with fractured midpieces. Segemental aplasia
of the mitochondrial sheath has the appear-
ance of gaps in the mitochondrial shealth, a
swollen sheath involves visible thickening of
the sheath, a pseudo-droplet is bunching of
the mitochondria on the midpiece with a cor-
responding region on the midpiece of miss-
ing mitochondria, fractured midpieces usual- Figure 3 - Sperm with broken midpieces (all)
ly have broken fibres extruding at the site of and proximal droplets (left and uppermost).
the break, a distal midpiece reflex involves a
hairpin turn of the principle piece with or
without a distal droplet. Proximal droplets
are made of cytoplasmic material and are defects are present on a sperm it is not as-
considered separately and are not a midpiece sumed that one defect is more important than
defect per se. another. The examiner is enabled to determine
the potential of the defects to interfere with
Summary: The Differential Spermiogram fertility.
System allows investigation of changes in the The enumeration all of the defects for each
frequency or the prevalence of the defects sperm allows the chronological pattern of the
recorded over time. All the sperm in a repre- changes over time to be considered (increase,
sentative field are categorized. When multiple decrease, stay the same).
6
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE
Comparing the Differential Spermiogram
to Other Classification Systems: The per-
centage of normal sperm is a constant in all of
the classification systems. Therefore this
measure should be same in all systems.
Review of Spermatogenesis in the Stallion:
During spermatogenesis diploid germinal
cells termed spermatogonia, replicate and ma-
ture to form haploid, mature sperm. A stallion
produces an estimated 16 million sperm per
gram of testicular tissue per day. The defective
sperm are eliminated through apoptosis and
phagocytosis by the Sertoli cells, and other
phagocytic cells in the excurrent ducts, while
others are passed into the ejaculate. The sem-
iniferous tubules contain the germinal or sper-
matogenic epithelium and the supportive Ser-
toli cells. Cross sections of the seminiferous
tubules show that 4 or 5 specific cell types are
consistently found together, and these are
present in different combinations called cellu-
lar associations. The successive progression in
the cellular associations requires 12.2 days in
the stallion. The repetitions of the 12.2 day pe-
riod means there is a progression of the cells
through 8 stages which are numbered I
through VIII. The time frame to pass though
each stage is not equal. The stages can be dif-
ferentiated by a detailed examination of the
spermatocytes, spermatogonia, and sper-
matids in a cross section. The succession is
evident when the viewing the rings of the ger-
minal cells in the tubule. The cells located in
the outer ring of the tubule replicate so that
over time more differentiated cells are formed,
which through successive generations, results
in the most differentiated cells being located
closer to the lumen13.
Time Frame: The time period from a com-
mitted spermatogonium to a mature to a
sperm that is released in the lumen of the sem-
iniferous tubule has been shown experimen-
tally to be around 55 - 57 days in the stallion.
This 55 - 57 day period may be divided into 3
phases: spermatocytogenesis where mitosis
and differentiation of spematogonia occur
(19.4 days); meiosis the period of replication
(primary spermatocytes) and then reduction of
7
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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Figure 4 - Sperm with a loose head (left),
pyriform head/vacuole/distal midpiece reflex
(centre) and a knobbed acrosome/pyriform
head (right).
genetic material into haploid spermatids (19.4
days), and spermiogenesis development and
differentiation (18.6 days). Spermiation is the
release of spermatids into the lumen of semi-
niferous tubule. Approximately 9 days are re-
quired for transportation of sperm through the
excurrent ducts such as the rete testis, and epi-
didymides. A new population of sperm are
available for ejaculation every 64-66 days. In
the absence of ejaculation sperm are eliminat-
ed in the urine. There is no seasonal influence
on the developmental intervals, in other words
in winter or summer a sperm takes the same
amount of time to produce sperm. There is a
seasonal influence and an influence of age on
total sperm production, sperm motility and
sperm morphology.
Stallion sperm morphology: A number of
authors have reported that the percentage of
morphologically normal sperm in the general
population of stallions is around 50%14,15.
Therefore in fertile horses there are a relative-
ly large number of morphologically defective
sperm9. This is important to remember, but
pasture mated and feral horses achieve high
fertility rates under good nutritional condi-
tions16. The question then becomes determin-
ing what constitutes a problem when the per-
centage of morphologically normal sperm is
lower than 50%, and / or mares are failing to
conceive. A high percentage of defects is as-
Published in IVIS with the permission of SIVE
sociated with lower fertility17,18. Selection for
fertility such as in Warmblood breeds does re-
sult in higher percentages of normal sperm19,
or in the case of inbred breeds such as:
Clydesdales, Shires, Tenneesee Walkers, and
Friesians lower fertility and poorer sperm
quality is present20.
