ENVIRONMENTAL INFLUENCES ON
THE CRITICAL OXYGEN TENSION, WATER BALANCE,
METABOLISM, AND GROWTH OF REPTILIAN EMBRYOS

BY

YEONG-CHOY KAM

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

OtllVOTTV OF FUfflM U»51ES

1992

This work is dedicated to my parents, brothers, sisters,

my wife, Iris, and my son, Michael

ACKNOWLEDGEMENTS

I gratefully acknowledge the support, guidance, patience, and facilities
provided by my major advisor, Dr. Harvey B. Lillywhite. Committee members,
Drs. John Anderson, Richard Kiltie, and Michele Wheatly from the Department
of Zoology and Dr. Charles Woods from the Department of Physiology, all
provided facilities and helpful comments.

I thank Dr. V. DeMarco for discussion and help, Dr. J. F. Anderson, Drs. V.
DeMarco, D. Evans, B. McNab, M. Wheatly, and T. J. Wronski for the loan of
equipment, and Dr. F. Percival and G. Masson (The Florida Cooperative Fish
and Wildlife Research Unit), W. Woodward (The Florida Game and Freshwater
Fish Commission) and B. Witherington for collecting turtle eggs. I also thank R.
Edwards for helpful comments on an earlier draft of the dissertation. Help and
advice were also given by Dr. K. A. Bjorndal, Dr. A. Bolten, L. Eberhardt, J.
Matter, Dr. J. Payne, Dr. F. Percival (The Florida Cooperative Fish and Wildlife
Research Unit), J. Pipken, and L. Somma.

This study was supported in part by a Sigma Xi Research Grant and the
Department of Zoology, University of Florida.

Thanks are also due to Jamie Lillywhite for making our stay in
Gainesville so enjoyable during the last four years.

I thank my parents, brothers, and sisters for their understanding,
patience, and support, without which I never would have had the chance to
study in the United States.

Finally, I owe many thanks to my wife, Iris, who not only gave me the
encouragement and support when I needed it during my early graduate student
career, but also helped me to make the very first step in achieving my goal. Of
course, life would not be perfect without Michael's smile and his sister's kicks.

IV

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

ABSTRACT vii

GENERAL INTRODUCTION 1

CHAPTERS

1 COMPARATIVE STUDY ON THE CRITICAL OXYGEN TENSION OF
REPTILIAN EMBRYOS

Introduction 5

Materials and Methods 7

Results 11

Discussion 12

2 TEMPERATURE EFFECTS ON METABOLISM AND CRITICAL
OXYGEN TENSION OF FLORIDA RED-BELLIED TURTLE
(PSEUDEMYS NELSONH EMBRYOS

Introduction 29

Materials and Methods 30

Results 32

Discussion 33

3 INFLUENCES OF EGG HYDRATION ON METABOLISM, CRITICAL
OXYGEN TENSION, AND HATCHLINGS OF FLORIDA RED-
BELLIED TURTLE (PSEUDEMYS NELSONh EMBRYOS

Introduction 49

Materials and Methods 50

Results 52

Discussion 53

4 PHYSIOLOGICAL RESPONSES OF FLORIDA RED-BELLIED TURTLE
(PSEUDEMYS NELSONh EMBRYOS TO CHRONIC HYPOXIA

Introduction 62

Materials and Methods 64

Results 66

Discussion 67

v

5 EFFECTS OF SIMULATED FLOODING ON METABOLISM, WATER
BALANCE, AND HATCHING SUCCESS OF FLORIDA RED-
BELLIED TURTLE (PSEUDEMYS NELSONh EMBRYOS

Introduction 83

Materials and Methods 84

Results 86

Discussion 88

GENERAL DISCUSSION 103

LITERATURE CITED 106

BIOGRAPHICAL SKETCH 114

VI

5 EFFECTS OF SIMULATED FLOODING ON METABOLISM, WATER
BALANCE, AND HATCHING SUCCESS OF FLORIDA RED-
BELLIED TURTLE (PSEUDEMYS NELSONh EMBRYOS

Introduction 83

Materials and Methods 84

Results 86

Discussion 88

GENERAL DISCUSSION 103

LITERATURE CITED 106

BIOGRAPHICAL SKETCH 114

)

VI

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENVIRONMENTAL INFLUENCES ON
THE CRITICAL OXYGEN TENSION, WATER BALANCE,
METABOLISM, AND GROWTH OF REPTILIAN EMBRYOS

By

Yeong-Choy Kam
May, 1992

Chairman: Harvey B. Lillywhite
Major Department: Zoology

The effect of hypoxia on gas exchange of reptilian embryos was studied.
Embryos of Alligator mississippiensis. Caretta caretta. Pseudemvs nelsoni.

Elaphe obsoleta quadrivittata. and Sceloporus undulatus undulatus maintained
normal levels of oxygen consumption, Vo2 . to some degree of hypoxia,

indicating that they are metabolic regulators. A low Vo2 and a high eggshell
conductance for 02 explain how reptilian embryos maintain their Vo2 in

hypoxic conditions. Hypoxic tolerance decreased during incubation, as
indicated by an increase in the critical oxygen tension, Pc. The Pc increased
presumably because oxygen transport capacity, reflected by the critical oxygen
gradient, Gc, did not offset the increase of Vo2 during development.

Embryos of the Florida red-bellied turtle, P. nelsoni. were used as model
organisms to further examine environmental influences on gas exchange and

physiological responses of embryos to hypoxia and flooding. Oxygen
consumption and Pc of embryos were significantly affected by temperature. The
Qio values remained the same (Qio = 2-3) at different incubation days, thus
indicating that the thermal sensitivity of metabolism does not change during
development. The Pc was temperature-dependent and increased in direct
proportion to temperature at each incubation day. Oxygen consumption and Pc
of embryos were not affected by egg hydration. Eggs incubated in 13 %
gravimetric water content gained mass at a faster rate than those in 3 %
gravimetric water content, and the respective egg masses were statistically
different at day 30 and 39. However, oxygen consumption, critical oxygen
tensions, and hatchling mass of the two groups were not different. Exposure to
chronic hypoxia (10 % air) resulted in retarded growth, depressed metabolism,
and reduced hatchling mass but comparable incubation period and hatchability.
Embryos are flexible, structurally and physiologically, in enhancing oxygen
transport to compensate for hypoxic effects. Simulated flooding has dramatic
effects on water balance, embryonic metabolism and development of embryos.
One-day submergence in water did not affect water balance and oxygen
consumption of embryos, but did increase egg mortality. On the other hand, 3-
and 6-day submergence treatments affected water balance, oxygen
consumption and hatching success of eggs.

