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The Possible Relationship between Embryo Orientation Opaque Banding and the
Dehydration of Albumen in Crocodile Eggs

Article  in  Copeia · February 1987

DOI: 10.2307/1446070

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 The Possible Relationship between Embryo Orientation Opaque Banding and the Dehydration of
Albumen in Crocodile Eggs
Author(s): Grahame J. W. Webb, S. Charlie Manolis, Peter J. Whitehead and Karen Dempsey
Source: Copeia, Vol. 1987, No. 1 (Feb. 11, 1987), pp. 252-257
Published by: American Society of Ichthyologists and Herpetologists (ASIH)
Stable URL: http://www.jstor.org/stable/1446070
Accessed: 02-02-2016 22:56 UTC

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252 COPEIA, 1987, NO. 1

vivorshipin the painted turtle, Chrysemys picta.Co- and 113.4 ? 2.6 g respectively (Webb et al.,
peia 1968:260-268. 1983a, 1983c).
HARRIS,L. D. 1984. The fragmented forest. Uni-
versity of Chicago Press, Chicago, Illinois. Embryo orientation.-Regardless of how croco-
JOHNSON,R., AND S. A. TEMPLE. 1986. Assessing dilian eggs become oriented at the time of lay-
habitatqualityfor birds nesting in fragmented tall
grassprairies,p. 245-249. In: Wildlife 2000: mod- ing, the embryos within them are almost in-
eling habitatrelationshipsof terrestrialvertebrates. variably found on the uppermost surface of the
J. Verner, M. L. Morrison and C. J. Ralph (eds.). yolk (Ferguson, 1985). They attach to the shell
Universityof WisconsinPress,Madison,Wisconsin. membrane at this position within 24 h of laying.
S.
TEMPLE, A., AND B. WILCOX. 1986. Introduction: If eggs are rotated experimentally after laying,
predicting effects of habitat patchiness and frag- but before the embryos attach to the shell mem-
mentation,p. 261-262. In:Wildlife2000: modeling brane, the embryos return to the uppermost
habitat relationships of terrestrial vertebrates. J.
part of the yolk, where they attach and develop
Verner, M. L. Morrison and C. J. Ralph (eds.).
normally (Table 1). If rotated after attachment,
Universityof WisconsinPress,Madison,Wisconsin. but while the
VOGT,R. C. 1981. Natural history of amphibians embryos are still young (2-9 d),
and reptiles of Wisconsin. MilwaukeePublic Mu- they remain at the attachment site and die (Ta-
seum, Milwaukee,Wisconsin. ble 1) (Ferguson, 1985). The survival of croc-
WILCOVE, D. S. 1985. Nest predationin forest tracts odilian embryos is thus partly dependent on
and the decline of migratorysongbirds.Ecology66: them attaching at the top of the egg, regardless
1211-1214. of their orientation at laying.
At the time of egg laying, the chorion, am-
STANLEYA. TEMPLE, Department of Wildlife Ecol- nion and yolk sac have already started devel-
ogy, Universityof Wisconsin,Madison, Wisconsin oping (Fig. 1) and attach the embryo (10-20
53706. Accepted 14 March 1986. somite stage of development) to the inside of
the vitelline membrane (Fig. 2A-D). Thus when
an embryo "moves" to the top of an egg, in a
wild nest or after an experimental manipulation
(Table 1), the vitelline membrane must also
Copeia, 1987(1), pp. 252-257 move. The only plausible explanation is that the
? 1987 by the American Society of
Ichthyologists and Herpetologists
yolk rotates within the surrounding albumen (as
in bird eggs; Romanoff and Romanoff, 1949;
THE POSSIBLE RELATIONSHIP BE- Romanoff, 1960) (Fig. 2A-D). The ability of
TWEEN EMBRYO ORIENTATION, OPAQUE the crocodilian yolk to "swing" within the al-
BANDING AND THE DEHYDRATION OF bumen was demonstrated by inserting strings
ALBUMEN IN CROCODILE EGGS.-Stud- of graphite granules through the albumen and
ies of crocodilian eggs and embryos have ad- into the yolk of a fresh egg (with watchmakers
vanced greatly in recent years, yet some very forceps) and then tilting or rotating the egg
basic aspects of egg function are poorly under- (Fig. 3). The granules also indicate that there
stood (Ferguson, 1985). The reorganization of is shearing between layers of albumen rather
egg contents that occurs during development than between the vitelline membrane or shell
has not been quantified, the mechanism by which membrane and albumen. As discussed below,
embryos appear on the upper surface of the yolk yolk rotation is probably caused by the influence
after laying does not appear to have been stud- of gravity on a density gradient through the
ied and the mechanism of opaque banding or yolk.
"chalking" of eggs is poorly understood. Our
studies of Crocodylusjohnstoniand C. porosusyield Egg content changes.-Crocodile eggs (16 C. po-
data on each of the above which suggest an rosus; 83 C. johnstoni) incubated at 30 C were
intimate association exists between them. opened and the major egg contents were
The eggs of all crocodilians are large (40- weighed separately.The patternof change (Fig.
140 g), elliptical and hard-shelled. They contain 4) through incubationwassimilarin both species
a spherical yolk mass bounded by a vitelline and closely parallels the situation described in
membrane, albumen, a shell membrane and a turtle (Agassiz, 1857) and bird eggs (Romanoff,
brittle shell composed mainly of calcite (Fer- 1967). Infertile eggs (no sign of embryonic de-
guson, 1982, 1985). Mean egg weights for C. velopment; no opaque banding; no build up of
johnstoni and C. porosus are 68.2 ? 1.4 g (SE) subembryonicfluid) were used to approximate