Serial evaluation of the Sperm Morpholo-
gy: Serial examinations of semen using the
Differential Spermiogram system, is a power-
ful clinical tool when it is possible to gain in-
sight into the fertility potential of an individ-
ual. The problems that arise as a result of the
stallion’s intrinsic fertility (genetic), a point
source problem such as a stress (fever), or a
long standing alteration (testicular degenera-
tion) may be diagnosed by serial examinations
of ejaculates spaced at least 1 month apart. The
clinical interpretation of semen samples is
challenging because an examiner must deter-
mine which parameters and abnormalities are
constitutive and reflect an individual stallion’s
age and intrinsic genetic ability, and which
changes are extrinsic due to a disturbance (nu-
tritional, hormonal, infectious, toxic, degener-
ative, idiopathic) in spermatogenesis21.
Age related changes: In general stallions
reach puberty at about 18 months of age, but
there is wide variability with stallions as
young as 8 months achieve puberty. Testicular
size is correlated with fertility22. Larger ma-
ture size breeds tend to mature later. Mature
stallions will have an increase in testicular
mass until around 4 years of age. Older stal-
lions tend to be more seasonal than younger
stallions, but in general fertility does not de-
cline with age in horses23,24.
Puberty - Young stallions at 2 years of age
may still be exhibiting signs of puberty, while
others may have poor intrinsic fertility. Until
a stallion is producing at least 100 million
sperm per ejaculate he is not considered to
have reached puberty. Stallions reaching 18
months of age in the winter may have a de-
layed onset of puberty related to photoperiod.
The process of puberty takes approximately 2
months to complete and involves the attain-
8
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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ment of patent seminiferous tubules. This ex-
plains why germinal epithelial cells appear in
the ejaculate at this time. The process of sper-
matogenesis is not highly efficient when pro-
duction first begins and there are increased
numbers of abnormal sperm. Four types of
defects are common in pubertal stallions; ger-
minal cells, head defects, midpiece defects,
and proximal droplets. A total scrotal width
of 8 cm and soft testes usually indicates that
the stallion is immature. A typical ejaculate of
a pubertal stallion has a low concentration,
low motility, high percentage of germinal
cells, and other defects such as head, mid-
piece and proximal droplets. The second ejac-
ulate shows similar parameters except the
concentration plummets revealing a low re-
serve capacity.
A pubertal stallion should be classified as
Questionable Breeding Prospect, and re-eval-
uated in 2-4 months time or longer depending
on the photoperiod conditions. A recheck ex-
amination should show the following an in-
crease in: total sperm numbers, sperm con-
centration, motility and percentage of mor-
phologically normal sperm with a decrease in
germinal epithelial cells, proximal droplets
and head/midpiece defects. If the stallion has
poor intrinsic fertility he will not show signif-
icant improvement over time. It is important
to determine if the stallions may be under
stress or is receiving anabolic steroids or glu-
cocorticoids which may negatively influence
sperm morphology.
Intrinsic Defects: In a population of male an-
imals examined for fertility there will be: be-
low average, average and above average levels
of fertility, which are reflected in their testicu-
lar size, intrinsic sperm number/concentration
and motility / morphology parameters. Intrin-
sic defects are consistent and there will be on-
ly minor seasonal fluctuations, and therefore
there is no improvement over time. The abili-
ty to produce sperm is determined by the stal-
lion’s genetics and testicular size. The consis-
tent appearance of a high percentage of mor-
phologically defective sperm in an otherwise
healthy young stallion who has no history of
illness and who is not receiving exogenous
Published in IVIS with the permission of SIVE
hormone therapy suggests that he has intrinsi-
cally low fertility, and he will exhibit only mi-
nor seasonal variations in his semen. The pres-
ence of intrinsically low fertility is accentuat-
ed by inbreeding, and is supported by the
practice of not selecting for fertility character-
istics in stallions. In some stallions these are
manifested by consistent defects present in
high percentage of sperm, visible at the light
and ultrastructural level, such as Dagg-like
sperm (fractured midpieces), or missing
dyneine arms (immotile live sperm)25,26.
Disturbances in Spermatogenesis: A distur-
bance in spermatogenesis is generally mani-
fested by changes in the sperm morphology as
quantified using a Differential Spermiogram.