VIII

GENERAL INTRODUCTION

Eggs incubated underground are subjected to hypoxia (low oxygen
tension) when O2 concentration is reduced in the nest chamber or when
embryos outgrow the O2 diffusing capacity of their exchange systems
(Ackerman 1977; Metcalfe et al. 1984). The O2 concentration within a nest
chamber depends on the balance between the rate of O2 uptake of eggs and
the rate of O2 diffusion through soil (Ackerman 1977, 1981b; Packard and
Packard 1989). Hypoxia that occurs in the crocodile Crocodvlus. the green
sea turtle Chelonia. and the loggerhead sea turtle Caretta nests during the
second half of incubation is probably due to the large increase in oxygen
consumption (Ackerman 1977; Lutz and Dunbar-Cooper 1984). In addition,
microbial respiration could also deplete oxygen in the nest, as in incubating
mounds of the brush turkey, Alectura lathami (Seymour et. al. 1 986). Finally,
wetting of the incubating media by rainfall (Chabreck 1975), flooding (Plummer
1976), and high tides, reduces soil O2 diffusion (Kam 1988; Seymour et al.
1986) and might cause short-term, periodic hypoxia in nests.

Even if the nest chamber is normoxic (normal oxygen tension), embryos
might experience internal hypoxia because of the limitation of the O2 diffusing
capacity of the eggshell. Partial pressure of oxygen (P02) *n air space of

chicken eggs decreases during incubation due to restricted diffusion across

the eggshell (Temple and Metcalfe 1969; Tazawa 1980). Oxygen consumption
(V02) increases when eggs are exposed briefly to hyperoxia (Metcalfe et al.

1

2

1981 ; Stock et al. 1985; Stock and Metcalfe 1987). It has been postulated that
during late incubation, chick embryos outgrow the oxygen diffusing capacity of
their gas exchange systems; thus, embryos become metabolic conformers
during late incubation as found in megapode and chick eggs, in contrast to
being metabolic regulators during early incubation (Visschedijk et al. 1980;
Seymour 1985).

A metabolic regulator can maintain a constant Vo2 to some degree of
hypoxia, whereas a metabolic conformer has Vo2 proportional to
environmental P02 (Prosser 1973). It is not known whether reptilian embryos
are metabolic regulators or conformers, or how their development influences
metabolic regulation. Embryonic development is an energy-demanding
process, and growing embryos consume more and more oxygen. Earlier
studies on adult turtles (Belkin 1965), lizards (Boyer 1966), and plethodontid
salamanders (Beckenbach 1975) demonstrated that increased metabolism
due to elevated body temperature increases critical oxygen tensions (Pc, the
Po2 at which the oxygen consumption is first reduced) proportionally. This may

not be true for embryos because their oxygen transport system, including
chorioallantoic membrane and blood, develops as they grow and increases
the ability to extract oxygen (Tazawa 1980). In theory, if the oxygen transport
system did not develop as embryos grow, then the Pc of embryos would
increase with increased metabolism. In contrast, if the oxygen transport system
developed in concert with embryonic development, then developing embryos
might tolerate the same hypoxic conditions even at a higher metabolic level,
i.e, no resultant change in Pc.

Environmental variables such as temperature and hydric conditions also
interact with embryonic development in determining the Pc of embryos. Adult
reptiles increase their Pc in direct proportion to any given metabolic level as

3

body temperature changes (Belkin 1965; Boyer 1966). Direct and indirect

evidence indicate temperature affects the metabolism of eggs (Zarrow and

Pomerat 1937; Packard and Packard 1989; Deeming and Ferguson 1989), and
the Pc presumably will increase in proportion to the \lo2- Also, reptilian eggs

take up water from their surroundings under favorable conditions. The O2
conductance of the egg is inversely correlated to the amount of water uptake,
suggesting that O2 transport capacity of embryos is reduced as they developed
(Black et al. 1984).

If hypoxia does occur, what is its effect on embryonic metabolism and
how do reptilian embryos respond? Chronic hypoxia causes slower growth,
longer incubation time, and lower hatching success in embryonic turtles of
Caretta caretta and Chelonia mvdas (Ackerman 1 981 a). However, he did not
measure the oxygen concentration in the artificial nests, and thus the hypoxic
effects were not estimated. Further, physiological adjustments of embryos to
minimize the hypoxic effect have not been studied.

One extreme case of hypoxia occurs when eggs are flooded. Reptilian
eggs incubated underground are potentially subjected to flooding when water
level is raised by rainfall, high tides, or other natural phenomena. Laboratory
and field studies have demonstrated that flooding is a major cause of egg
mortality of Crocodvlus porosus (Webb et al. 1977), Alligator mississipiensis
(Hines et al. 1968, Joanen 1969, Kushlan and Kushlan 1980), and Trionyx m.
muticus (Plummer 1976). However, no attempt has been made to investigate
flooding influence on water balance, metabolic rate, or embryonic development
of eggs. When eggs are inundated, oxygen availability drops to a minimum
level. Eggshells are saturated with water, which favors water exchange but not
gas exchange. It is not known how embryos respond to a sudden change from
an aerial environment to an aqueous environment; and for those embryos that

4

survive, it is unclear if flooding has any effect on embryonic development and
metabolism.

The present study investigates gas exchange of reptilian embryos

exposed to hypoxic conditions. Specifically, I compare the effect of acute
hypoxia on Vo2 of embryos among five different species. Then I choose the

Florida red-bellied turtle, Pseudemvs nelsoni. as model organisms to
investigate (1) the environmental influences on the critical oxygen tensions of
eggs, (2) the physiological responses of embryos to hypoxia, and (3) the effect
of flooding on water balance, embryonic metabolism and growth of embryos.

CHAPTER 1

COMPARATIVE STUDY ON THE CRITICAL OXYGEN TENSION

OF REPTILIAN EMBRYOS

Introduction

Eggs incubated underground are subjected to hypoxia when O2
concentration is reduced in the nest chamber or when embryos outgrow the O2
diffusing capacity of their exchange systems (Ackerman 1977; Metcalfe et al.

1 984). The O2 concentration within a nest chamber depends upon the
balance between the rate of O2 uptake of eggs and the rate of O2 diffusion
through soil (Ackerman 1977, 1981b; Packard and Packard 1989). Hypoxia
that occurs in Crocodvlus. Chelonia. and Caretta nests during the second half
of incubation is probably due to the large increase in oxygen consumption
(Ackerman 1977; Lutz and Dunbar-Cooper 1984). In addition, microbial
respiration could also deplete oxygen in the nest, as in incubating mounds of
the brush turkey. Alectura lathami (Seymour et. al. 1986). Wetting of the
incubating media by rainfall (Chabreck 1975), flooding (Plummer 1976), and
high tides reduces soil O2 diffusion (Seymour et al. 1986; Kam 1988) and
might cause short-term, periodical hypoxia in nests.

Even if the nest chamber is normoxic, embryos might experience

hypoxia because of the limitation of O2 diffusing capacity of the eggshell. The
PO2 in air space of chicken eggs decreases during incubation due to restricted

diffusion across the eggshell (Temple and Metcalfe 1969; Tazawa 1980). Also,
embryonic oxygen consumption (Vo2) increases when the egg is exposed

briefly to hyperoxia (Metcalfe et al. 1981 ; Stock et al. 1985; Stock and

5

6

Metcalf el 987). It has been postulated that during late incubation chick
embryos outgrow the oxygen diffusing capacity of their gas exchange systems
(Metcalfe et al. 1984); thus, embryos become metabolic conformers during late
incubation, as found in megapode and chick eggs, in contrast to being
metabolic regulators during early incubation (Visschedijk et al. 1980; Seymour
1985).