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 HERPETOLOGICAL NOTES 253

t _:
A ,,.rr

.
. ,7
X'*
.2 v
,,.__

V
*'_,,* ~ . 7 :

" I

I
"~~~./^ X
I'^ Sr

.'A~~~,~ I*o _* ;.I ttr.A*,, r 4.

ys.,X
YS'

.,. -,

it-~~~~ ~ . ...

tEs.
.
s-4.,;*
^J.

1I Lo
0 4
s
Fig. 1. Low power (upper)and high power (lower)views of a transversesection through the albumen(A),
vitelline membrane (V) and attached Crocodylus
porosusembryo preserved on the day of laying. Globules of
yolk (Y)remainattachedto the developingyolk sac tissue.The chorionicectoderm (CE)is fused to the vitelline
membraneand is separatedslightlyfrom the chorionicmesoderm(CM).The yolk sacmesodermand endoderm
(YS) and amniotic mesoderm (AM) and ectoderm (AE) are present.

the condition which exists in the oviducts before
significant embryonic development occurs (Fig.
2A), so that a chronology of developmental
events could be constructed.
Within the oviducts the egg contents are ini-
tially albumen, yolk and a small mass of embry-
onic tissue which is probably located at the top
of the yolk sphere, as in birds (Romanoff and
Romanoff, 1949). As development proceeds in
the oviducts (Agassiz, 1857), water is drawn from
the water-laden albumen, presumably by the
action of the ectodermal surfaces of the em-
bryonic disc as in birds (New, 1956), and is se-
creted beneath the embryo, on the inside of the
vitelline membrane. This subembryonic fluid is
appreciably lighter than the yolk and settles
above it (Fig. 2B; Table 2) and beneath the
embryo. There is thus a density gradient

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254 COPEIA, 1987, NO. 1

TABLE 1. SOME RESULTS OF ROTATING Crocodylus johnstoni EGGS DURING ARTIFICIAL INCUBATION. The up-
permost point of each egg was marked in the nest and they were rotated (about the major axis) relative to
that mark. Eggs were rotated before (B) or after (A) embryo attachment as determined by opening an egg
from the clutch used. Controls were not rotated. Incubation temperatures were between 30.0 and 31.5 C.
N = number of eggs in the sample.

Embryo age
incubation started
(days) N Rotation (degrees) Days until opened Embryo position Notes

0.5 B 13 control 17-33 top all alive

0.5 B 6 45-225 6 top all alive; yolk rotated
2-10 A 33 control hatched top 32 alive; 1 dead (at 45 d)
2-9 A 9 180 13-16 bottom all dead

vitelline membrane subembryonic fluid through the yolk sphere at the time of laying.
A embryo~ If eggs are laid with the embryo displaced from
albumen
nolk the top, and hence the yolk's center of gravity
shell
shell membrane displaced from its equilibrium position, then
gravity could and probably does cause the yolk
to rotate (Fig. 2C-D).
:0 D. The amount of subembryonic fluid increases
greatly after laying (Fig. 4) and this causes the
volume within the vitelline membrane (which
contains embryo, subembryonic fluid and yolk)
to expand. It occupies space created by the de-
E 1
hydration of albumen (Fig. 4). The albumen
immediately above the embryo is dehydrated
first and the vitelline membrane and embryo
adhere to the shell membrane (Fig. 2E) in that
allantois area (a layer of dehydrated albumen may be
between them). The albumen between the yolk
sphere and shell membrane around the minor