Insults to spermatogenesis may be transient
and result in temporary fertility impairment,
or may be long standing and result in perma-
nent fertility impairment. Temporary insults to
spermatogenesis for example may result from
fever, stress, exposure to toxins, trauma, nutri-
tional deficiencies/toxicities. Damage to the
progenitor spermatogonia, which are required
to replicate in order to replenish the popula-
tion of dividing cells, may be severe enough to
result in a permanent reduction or loss of
sperm production.
In general the age of the stallion, season of the
year, nutrition, health status, medications, and
the general stress the horse is under impact
sperm morphology27. Adverse exposure to tox-
ins, radiation, dietary deficiencies or excesses,
heat stress, trauma, parasite migrations, tu-
mours etc also influence sperm morphology. A
disturbance in spermatogenesis is therefore su-
perimposed on the other factors that influence
sperm production. The features of the sperm in
the ejaculate provide a snapshot of the cumu-
lative health of the seminiferous epithelium,
sertoli cells and epididymides. The intensity,
nature, duration, and sensitivity of the sperm
cell types to an insult will determine the im-
pact on the Differential Spermiogram. Note
that all of the sperm cell types, stages, and cel-
lular associations are present in all stallions.
By integrating the information on history, age,
season, environment, nutrition, ejaculatory
characteristics, such as concentration, total
9
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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number of sperm, progressively motility of the
sperm, and the features of the Differential
Spermiogram the status of the stallion’s fertil-
ity and progression through the breeding sea-
son may be followed. During the examination
of a stallion the morphology of the sperm
must be integrated with the rest of the infor-
mation about the patient. This includes the
historical information on per cycle pregnancy
rates, early pregnancy losses, the testicular
size / consistency, and the features of the ejac-
ulates such as total sperm number, concentra-
tion and volume, percentage of progressively
motile sperm and percentage of morphologi-
cally normal sperm.
Temporary fertility impairment: Temporary
fertility impairment may arise from a point
source insult. These types of injuries represent
Extrinsic influences on spermatogenesis. Typi-
cally a change is noted over time or historical-
ly fertility was acceptable in the recent past28.
Examples of point source insults include tran-
sient high fever or scrotal heat stress29,30. Seri-
al evaluations of multiple ejaculates and an
examination of the changes in the proportions
or prevalence of the various sperm morpho-
logic defects provide the best information on
the nature of and subsequent recovery from a
disturbance in spermatogenesis7. A point
source insult creates damage to the cells pres-
ent at the time, generally producing a variety
of head and midpiece defects. Due to the
length of the time required for spermatogene-
sis at least 64 days plus the time for transit
through the excurrent ducts must elapse be-
fore a new population of cells is ejaculated.
Sperm that are injured that are close to matu-
rity appear first in the ejaculate, while sperm
that are immature would appear later. This has
been shown by studies that examined semen
characteristics after steroid administration
(dexamethasone) or thermal insults (scrotal
insulation)31,32. Changes noted in sperm mor-
phology after scrotal insulation began to ap-
pear 10 days after insulation, and then peaked
at about 30 days. Preinsulation levels of nor-
mal sperm were not reached until 50-75 days
has elapsed after insulation32. During a serial
examination of sperm morphology if the per-
Published in IVIS with the permission of SIVE Close window to return to IVIS

centage of normal sperm increases and the

other categories of defects decrease, this
would indicate a recovery from the problem,
but a lack of change or a decline in the per-
centage of normal sperm indicates an ongoing
problem. The presence of specific cellular as-
sociations in the stallion means that certain
cell types may be damaged at the same time.

Stress - Athletic horses are subject to a num-

ber of stressors such as high environmental
temperatures, transport stress, chronic pain
due to lameness, and medications such as an-
abolic steroids. Generally this type of stress is Figure 5A
manifested as a decline in the percentage of
motile and normal sperm, which is reversible
if the stress is removed.

Permanent Fertility Disturbances: A vari-

ety of problems may result in permanent fer-
tility impairment such as tumours of the testis,
testicular torsion/infarction, heavy metal ex-
posure, or unilateral castration. Severe nutri-
tional, hormonal, traumatic, infectious, toxic,
or degenerative insults may permanently im-
pair fertility. These conditions may alter scro-
tal thermoregulation, cause intense inflamma-
tion, may breach the blood testis barrier, inter-
fere with endocrine function, or damage the
Figure 5B
spermatogonial progenitor cells. More often
than not in stallions the cause for the decline
Figure 5 - Top panel A shows an Eosin Ni-
in fertility is not identified and is therefore id-
grosin stain of an ejaculate, the dark round
iopathic. The most common cause of idio-
cells are germinal epithelial cells. The bottom
pathic sub or infertility in stallions is testicu-
panel B is a Diff Quik stain of the same sam-
lar degeneration. Occasionally testicular de-
ple, demonstrating that the cells are not neu-
generation is transient following a dramatic
trophils.
insult, but in most cases it results in perma-
nent alterations.