A metabolic regulator can maintain a constant \Zo2 to some degree of
hypoxia, whereas a metabolic conformer has Vo2 proportional to the
environmental Po2 (Prosser 1973). It is not known whether reptilian embryos

are metabolic regulators or conformers, or how their development influences
their metabolic regulation. Black et al. (1984) reported that P02 gradients

across shells of python eggs decrease during incubation, suggesting that
embryos are subject to hypoxia as are avian embryos (Tazawa 1980).
Embryonic development is an energy-demanding process, and growing
embryos consume more and more oxygen. Earlier studies on adult turtles
(Belkin 1965), lizards (Boyer 1966), and plethodontid salamanders
(Beckenbach 1 975) showed that increased metabolism due to elevated body
temperature increases critical oxygen tensions (Pc). However, this may not be
true for embryos because their oxygen transport system, including
chorioallantoic membrane and blood, develops as they grow and increases
the ability to extract oxygen (Tazawa 1980). In theory, if the oxygen transport
system did not develop as embryos grow, then the Pc of embryos would
increase with increased metabolism. In contrast, if the oxygen transport system
developed in concert with embryonic development, then developing embryos
might tolerate the same hypoxic conditions even at a higher metabolic level,
i.e., no resultant change in Pc.

7

The purpose of this chapter is to examine the metabolism of reptilian
embryos as related to environmental oxygen and to investigate how embryonic
development influences the metabolic regulation. Critical O2 tension curves of
five species of reptilian eggs were determined throughout incubation.

Materials and Methods

Animals

Sixteen to twenty-four eggs of each of five species, the American
Alligator, Alligator mississippiensis. the loggerhead sea turtle, Caretta caretta.
the Florida red-bellied turtle, Pseudemvs nelsoni. the yellow rat snake, Elaphe
obsoleta quadrivittata. and the southern fence lizard. Sceloporus undulatus
undulatus. were used (Table 1-1 ). Caretta caretta eggs were collected directly
after oviposition at Melbourne Beach, Florida. Elaphe quadrivittata and fL. IL
undulatus eggs were oviposited in laboratory terraria by gravid females
collected in Alachua County, Florida. Alligator mississippiensis and P. nelsoni
eggs were collected from local alligator nests, and one egg from each clutch
was dissected and its age was estimated.

Eggs for each species were assigned equally to two or three boxes. All
except A. mississipiensis eggs were half-buried in sand with 3% gravimetric
water content (g water / g sand) and incubated at 30 °C. Alligator
mississippiensis eggs, which were housed in the Fish and Wildlife
Commission Laboratory at Gainesville, Florida, were half-buried in sphagnum
moss with 90-1 30 % gravimetric water content and incubated at 32 °C. The
boxes were covered with plastic sheets to reduce evaporative water loss, and
water was added as necessary.

8

Oxygen consumption measurements at different Pn2

The Vo2 was measured using a closed-system. Different sizes of glass

jars (Kerr Glass Inc.) fitted with two rubber stoppers and three-way stopcocks
were used as metabolic chambers. Chamber volume was determined by
weighing the jar empty and filled with water, then dividing the difference
between measurements by water density at room temperature.

Four to six eggs were randomly selected at early, middle, and late
stages of incubation (Table 1-1). One day before each experiment, eggs were
weighed to the nearest 0.001 g, and the length and width were measured to the
nearest 0.01 mm. An egg was placed in a cup inside a metabolic chamber
containing 5 ml of distilled water and a piece of wet filter paper to keep the
chamber at 100% relative humidity. The chambers were equilibrated at 30 °C
overnight.

At the onset of the experiment, several metabolic chambers were
submerged in a water bath at 30 °C except for the stopcocks . One empty
chamber served as a control. The chambers were connected via stopcocks by
Tygon tubing. Gas mixtures were made from nitrogen and air by means of a
Digamix gas mixing pump (Type M/300 a) and humidified by bubbling through
a gas-sealed flask containing distilled water at 30 °C. Embryos were allowed
to equilibrate at each gas mixture for 15-20 min after the chambers were
flushed. Preliminary experiments demonstrated that 15-20 min of equilibration
time produced the same results as 40 and 60 min of equilibration time.
Metabolic chambers were then re-flushed thoroughly, and the three-way
stopcocks of each chamber were closed quickly. The interval for gas sampling
depended upon the developmental stage of embryos such that no more than
1-2% of the total oxygen was consumed (Vleck 1987).

9

About 15-20 min before gas sampling an Applied Electro-chemistry S-3A
O2 analyzer was flushed with the gas mixture until a baseline was obtained on
a chart recorder and was maintained during measurements. Because the
delivery rate from the mixing pump was higher than needed, the gas mixture
was sampled by a pump located downstream with a smaller Tygon tubing
inside the delivery tubing. The gas mixture was directed through soda lime and
silica gel to absorb CO2 and water vapor respectively before being fed into the
O2 analyzer.

A gas sample from each chamber was taken using two 60 ml gas tight
syringes, each tipped with a three-way stopcock. One of these syringes was
filled with 25 ml of saturated gas mixture. The syringes were fitted on the
stopcocks of the chamber and clamped tightly onto an iron stand. As the 25 ml
gas mixture in one syringe was injected gradually into the chamber, 25 ml of
gas mixture was withdrawn simultaneously from the chamber into the other
syringe. This simultaneous 'push and puli' mixing movement was repeated
about ten times before a final gas sample was withdrawn and injected quickly
into the oxygen analyzer to measure oxygen concentration. This sampling
technique allowed the withdrawal of gas samples from the metabolic chamber
without creating negative pressures.

Immediately after the O2 concentration of all chambers was measured,
eggs were subjected to the next gas mixture, ranging from 90 to 20% air, and
the oxygen concentrations were determined by repeating the above protocols.
Each new gas mixture was 1 0 or 20% lower in air content than the previous
treatment. Every experiment was started at the same time of a day and
completed in the same day. A complete experiment usually lasted 10-16 h.

Each treatment did not last more than 2-3 h, and each egg was treated with at
least six gas mixtures at each of the three stages of incubation.

10

The egg volume of ellipsoid eggs was calculated by use of the equation,
V=4/3T a2b, where V is volume (ml), a is the shortest diameter (cm), and b is

the longest diameter (cm); whereas volume of spherical eggs was calculated
by the equation, 4/3 ¥ r3, where r is the radius (cm). Oxygen consumption, Vo2

(ml STPD/ h-egg), was calculated using the equation, Vo2 =V(FI()2*FEo2)/0*
FEo2)t. where V is the dry, CC>2-free air (ml), Flo2 and FE02 are the initial and
final oxygen fraction of a measurement (%), and t is the duration (h) from start
to end of a measurement (Vleck 1987).

Experimental analysis

The surface areas of spherical eggs and ellipsoid eggs were
respectively determined using the equation A=4T r2, where A is surface area
(cm2) and r is radius (cm) and the equation A=2TT b2 + 2¥ (ab/e)(sin’1e),
where a = 1/2 length of the egg, b = 1/2 width of the egg, and eccentricity e =

[1 -(b/a)2]0-5 (Ackerman et al. 1 985) respectively.