95 d. A: Oviducal egg prior to subembryonicfluid

chorioallantois
45
65
Fig. 2. The chronology of development in croc-
odile eggs. Numbersrefer to daysof incubationat 30
C for Crocodylusporosusand if multipliedby 0.95 ap-
proximatesthe situationin C.johnstoni.Yolk is inter-
nalizedafter about 90 d and pipping and hatching at
formation.B: Oviducalegg at the time of laying with
< 2 ml of subembryonicfluid. C: If necessarythe yolk
rotateswithin the albumen.D: Withinhours of laying
the embryo is on the upper surface of the yolk. E:
The volume of subembryonicfluid increases at the
expense of the albumen; within 24 h the vitelline
membrane and embryo adhere to the shell mem-
brane. F: The allantoisforms and as it expands sub-
embryonic fluid formation ceases. G: The chorioal-
lantois spreadsaround the midpoint axis of the egg
and the majorityof subembryonicfluid becomes en-
closed with yolk within the embryonic vitelline sac;
hydratedalbumen remains in both poles of the egg.
H: The chorioallantoisexpands from the midpoint
band around the shell membrane, separatingthe re-
maining albumen from the shell membrane. I: The
chorioallantoiscompletelysurroundsthe egg and the
remaining albumen is dehydrated.J-N: Changes in
opaque bandingassociatedwith the internaldevelop-
ments depicted in E-I.

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 HERPETOLOGICAL NOTES 255

A. 80 a)
graphite shell window
EMBRYO
/
-shell 60 o
<^^^'yolk she shell membrane ?0--'-?--. -..- i

\ albumen - YOLKK
' so,!
vitelline membrane
40'\ ALBUMEN s /
B. _ \
\ SUBEMBRYONIC
A +\
Xx
X FLUID
D ALLANTOIC /
LUx FLUID /

20

Fig. 3. Transversesection through the upper part I . I

20 30
. . .
0 50 60 70 80 90 100
*II. .

of the minor axis of a crocodilian egg (with a shell **"

window removed). Graphite granules inserted into -f ^..,' . ....
0 ' ' '
the albumen and yolk (A) adopt new positions when 40 o' o700' o0 100o
0 10 20 '30
the egg is rocked from side to side (B); the vitelline
membraneand yolk move together and shearingtakes
40-
place within the albumen. b)
/

EMBRYO/
axis of the egg is dehydrated next and the vi- 30- /
telline membrane attaches to the shell mem- '-
brane in a band around the egg. Hydrated re- ^YOLK-' /