Testicular Degeneration: The history of the (ribs)33. A Differential Spermiogram will

stallion generally includes that previously ac-
ceptable (45-55%) per cycle pregnancy rates
has declined to a lower per cycle pregnancy
rate (<30%). This history should trigger a full
breeding soundness examination including an
evaluation of the stallion’s testicular size and
consistency. Testicular degeneration results in
a loss of germinal epithelial cells, and will re-
sult in small testes, with a soft consistency
with prominent connective tissue bands
10
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
show low numbers of normal sperm, many
germinal epithelial cells (>10%), and high
percentages of abnormal sperm. Head and
midpiece defects may exceed 25-30% see
figure 5. The potential for improvement is low
based on testicular consistency and size. The
poor motility of the sperm often relate to un-
derlying morphologic problems of the mid-
piece. An excess number of germinal epithe-
lial cells, high percentages of head and mid-
Published in IVIS with the permission of SIVE
piece defects should suggest testicular degen-
eration. Serial examinations will show persist-
ence of the problem or a continued decline.
These stallions would be classified as having
an Unsatisfactory Breeding Potential. The
stallions with testicular degeneration will tend
to stay the same or decline in the percentage
of normal sperm over time. True testicular de-
generation is not reversible, and improved fer-
tility relies predominately on improving the
management of the stallion.
Subfertility: A subfertile stallion may be in-
trinsically an inefficient breeder, or he may
have lost fertility over time. Therefore the his-
tory is very important is determining the na-
ture of a stallion’s subfertility. Stallions loose
commercial viability when their per cycle
pregnancy rates drop below 30%. There are a
host of reasons why stallions become subfer-
tile, including psychological and medical
problems. However the majority of subfertili-
ty problems are manifested by high percent-
ages of morphologically abnormal sperm, and
the stallion may or may not have low sperm
motility. The presence of germinal epithelial
cells indicates the stallion may have testicular
degeneration, but the absence of germinal ep-
ithelial cells indicates the stallion has poor
sperm quality. Ultrastructural defects may be
present in the sperm. Problems with the head,
annulus, midpiece and axoneme have been re-
ported25,34-37.
Defects: For example severely knobbed acro-
somes, midpiece defects, DNA condensation
defects, and nuclear vacuoles may be present
in sperm that are alive and progressively
motile in stallions with a testicular size/mass
and consistency within normal limits. Sper-
matogenesis is a complex process, which in-
cludes dramatic changes in the chromatin,
which refers to DNA and the complex of pro-
teins that associate with it. During maturation
in the testes and epididymides there are a num-
ber of protein substitutions, often with zinc
containing proteins, that neutralize the charges
on the DNA, and allowing it to compact or fold
up. This process is known as DNA condensa-
tion. The proteins need to be substituted in a
11
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Close window to return to IVIS
specific order and in a specific time frame. We
know that immature sperm, such as those with
proximal droplets, are not able to fertilize be-
cause their DNA is not fully condensed. The
droplet present on sperm begins is in a proxi-
mal location on the midpiece as the sperm en-
ters the epididymis, but is shed from the distal
piece just prior to ejaculation. Proximal
droplets in a high percentage of sperm indicate
that these sperm are immature and have not
completed chromatin compaction.
The formation of the acrosome occurs concur-
rently with some stages of chromatin com-
paction. The presence of acrosomal defects
suggests that additional tests should be con-
sidered (chromatin assays), as the sperm with
knobbed acrosomes have been shown to have
more immature chromatin38. Sperm with acro-
somal defects are a heterogenous population
and morphologic problems such as mild knob-
bing or folding of the acrosome may not in-
fluence fertility, but extremely severely
knobbed acrosomes or those that contain
vesicular material represent a more serious
form of the defect, and are more likely to have
an underlying problem in chromatin matura-
tion. The knobbed acrosome defect may have
a heritable basis, and may be a constitutive
problem in some stallions. Therefore one ab-
normality may signal the presence of another,
as acrosome defects are associated with chro-
matin defects. Serial semen evaluations may
be used to determine the persistence of the de-
fect39. There is some suggestion that this con-
dition is heritable in many species40.