Measurements from eggs that did not hatch were discarded from
analysis. Three caretta eggs were repeatedly used on days 39 and 45.

The results were not different from non-repeated measurements; therefore,
data were pooled. The V o2 of tw0 £L UL undulatus eggs were measured at

day 1 8 in one chamber, and the average value was used. Experimental eggs
hatched at the same time as controls and no external deformities were visible.
The critical oxygen tension, Pc (the Po2 at which the oxygen

consumption is first reduced) and the critical oxygen gradient, Gc (the slope of
regression line below the Pc) were determined using a BASIC program that
fits a two-segmented straight line (Yeager and Ultsch 1989). These data were
analyzed individually and averaged. All means are expressed as mean (+

SE) unless otherwise noted. Reduced major axis regression analyses were

1 1

used because variables in regression analyses were random measurements
of a population with error.

Results

All embryos maintained Vo2 to some degree of hypoxia, although the

degree of hypoxic tolerance varied among species (Figs. 1-1 to 1-5; Table 1-2).

Sceloporus. u. undulatus was the most hypoxic-tolerant species, maintaining
its Vo2 when P02 was 64 mmHg at day 25 of incubation. In contrast, C. caretta

had the lowest hypoxic tolerance and did not maintain its Vo2 when P02 was

lower than 123 mmHg following day 22 of incubation.

The degree of hypoxic tolerance of embryos decreased significantly

during incubation except in C. caretta. as indicated by the increase of the
critical oxygen tensions (Pc) (Table 1-2). The V02 and Gc also increased

significantly during incubation, but the latter increased at a slower rate than the

former. In some cases, the rate of increase of Gc slowed down at late
incubation. For example, the Vo2 of F\ nelsoni increased 249%, 191%, and

71 % from day 1 2 to 26, day 26 to 34, and day 34 to 40 respectively, but the Gc
increased 222%, 132%, and 37 % at the same periods (Table 1-2).

The correlation between log surface area/volume ratio (S/V) of eggs and
log Pc was time-dependent (Table 1-3). The Pc was significantly correlated

with S/V at early (P < 0.01) but not at middle and late the incubation period (P
> 0.05). Further, the relative Vo2 (Vo2 at any incubation period expressed as a

fraction of that at late the incubation period) was calculated to compare

interspecifically the sensitivity of Pc to metabolic rate (Table 1-4). The Pc of 3^
y L undulatus was most sensitive to Vo2. whereas A,, mississippiensis. EL (L.

12

guadrivittata. and EL nelsoni were the least sensitive. At any given level of
relative V02. CL caretta had a Pc higher than that of the other species.

Discussion

Effects of development on oxygen consumption and P£

The fact that reptilian embryos could maintain constant Vo2 over a

range of environmental Po2 during development indicates that they are
metabolic regulators (Figs. 1-1 to 1-5). Different patterns have been reported
for bird embryos in that embryos of the Mallee fowl, Leipoa ocellata. are
metabolic regulators during early and middle incubation, but become
metabolic conformers at late incubation (Seymour 1985). Embryos of chickens
at day 16-19 could not maintain normal rates of metabolism when exposed to
acute hypoxia (Visschedijk et al. 1980; Ar et al. 1991). A similar finding has
been reported for the Canada goose fBranta canadensis) where embryos
could not maintain normal metabolism when air cell oxygen tensions were
reduced more than 5 mmHg (Snyder et al. 1 982). But results from that same
study also show that embryos of the bar-headed goose (Anser indicus) are
metabolic regulators and have critical oxygen tensions 33 mmHg lower than
that of the Canada goose. It appears that eggs of the bar-headed goose, laid
at high altitude (as high as 5500 m), possess genetic adaptations which allow
them to maintain normal oxygen consumption in hypoxic conditions (Snyder et
al. 1982).

Reptilian embryos tolerate hypoxia better than bird embryos, and this
difference is mainly attributed to the low Vo2 of embryos (Dmi'el 1970;

Ackerman 1 980;1 981 b) and, in some cases, the high O2 conductance of

13

reptilian eggs (Ackerman and Prange 1972; Lutz et al. 1980; Feder et al.

1982). Bird and reptilian eggs with similar masses have different ^02^ *or

example, the Vo2 of the green turtle (Chelonia mvdasl is only one-fourth that of

chicken eggs at late incubation (Ackerman 1980). This difference is due to a
lower incubation temperature and rate of growth in green turtle eggs
(Ackerman 1980). Conversely, O2 conductance of reptilian eggs varies among
shell types and egg hydrations (Lutz et al. 1980; Feder et al. 1982; Black et al.
1984). American crocodile (Crocodvlus acutusl eggs have a rigid eggshell
which contributes the majority of the resistance to oxygen diffusion. Oxygen
permeability of moist and dry eggshell-membrane complexes of C. acutus is
similar to that of chicken eggs (Lutz et al. 1 980). In contrast, the moist and dry
eggshell-membrane complexes of flexible eggs, as found in the snapping turtle
(Chelvdra serpentinal. have O2 permeability ten times higher than that of
chicken eggs (Feder et al. 1982).

The increase of Pc during incubation in this study resembles a finding

previously reported for Mallee fowl eggs. This similarity suggests that both

Mallee fowl and reptilian eggs have difficulty taking up sufficient oxygen to

maintain their metabolism. Any Pc level can be described by the interaction of
Vo2 and Gc. Embryonic development increases Vo2 which increases the Pc,

whereas circulatory and respiratory developments promote the Gc which
reduces the Pc. If oxygen diffuses across the respiratory surface at the
maximum rate at a Po2 below the Pc, Gc is a measure of the maximum overall

oxygen transport capability (Belkin 1965; Beckenbach 1975; Bradford and
Seymour 1988). An increase of Pc over incubation indicates that Gc does not
develop in concert with embryonic development; in other words, although the
Gc increases during development, it approaches its upper limit at late

14

incubation such that it cannot catch up with the pace of the increase of Vo2.

Therefore, the embryos have to increase their Pc at a higher metabolic level.
Evidence suggests that the inability of Gc to match the increased Vo2 is

due to diffusion limitation of the gas exchange surface. The Pc of hatchlings (P*
nelsoni. 70±4.23 mmHg, n=4: C. caretta. 44.5±1.80 mmHg, n=5) is significantly
lower than those of embryos at late incubation (Table 1-2) (P<0.001). The
principal factor responsible for these differences may be the types of gas
exchange organs (lung vs chorioallantoic membrane) (personal observation).