sidual albumen becomes restricted to the poles ALBUMEN \ /

of the egg. Albumen dehydration and subem- 20-
\ SUEMBRYONICALLANTOIC
bryonic fluid production peak when the allan- \ FLLD FLUID \
tois is expanding. Part of the allantoic mem- !' /\ ,J ...
-/ .. "'. ,/
brane fuses with the chorion, creating the 10- 2 3 4 5 6 7 8 9
1..
chorioallantois (Fig. 2G), while the remainder
.1983a).
al., .
is within the subembryonic space. At this stage,
contact between the chorioallantois and the shell o.-I .?-; \ -
membrane is restricted to the minor axis of the
egg, where the albumen has been dehydrated, H 30 40 50 60 70 s80
0p 10 20 t 90 00
and where the opaque band occurs. When the DAYS
OFINCUBATION
AT30?C
chorioallantois spreads outside this band (Fig.
Fig. 4. Changes
Fig. Changes in the weights of egg
the weights egg components
components
2H-I) the remaining albumen (in the poles) is with progressivedevelopmentof Crocodylus porosus(a)
dehydrated. Yolk is primarily utilized later in and C.johnstoni(b) eggs at 30 C. C. porosuseggs (N =
development (Fig. 4) as a source of materials 16) were
same from
the a single
as that clutch (121.7
dehydrating ? 0.55 g, SE,
the albumen. As
for the rapidly growing embryo. N = 46). C. johnstoni eggs (N = 83) were from 16
clutches and the weights of components were scaled
Opaquebanding.-The pattern of opaque band- to the mean egg weight (68.2 ?+1.4 g; SE; Webb et
ing on the shell in both species (Fig. 2J-N) has al., 1983a).
been described previously (Webb et al., 1983a,
1983b). The opacity is associated with structur- The mechanism responsible for the dehydra-
al changes between the shell and shell mem- tion of the shell/shell membrane appears to be
brane and dehydration (Ferguson, 1982; the same as that dehydrating the albumen. As
Thompson, 1985). It would appear that this de- albumen is dehydrated around the minor axis
hydration is at least partly intrinsic. of the egg, so the opacity spreads in an identical
When the albumen above the embryo is being pattern (Fig. 2K), preceding the spread of the
dehydrated, the opaque patch forms at the site chorioallantois. The chorioallantois eventually
in which the embryo will attach (Fig. 2J). Ini- surrounds the opaque band and when it even-
tially a thin layer of partly dehydrated albumen tually expands outside that band, along the in-
remains between the shell membrane and vi- ner surface of the shell membrane of the egg
telline membrane, but this rapidly disappears. poles (Fig. 2H), the opacity mirrors its spread

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256 COPEIA, 1987, NO. 1
TABLE 2. THE DENSITY AND WATER CONTENT OF
MAJOR EGG COMPONENTS IN TWO CROCODILIAN SPE-
CIES.
C. johnstoni C. porosus
DensDensityDensit
% water (g/ml) % water
Albumen 95.0 1.024 96.4
Yolk 58.1 1.058 56.4
Subembryonic
fluid 98.8 1.005
(Fig. 2M) and the egg shell becomes completely
opaque (Fig. 2N). The increase in allantoic fluid
during this period suggests that the chorioal-
lantois is taking up water from both the albu-
men and shell/shell membrane (Ferguson, 1982,
1985). The pattern of opaque banding de-
scribed occurs in air fully saturated with water
vapor, even if droplets of free water are present
on the shell surface. However if crocodilian eggs
containing live embyros are partly or complete-
ly submerged, the shell does not go opaque in
the region in contact with water (as in turtle
eggs; Ewert, 1979). Immersion in water may
permit bulk flow through liquid filled pores in
the shell, exceeding the capacity of intrinsic
mechanisms to remove water from the shell/
shell membrane.
The generalized model of development de-
scribed above requires further study, but in its
present form is consistent with the available in-
formation on crocodilian eggs and embryos
(Ferguson, 1985). Movement of fluids within
the egg during development appears to be in-
timately associated with both embryo orienta-
tion and opaque banding. The model is also
consistent with most observations on the de-
velopment of large, hard-shelled, chelonian eggs
(Agassiz, 1857; Ewert, 1985). The spherical eggs
of the pitted-shell turtle Carettochelysinsculpta
(33.7 ? 0.9 g; SE; Webb et al., in press), de-
veloped as the model would predict for the de-
velopment of a spherical crocodile egg. [In
spherical eggs the shell opacity ("chalking") and
albumen dehydration proceed as a field from
the top to the bottom of the egg rather than as
a band spreading from the midline.]
Yolk rotation appears essential for normal
embryonic development, and we suspect that
variation in both the water content and quantity
of albumen supplied to the eggs of different
crocodilian and chelonian species may be con-
strained by the need to facilitate yolk rotation.
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For example marine, freshwater and terrestrial
chelonians supply their ova (yolk) with similar
proportions of albumen (Ewert, 1979, 1985),
the bulk of which is a water supply, even though
water availability in the three environments dif-
(g/ml)
fers greatly. This is surprising, because many
turtle eggs can absorb appreciable amounts of
1.020 water after laying (Packard and Packard, 1984)
1.042 and a less hydrated albumen would seem con-
sistent with water economy in some species. On
1.004
the other hand, yolk rotation requires the ovum
to be suspended in a low friction medium at the
time of laying. The water-laden albumen pro-
vides that medium. For mechanical purposes
alone, a minimum surrounding of thin, watery
albumen may be needed for embryo survival,
regardless of the water and chemical constitu-
ents of albumen needed for later embryonic
development.
Acknowledgments.-We would like to thank A.
and R. Bellairs, M. Beal, M. Ferguson, M. Ew-
ert, A. Smith, V. Onions,J. Joss and L. Melville
for their assistance in carrying out the work,
discussing the results and/or commenting on
an early draft of the paper. M. Ferguson and
M. Ewert kindly supplied pre-prints of their re-
cent review articles. R. Bellairs kindly examined
the embryo sections and advised on the likely
disposition of extraembryonic membranes. Fi-
nancial assistance came from the Australian Re-
search Grants Scheme (Grant No. D1/831604
9), the Conservation Commission of the North-
ern Territory and the University of New South
Wales, and the University Planning Authority
for the Northern Territory.
LITERATURE CITED
AGASSIZ,L. 1857. Contributions to the natural his-
tory of the United States of America. Monograph
1, Vol. 2. Little Brown Co., Boston, Massachusets.
EWERT, M. A. 1979. The embryo and its egg: de-
velopment and natural history, p. 333-413. In:
Turtles: perspectivesand research. M. Harlessand
H. Morlock(eds.).JohnWileyand Sons, New York,
New York.
. 1985. Embryologyof turtles, p. 75-267. In:
Biology of the Reptilia,Vol. 14. C. Gans, F. Billett
and P. F. A. Maderson(eds.).John Wiley and Sons,
New York, New York.
FERGUSON, M. W. J. 1982. The structure and com-
positionof the eggshell and embryonicmembranes
of Alligatormississippiensis.
Trans. Zool. Soc. Lond.
36:99-152.
.1985. Reproductivebiology and embryology
 HERPETOLOGICAL NOTES 257