Spermiostasis: Spermiostasis is usually an
acquired and persistent idiopathic condition in
the stallion. In these stallions one or both am-
pullae fail to function normally and sperm ac-
cumulate. The ampullae may be partially ob-
structed or completely blocked. In the case of
complete obstruction alkaline phosphatase
will still usually be elevated in the semen sam-
ple indicating the horse is ejaculating. The
typical spermiogram shows extremely high
numbers of sperm with detached usually pink
staining heads, either in every ejaculate or re-
leased intermittently. The ampullary plugs are
occasionally released and grossly visible
Published in IVIS with the permission of SIVE
Figure 6 - Abnormal sperm in a stallion with
spermiostasis, note the presence of many de-
tached heads.
within the ejaculate. The detached heads are
believed to belong to senescent sperm that are
then improperly stored and intermittently re-
leased. Cannulation and flushing of the am-
pullae through a urethral approach has not
been successful. The underlying pathogenesis
of the condition remains obscure. Rectal mas-
sage of the ampulla, and oxytocin before col-
lection have been used along with frequent col-
lection to manage some stallions (Figure 6).
Supplemental tests: When sperm appear to
have normal morphology but do not get mares
in foal it is time for a different level of exam-
ination13,33. Transrectal ultrasound and testicu-
lar ultrasound to evaluate testicular echotex-
ture, and Doppler analysis of blood flow may
be used. A variety of stains such as coomasie
blue, or fluorescent stains may be used to
evaluate the acrosome, and tests to provoke
the acrosome reaction may be performed. En-
docrine tests are used to characterize the prob-
lem and responsiveness of the hypothalamo-
pituitary-testis axis. A Feulgen stain may help
identify nuclear vacuoles and chromatin con-
densation defects. A sperm chromatin struc-
ture assay may be used to investigate chro-
matin stability. An antisperm antibody assay
may be indicated if sperm are observed to ex-
hibit head to head agglutination. Scanning or
Tranmission electron microscopy may show
or reveal the nature of a defect through ultra-
12
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Close window to return to IVIS
structural analysis12,39, such as with immotile
sperm that stain alive (primary ciliary dyski-
nesia)25,34,36,37,40,41. Occasionally testicular
biopsy is used to determine if the appropriate
cell types are being produced and to confirm
where the defective sperm are forming, or if a
tumour is present42-44.
Treatments to improve sperm morphology:
There continues to be interest in improving se-
men quality through the use of supplements
such as docosahexaenoic acid (DHA), and
omega 3,6 containing neutriceuticals. Others
suggest supplements such as carnitine, and vi-
tamin E selenium may be beneficial. The
DHA products are derived from fish meal and
are fed for months before a desired effect is
reached. Some reports show improvements in
sperm morphology, motion characteristics,
and ability to survive cooling and freezing45,46.
Many times efforts are aimed at enriching the
stallion’s environment psychologically so he
achieves harem status, and are focused on
medical management to remove stress, im-
prove health and ensure the stallion is fully
ejaculating.
Adjustments to Breeding Doses Manage-
ment Routines: Stallions with high percent-
ages of abnormal sperm may still successfully
get mares in foals at acceptable rates if they
have enough vigorous normal sperm to com-
pensate, especially when breeding at pasture.
Planned multiple mating have been reported
to increase pregnancy rates in some stallions47.
Insemination with whole ejaculates in stal-
lions with very low numbers of normal sperm
has also been used as a strategy to increase
pregnancy rates25. Therefore when defects are
compensable increasing the number of sperm
in an ejaculate will often achieve the desired
result. Sperm samples with high percentages
of abnormal sperm on the average do not cool
as well or freeze very well. Veterinarians may
need to adjust the number of sperm in a breed-
ing dose upwards to compensate for poorer
morphology. On the average stallions produce
around 50% normal sperm, a recommendation
is adding at least 100 million more motile
sperm for every 10% decrease below 50%
Published in IVIS with the permission of SIVE
in morphologically normal sperm9. Stallions
generally will begin to leave unacceptably
high numbers of mares open when they fall
below 100 million morphologically normal
and progressively motile sperm per breeding
dose. There are some papers that suggest that
additional processing steps such as density
gradient centrifugation, glass wool sephadex
separation, or pelleting sperm followed by
swim up may assist in harvesting the best
sperm from an ejaculate48,49. The percentage
of morphologically normal sperm and sperm
with intact chromatin was increased by col-
loidal centrifugation. The authors reported a
decrease in the incidence of proximal cyto-
plasmic droplets and midpiece defects after
centrifugation through colloids. Whether fer-
tility is improved by such processing steps
will vary with individual stallions, and likely
would only apply to subfertile stallions, and of
these stallions only a proportion of the them
would realize an increase in fertility49,50.
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