A similar finding has been reported for the frog, Pseudophrvne bibroni. in that
eggs have much higher critical oxygen tensions than tadpoles (111 and 31
mmHg respectively, Bradford and Seymour 1 988). This difference is due to the
fact that tadpoles can ventilate gills and surface area to increase oxygen
uptake in response to hypoxia, whereas eggs cannot. An earlier study of
Burmese python eggs showed that the P02 beneath the eggshell decreases

during development, suggesting that the eggshell-membrane complex poses a
significant resistance to O2 diffusion (Black et al. 1984). Similar results were
found in chicken eggs where the air cell P02 drops during incubation due to

restricted diffusion through the shell (Tazawa 1980). The oxygen diffusing
capacity of eggs reaches a plateau at late incubation, and the supply of oxygen
to tissues is maximized (Temple and Metcalfe 1970; Metcalfe et al. 1979;
Metcalfe et al. 1 984; Ar et al. 1 991 ). Thus, when embryos are exposed to
hypoxia, they are no longer able to maintain normal rates of oxygen
consumption and become metabolic conformers (Visschedijk et al. 1980; Ar et
al. 1991).

15

Interspecific comparisons of

The Pc of turtle eggs did not differ from that of squamate and alligator
eggs, which is opposite to the observations on adults. The Pc of adult turtles is
about 8-24 mmHg when compared to that of lizards, snakes, and crocodilians
of about 70 mmHg (Bennett 1976; Ultsch and Anderson 1988). Adult turtles
exhibit a larger change in ventilation than do other reptiles when exposed to
hypoxia (Boyer 1966; Glass et al. 1983), and this types of adjustment is not
possible in eggs where oxygen is exchanged via diffusion over the
chorioallantoic membrane.

The Pc of eggs was significantly correlated with SA/ ratio at early

incubation in that smaller eggs had a lower Pc than did larger eggs (Table 1-3).
At a given relative Vo2 level, smaller eggs had larger Gc than larger eggs; thus

more oxygen diffused across the eggshell in a given amount of time. Similar
findings have been reported for lungless salamanders where the Pc is
correlated with S/V ratio so that a smaller one has a lower Pc than a larger one
(Beckenbach 1975). Interestingly, the correlationship between SA/ ratio and Pc
of eggs decreased over the incubation period and became insignificant during
middle and late incubation (Table 1-3). This difference may relate to possible
differences in development of respiratory and circulatory systems, water relation
of eggs and other factors.

In this study, the sensitivity of Pc to metabolic rate differs among species

(Fig. 1 -6). The Pc of S.. iL undulatus increased 83% from day 25 to day 42,

whereas those of £L caretta and mississippiensis increased 9% from day 22

to day 45 and 32% from day 29 to day 56 respectively. The Pc of

undulatus may be more sensitive to Vo2 than those of other species is because

it has a higher water uptake. Eggs of S. u. undulatus increased in mass and
attained 150.2 ± 5.78 % of initial mass at day 42, which is about 5-50 fold higher

16

when compared to eggs of other species ( £* quadrivittata. 33.7 ± 1 .1 8 % at
day 47, £* caEfitia, 5.9 ± 0.91 % at day 45, A*, mississippiensi. 2.84 ± 1 .22 % at
day 57, and P. nelsoni.12.99 ± 2.00 % at day 40). The tremendous amount of
absorbed water in ll undulatus presumably forms a diffusion barrier which
significantly reduces the O2 conductance (Black et al. 1984) and increases Pc.
The large change of Pc, subsequently, reduces the correlation between the Pc
and S/V ratio (Table 1-3).

Ecological Considerations

All five species exhibited a similar pattern of metabolic regulation during
incubation, i.e., increasing dependence of embryonic oxygen consumption on
ambient P02- The similarity of Pc of embryos between species late in incubation

suggests hypoxic tolerance of reptilian embryos is independent of shell types
and egg size. Also, the ability of embryos to tolerate hypoxia does not correlate
with their habitats. For example, caretta embryos are the least hypoxia
tolerant species even though they clearly experience hypoxic stress when the
P02 of nest chambers decreases rapidly during the second half of incubation

(Ackerman 1 977)(Table 1 -2). In contrast, quadrivittata and u. undulatus
embryos are the most hypoxia tolerant species even though they probably do
not experience hypoxic stress during incubation. These species are shallow
nesters, and they lay eggs in hollow, rotting logs and vegetation, under railroad
ties, beneath rocks, at the base of grass clumps and sawdust piles (Ashton and
Ashton 1 985, 1 988; Ernest and Barbour 1 989).

However, the generalizations about the ecological adaptation of reptilian
embryos to hypoxic conditions are far from resolved due to extremely limited
information concerning the responses of embryos to chronic hypoxia. Earlier
studies on embryos of other animals show much flexibilities in adapting to their

ft

17

hypoxic environments structurally or physiologically. Bullfrog tadpoles exhibit
morphological changes in response to chronic hypoxia, including thinning the
blood-water barrier by 50% and vasoproliferation in the skin, both of which
probably facilitate gaseous diffusion (Burggren and Mwalukoma 1983). In
chicken eggs, chronic hypoxia induces vasoproliferation in the chorioallantoic
membrane, and thus increases surface area for gas exchange (Dusseau and
Hutchins 1988; Strick et al. 1991). Exposure to hypoxia also increases
hemoglobin-oxygen affinity in chicken and domestic fowl embryos (Black and
Snyder 1980; Baumann et al. 1983) and heart mass of domestic chicken and
Canada goose hatchlings (Black and Snyder 1980; Snyder et al. 1982).
Hypoxia probably induces a redistribution of blood flow to embryonic tissue
such that blood is preferentially distributed to an area with a high priority (e.g.
heart) than to an area with a low priority (e.g. limb muscle). As a result, chicks
that hatched in the hypoxic conditions have heavier hearts and lighter
gastrocnemius muscles when compared to those hatched in the normoxic
conditions, even though their body masses are the same (Black and Snyder
1980).

18

Table 1-1 Initial mass, incubation time, sample size, and sampling day of five

species. Eggs were incubated at 30 °C with the exception of A,
mississippiensis eggs (32 °C.). Egg mass of A, mississippiensis
and P. nelsoni was measured at day 29 and 12 respectively.
Values are reported as mean with standard error in parenthesis.

Species

no.

egg

Initial egg
mass (g)

Inc.

time

(days)

Sampling day
(sample size)

A. mississippiensis

18

84.25

(1.39)

60

(0.3)

29(4), 40(4), 56(5)

C. caretta

24

43.68

(1.50)

57

(0.7)

22(5), 32(5), 39(6), 45(5)

P. nelsoni

24

13.02

(0.19)

50

(0.4)

12(4), 26(6), 34(4), 40(4)

E. o. auadrivittata

16

7.33

(0.09)

61

(0.7)

14(4), 31(4), 47(4)

S. u. undulatus

20

0.336

(0.004)

44

(0.3)

16(6), 33(6), 42(5)

19

Table 1-2. Critical oxygen tensions (Pc), critical oxygen gradients (Gc), and

oxygen consumptions (V02 ) of five species. ANOVA tests show

that all Pc, V02. and Gc of each species are at least significantly

different at the 0.05 level over incubation with the exception of Pc
of C. caretta (P > 0.05). Values are reported as mean with SE in
parenthesis.