of the crocodilians,p. 329-491. In: Biology of the N.T. VI. Nestingbiology.Aust. Wildl.Res. 10:607-
Reptilia, Vol. 14. C. Gans, F. Billett and P. Mad- 637.
erson (eds.).John Wiley and Sons, New York, New ,AND G. C. SACK. 1983b. An
York. interimmethod for estimatingthe age of Crocodylus
NEW,D. A. T. 1956. The formation of sub-blasto- porosusembryos.Ibid. 10:563-570.
dermic fluid in hens' eggs. J. Embryol.Exp. Mor- , D. CHOQUENOT, AND P. J. WHITEHEAD. N.d.

phol. 4:221-227. Nests, eggs and embryonic development of Caret-

PACKARD, G. C., AND M.J. PACKARD. 1984. Coupling
of physiology of embryonic turtles to the hydric
environment, p. 99-119. In: Respirationand me-
tabolismof embryonic vertebrates. R. S. Seymour
(ed.). Dr. W. Junk, Dordrecht, Netherlands.
ROMANOFF,A. L. 1960. The avian embryo. The Mac-
millan Co., New York, New York.
1967. Biochemistryof the avian egg. John
Wiley and Sons, New York, New York.
, AND A. J. ROMANOFF. 1949. The avian egg.
John Wiley and Sons, New York, New York.
THOMPSON, M. B. 1985. Functional significance of
the opaque white patch in eggs of Emyduramac-
quarii,p. 387-395. In: Biologyof Australasianfrogs
and reptiles. G. Grigg, R. Shine and H. Ehmann
(eds.). Royal ZoologicalSociety, New South Wales,
Surrey Beatty & Sons, Sydney, Australia.
WEBB, G. J. W., R. BUCKWORTH, AND S. C. MANOLIS.
1983a. Crocodylusjohnstoniin the McKinlay River,
tochelysinsculpta(Chelonia: Carettochelidae)from
Northern Australia.J. Zool. Lond. suppl. Ser. B
1(3). In press.
, G. C. SACK, R. BUCKWORTH, AND S. C.
MANOLIS.1983c. An examination of Crocodyluspo-
rosusnests in two northern Australianfreshwater
swamps,with an analysisof embryomortality.Aust.
Wildl. Res. 10:571-605.
GRAHAMEJ. W. WEBB, S. CHARLIE MANOLIS,
School of Zoology,University of N.S.W., PO Box
1, Kensington, N.S.W. 2033, Australia; PETER
J. WHITEHEAD, ConservationCommissionof the
Northern Territory, PO Box 38496, Winnellie
5789 N.T., Australia; and KAREN DEMPSEY,
University of N.S.W., PO Box 1, Kensington,
N.S.W. 2033, Australia. Accepted 18 March
1986.

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