SPECIES

Age

(Days)

Pc

(mmHg)

Vo2

(ml/h)

Gc x 1 0-3
(ml/h mmHg)

A. mississiDDiensis

29

86.6

(3.80)

1.04

(0.11)

12.11

(2.11)

40

103.8

(1.18)

2.91

(0.27)

25.59

(2.46)

56

114.8

(5.28)

9.43

(0.58)

80.6

(2.28)

C. caretta

22

123.6

(3.23)

0.33

(0.01)

2.81

(0.10)

32

122.6

(4.16)

1.32

(0.03)

12.35

(0.49)

39

127.5

(4.65)

2.99

(0.10)

29.50

(1.72)

45

135.8

(4.96)

4.60

(0.27)

42.85

(3.49)

20

Table 1-2-continued

SPECIES

Age

(Days)

Pc

(mmHg)

V02

(ml/h)

Gc x 1 0-3
(ml/h mmHg)

P. nelsoni

12

74.6

0.11

1.48

(7.56)

(0.001)

(0.08)

26

86.0

0.39

4.76

(4.23)

(0.01)

(0.39)

34

106.3

1.13

10.96

(2.41)

(0.02)

(0.36)

40

120.5

1.93

14.98

(0.94)

(0.04)

(0.03)

E. o. quadrivittata

14

100.2

0.37

2.57

(3.12)

(0.003)

(0.19)

31

108.0

0.63

5.24

(2.75)

(0.01)

(0.19)

47

117.6

1.16

9.20

(4.09)

(0.05)

(0.50)

S. u. undulatus

16

64.3

0.05

0.52

(7.30)

(0.003)

(0.11)

33

92.1

0.09

0.76)

(2.51)

(0.002)

(0.01)

42

117.0

0.14

1.14

(4.52)

(0.002)

(0.08)

21

Table 1-3. Reduced major axis regressions for the critical oxygen tension (Pc)

in relation to the surface area/volume ratio of eggs. Data for all
species were pooled at each incubation stage. Data of day 39 of
£L caretta and day 34 of IL nelsoni were omitted. N = sample size;
r = correlation coefficient ; P = probability of the correlation
coefficient.

N

Regression equations

r

P

Early incubation

18

Log Pc = 1 .79 - 0.523 Log S/V

0.64

<.01

Middle incubation

24

Log Pc = 1.86 - 0.395 Log S/V

0.32

>.05

Late incubation

23

Log Pc = 2.02 - 0.195 Log S/V

0.20

>.05

22

Table 1 -4. Reduced major axis regressions for the critical oxygen tension

(Pc) in relation to the relative oxygen consumption (RVo2)of eggs.
N = sample size; r = correlation coefficient; P = probability of the
correlation coefficient.

N

Regression equations

r

P

A. mississiDDiensis

12

Log Pc= 2.07 + 0.145 Log RV02

0.85

<.001

C. caretta

21

Log Pc = 2.14 + 0.075 Log RV02

0.35

>.05

P. nelsoni

18

Loq P c = 2.08 + 0.208 Loq RV02

0.84

<.001

E. o. quadrivittata

11

Log Pc = 2.08 + 0.178 Log RVo2

0.78

<.01

S. u. undulatus

12

Log Pc = 2.09 + 0.770 Log RVq2

0.90

<.001

23

Q H 1 1 1 1 > 1 1 i

0 40 80 120 160

P02 (mmHg)

Figure 1-1. Oxygen consumption of Alligator mississippiensis embryos as a

function of oxygen tension at different incubation days. Values are
means + SE.

24

o

CL

CO £

O

O

CM

o

Figure 1-2.

Oxygen consumption of Caretta caretta embryos as a function of
oxygen tension at different incubation days. Values are means ±

CONSUMPTION

25

_i “i — “ — i 1 1 1 1 1

0 40 80 120 160

Pq2 (mmHg)

Figure 1 -3. Oxygen consumption (ml / h) of Pseudemvs nelsoni embryos as a

function of oxygen tension at different incubation days. Values are
means ± SE.

CONSUMPTION

26

Figure 1 -4. Oxygen consumption (ml / h) of Elaphe obsoleta quadrivittata

embryos as a function of oxygen tension at different incubation
days. Values are means ± SE.

CONSUMPTION

27

C\J

1 1 1 r ) I

0 40 80 120 160

P o2 (mmHg)

Figure 1 -5. Oxygen consumption (ml / h) of Sceloporus undulatus undulatus

embryos as a function of oxygen tension at different incubation
days. Values are means ± SE.

28

140-

al

_120‘

CD

E 100-
£

A

o 80-

■

60-
40 J

A

.01

o o
A □

□ ■

□

n r

I I' I I I I I I - T

.1

1

» I III T|

1 0

02 CONSUMPTION (ml / h)

Figure 1-6. Relationship between critical oxygen tension (Pc) and oxygen

consumption in embryos of reptilian eggs. A. mississiopiensis
(closed circles), C. caretta (open circles). P. nelsoni (closed
squares), E q* quadrivittata (open squares), and 3,. IL unduiatu?
(open triangles).

CHAPTER 2

TEMPERATURE EFFECTS ON METABOLISM AND CRITICAL OXYGEN
TENSION OF FLORIDA RED-BELLIED TURTLE (PSEUDEMYS NELSONH

EMBRYOS

Introduction

Many female red-bellied turtles (Pseudemys nelsonil that are found in
wetlands or wet prairies in Florida lay their eggs in nest mounds of the
American alligator (Alligator mississippiensist. Most alligator nest mounds are
dome-shaped and in close proximity to permanent water (Goodwin and Marion
1978). Oxygen content in a nest cavity depends on nest location and rainfall
during the nesting season and could be reduced by microbial respiration, egg
metabolism, and a low gas conductance of the nest due to wetting of incubation
medium by rainfall or flooding.

The hypoxic tolerance of red-bellied turtle embryos has been assessed
by measuring the critical oxygen tension (see Chapter 1). Turtle embryos are
metabolic regulators during incubation, i.e., they can maintain a constant level
of aerobic metabolism over a range of Po2. Hypoxic tolerance, however,

decreases during incubation, as indicated by an increase in critical oxygen
tension, Pc, suggesting that the hypoxic tolerance is influenced by embryonic
development.

The hypoxic problem is complicated by the thermal condition of nest
mounds. About 54% of nests are located in the open field, and receives direct
solar radiation during the day (Goodwin and Marion 1978). Depending on the
location in the nest mound, the turtle eggs could experience a wide range of

29

30

temperatures. The influence of temperature on hypoxic tolerance of reptilian
embryos has not been studied. Lungless plethodontids decrease their hypoxic
tolerance in direct proportion to any given metabolic level as body temperature
changes (Beckenbach 1975). In contrast, the hypoxic tolerance of some
freshwater fishes is independent of temperature, suggesting the presence of
adaptive adjustment(s) in response to temperature changes (Ultsch et al. 1978;
Ott et al. 1 980).

The objectives of this study were to investigate the effects of temperature
on the hypoxic tolerance and metabolism of embryos of the Florida red-bellied
turtle. Hypoxic tolerance was determined by measuring the critical oxygen
tension of embryos. Embryonic metabolism was also reported at different
temperatures to reveal the thermal sensitivity of metabolism of embryos during
development.

Materials and Methods

Animals

A total of three clutches of red-bellied turtle eggs were collected from
alligator nests in Florida during June and July, 1991 . One egg from each clutch
was opened to estimate age. Twenty eggs were assigned to each of three
temperatures: 22, 27 and 32 °C. All eggs were half-buried in sand with 4%
gravimetric water content and incubated at 30 °C. Boxes were covered with
plastic sheets to reduce evaporative water loss, and water was added as
necessary.

31

Oxygen consumption measurements at different Pn2

The critical oxygen tension was determined by a method described
elsewhere (see chapter 1). Briefly, each egg was placed in a cup inside a
metabolic chamber containing 5 mi of distilled water, and the Pc was
determined at the assigned temperature. Two metabolic chambers were
submerged in each of the three water baths at 22, 27, or 32 °C for 3 hours
before measurements were taken. Chambers were flushed and reflushed with
a humidified gas mixture before stopcocks were closed. A 30 ml gas sample
was collected from each chamber at the end of each measurement, and the
final oxygen fraction was determined by injecting the sample into an Applied
Electro-chemistry S-3A O2 analyzer. Immediately after the O2 concentration of
all chambers was measured, eggs were subjected to the next gas mixture,
ranging from 90 to 10% air, and the oxygen concentrations were determined

by repeating the above protocol. Each new gas mixture was 5 to 20 % lower in
air content than the previous treatment. Oxygen consumption, Vo2 (ml. STDP /

h egg), was calculated using the equation, Vo2 = V(Flo2-FEo2)/(1-FEo2)t,

where V is the dry, C02*free air (ml), Flo2 anc* FEo2 are the initial and final

oxygen fraction of a measurement, and t is the duration (h) from start to end of

a measurement (Vleck 1987).

Experimental analysis

The critical oxygen tension, Pc (the P02 31 which the oxygen
consumption is first reduced) was determined using a BASIC program that fits
a two-segmented straight line (Yeager and Ultsch 1989). Data were analyzed
individually and averaged. Oxygen consumption was adjusted using analysis
of covariance (ANCOVA) and the initial mass was used as covariate. Data

32

were analyzed individually and then averaged. All means are expressed as
mean (± SE) unless otherwise noted.

Results

The critical oxygen tension curves of red-bellied turtle embryos at

different temperatures and three incubation days are illustrated in Figures 2-1
to 2-3. Embryos at all treatments maintained Vo2 to some degree of hypoxia,

although the hypoxic tolerance varied among temperature treatments and
developmental conditions. Embryos exposed to 22 °C at day 39 were the
most hypoxia-tolerant individuals, maintaining constant metabolism when P02

was at or above 47.7 mmHg. In contrast, embryos that were exposed to 32 °C
at day 39 had the lowest hypoxic tolerance, and metabolism declined when
P02 was lower than 117 mmHg.

A two-way ANOVA shows that oxygen consumption was significantly

different among incubation days and temperatures (P < 0.001 and P < 0.001
respectively) (Fig. 2-4 and 2-6). Embryos increased Vo2 linearly during

development at all three temperatures. Embryos exposed to high
temperatures increased Vo2 faster than when exposed to low temperatures,

suggesting an interaction between the two variables (P < 0.01 )(Fig. 2-6). At a

given incubation day, increasing temperature also produced a linear increase
in V02 (Fig- 2-5).

Similar analysis shows that Pc was significantly different among
temperatures (P < 0.001 , Fig. 2-5) but similar among incubation days (P =
0.804, Fig. 2-7). Embryos exposed to 27 and 32 °C showed a slight increase
or no change, respectively, in the Pc during development. In contrast, the Pc

33

of embryos exposed to 22 °C decreased during development (Fig. 2-7).

Increasing temperature increased the Pc at each day of incubation (Fig. 2-5).
The sensitivity of Pc to Vo2 was highest when embryos were 16 days

old, followed by embryos of 30 and 39 days (Fig. 2-8). The Qio values, that

were between 2-3, were higher at low temperatures than at high temperatures
(Table 2-1). Thermal dependence of Vo2 remained the same with increasing

age (Table 2-1). The Qio values between 22-32 °C for embryos at day 16, 30,
and 39 were 2.58, 2.28, and 2.48 respectively.

Discussion

Temperature and oxygen consumption

Temperature sensitivity of the metabolic rates of turtle embryos agrees
with the study of Zarrow and Pomerat (1937) on the effect of temperature on egg
metabolism of the smooth green snake (Qpheodrvs vernalisl. There are
numerous studies on the effects of temperature on the embryonic development
of reptiles where eggs have been incubated in two or more constant
temperature regimes. Eggs that are incubated at warm temperatures grow
faster and hatch earlier than those incubated at cool temperatures. These
studies have assumed that egg metabolism is temperature-dependent, but this
assumption has never been tested extensively. Measurements of snake
embryos by Zarrow and Pomerat (1937) and turtle embryos (this study) provide
direct evidence for thermal dependence of embryonic metabolism in these two
species. The assumption is probably true for species that grow and develop
faster in a warm temperature than in a cool temperature simply because rapid

34

growth and short incubation period most certainly reflect higher rates of
metabolism (Ewert 1979; Packard and Packard 1989).

The Qio values of 2-3 measured in red-bellied turtle embryos are rather
typical of biological reactions. Similar Qio values for embryos at day 16, 30,
and 39 indicate that thermal dependence is similar at different incubation
days.

Surprisingly little is known about the thermal dependence of embryonic
metabolism in birds. Embryos of domestic fowl have a low Qio of 1 .0 for
temperatures between 34-40 °C (Greiff 1952), whereas embryos of
Heermann’s gull (Larus heermanni) have Qios of 2.05 between 20-30 °C, and
1.0 between 30-40 °C (Bennett and Dawson 1979). The Qio values of avian
embryos are therefore generally lower than those of red-bellied turtle
embryos, but the reasons are unclear.

The biological significance of a Qio value of 1.0 in Heermann's gull
embryos may be that thermal independence within a range of temperature
associated with incubation minimizes developmental and physiological
disruptions (Bennett and Dawson 1979). The thermal environments of
Heermann’s gull's nests vary tremendously due to intense solar radiation
during the day, and unattended eggs may undergo a wide thermal fluctuations
(Bennett and Dawson 1979). Low metabolic sensitivity to acute changes of
temperature within certain ranges is commonly found in invertebrates that
encounter abrupt changes in their environmental temperature (Newell and
Pye 1971; Newell 1979). Compared with Qio values of Heermann’s gull
embryos, the relatively high Qio of red-bellied turtle embryos indicates that
such an adaptation does not occur in this species.

Although metabolic Qio values of reptilian embryos have not been
measured before, indirect evidence suggests that Qio values of reptilian

35

embryos varies interspecifically. Temperature effect on incubation period of
turtle embryos was calculated based on the data compiled in Table 4 from
Ewert's paper (1979). The incubation period of eggs incubated at 25 and 30 °C
was compared. An increase of incubating temperature by 5 °C shortens the
incubation period from 2.5 days in Kinosternon flavescens to 86.1 days in
Kinosternon scorpioides. Of the 29 species examined, 24 species have
incubation periods of 20-40 days shorter at 30 °C than at 25 °C. These
interspecific differences in incubation period imply that the thermal dependence
of embryonic metabolism is different.

Intraspecifically, populations from different latitudes have different
responses to a 5 °C change in incubation temperature. The difference in
incubation period between 25 and 30 °C increases as latitude decreases is
found in Sternotherus odoratus. Chelvdra serpentina. Terrapens Carolina,
Chrvsemvs picta. and Pseudemvs scripta. In others, the difference remains the
same at different latitudes, e.g. in Graptemvs pseudoaeograohica.

The interspecific and latitudinal differences in incubation period in
response to an increase of incubating temperature by 5 °C provide excellent
opportunities to examine thermal relations of reptilian embryos associated with
different environments. For example, if the above observations reflect the
differences in thermal dependence, then the Qio of flavescens would be very
small when compared to that of K. scorpioides. and Qio of northern populations
would be smaller than that of southern populations.

Temperature and critical oxygen tension

Any Pc level can be described by an interaction of Vo2 and critical

oxygen gradient, Gc (the slope of the regression line at P02 below the Pc). If
oxygen diffuses across the respiratory surface at the maximum rate at P02

36

below the Pc, then Gc is a measure of the maximum overall oxygen transport
capability (Belkin 1965; Beckenbach 1975; Bradford and Seymour 1988).

Any enhancement of the circulatory or respiratory capability of an animal that
increases oxygen transport will increase the Gc.

At each incubation day, Pc increases directly proportional to
temperature (Fig. 2-1 to 2-3 and 2-5), indicating Pc is temperature-dependent.
Evidently, acute exposure to temperature changes does not allow time for
major structural and biochemical adjustments. In other words, the Gc could
not be altered much in a short duration of temperature exposure. Thus, a
further increase in metabolism due to elevating temperature results in an
increase of Pc (Fig. 2-5). This findings agree with earlier studies on
plethodontid salamanders (Beckenbach 1975), but there are no data
available to allow comparisons with embryos of other reptiles or birds.

The finding that Pc is temperature-dependent is opposite to earlier
studies in fishes where the Pc of most species examined remained constant or
decreased with increasing temperature ( Ultsch et al. 1978; Ott et al. 1980) .
The discrepancy is probably due to differences in experimental protocols.
Fishes were well acclimated to test temperatures, whereas plethodontids and
turtle embryos were not exposed to test temperatures only at the day of
experiment. Chronic exposure to a warm temperature could induce
physiological changes such as ventilation, heart rate, and hematocrit to
facilitate oxygen transport (Spitzer et al. 1968).

Reptilian and avian embryos obtain air through eggshell pores or
spaces by diffusion. Depending upon the resistances of diffusion pathways,
the diffusion rate of a gas could vary among or within species (Carey et al.
1982). At each day of incubation, embryos are able to maintain resting
metabolism when the ambient P02 is as low as 110-120 mmHg. In contrast,

37

Temple and Metcalfe (1969) and Ar et al. (1991) reported that the gas
exchange of avian eggs is near maximum under normoxic conditions.

Embryos of 16 days could not maintain resting metabolism when subjected to
acute hypoxia (below 159 mmHg), indicating that they are metabolic
conformers (Ar. et al. 1991). What happens to embryonic metabolism if the
egg temperature is elevated above normal? Because avian embryos operate
at a maximum rate of gas diffusion under normal conditions, the additional
oxygen demand due to increased temperature could probably not be
achieved. This would not produce a change in oxygen consumption, which
might offer an alternative explanation for why embryos of Heermann's gull
have a Qio of 1 at 30-40 °C (Bennett and Dawson 1979).

Development and critical oxygen tension

Any changes in Pc during development result from dynamic interaction
between Vo2 and Gc. Embryonic development increases Vo2. which

increases the Pc. Conversely, development of circulatory and respiratory
systems promotes oxygen transport and reduces the Pc by changing the Gc.

The effect of incubation day on Pc varies among temperatures. At 27 °C,
the Pc increases as embryos develop, which is similar to findings in an earlier
study (see chapter 1 ). However, the Pc of embryos at 32 °C is maintained high
at between 110 and 117 mmHg, and does not change much during incubation.
It is surprising to find that the Pc of embryos at day 16 is 1 16 mmHg, while an
earlier study showed that values for Pc on days 12 and 26 at 30 °C were only
75 and 86 mmHg respectively (see chapter 1). Two possible reasons might
account for the high Pc at day 16. Firstly, the chorioallantoic membrane has
not developed completely, and oxygen transport capacity is not as high as that
in older embryosis. Secondly, embryos do not function normally at such high

38

temperatures. For most embryonic turtles, 32 °C represents a high limit of
thermal tolerance (Packard and Packard 1989). In bird embryos the tolerance
to acute heat exposure is low at early development (Moreng and Shaffner
1951; MacMullan and Eberhart 1953; Romanoff 1960).

At 22 °C, the Pc of embryos was low, and it decreased at the older age
(Fig. 2-7). As embryos grew, the oxygen transport system, including
chorioallantoic membrane and circulatory system, developed to meet
increasing oxygen demand. Turtle eggs in this study were incubated at
constant 30 °C, so at any given time, the oxygen transport system developed in
response to the metabolic demand of embryos at 30 °C. However, when
embryos were acclimated at 22 °C, oxygen consumption decreased and the
metabolic demand was low. As a result, embryos at 22 °C could maintain
resting metabolism at even lower hypoxic conditions. Similarly, ectothermic
animals behaviorally lower body temperature when exposed to hypoxia,
thereby lowering the metabolic demand and enabling the animals to maintain
resting metabolism at more severe hypoxic conditions (Hicks and Wood 1985;
Wood et al. 1987).

Ecological considerations

Results of this study have shown that turtle embryos are hypoxia-
tolerant organisms, and their hypoxic tolerance decreases with increased
temperature (Table 2-1). Thus, it appears to be advantageous to bury eggs
deep in the nest mounds of alligators. This is because the location of the turtle
nest cavities in alligator nest mounds is a compromise between hypoxia and
temperature. A shallow nest in the upper part of a nest mound experiences
little or no hypoxia but a wide range of temperature. The situation is just the
opposite for a nest located deeper inside a nest mound.

39

This speculation is supported by an earlier observation that eggs are
usually located either level with or below the alligator nest cavities (Goodwin
and Marion 1977). Alligator nest cavities have temperatures that vary only 1 .2
°C daily, while the mean hourly air temperature fluctuates 9.3 °C daily
(Chabreck 1973). Thus, by putting eggs at the vicinity of alligator nest cavities,
turtle embryos experience a rather stable thermal environment.

40

Table 2-1. Oxyen consumption (Vo2) and critical oxygen tension (Pc) at

different temperatures and incubation times. Values are reported
as means with standard error in parentheses. Qio is calculated for
each successive increase in temperature. Temp = temperature; N
= sample size; W = egg mass.

Temp

(°C)

N

W (g)

V02

(ml / h)

Pc

(mmHg)

Qio

DAY 16

22

5

13.10

0.092

62.5

(0.11)

(0.004)

(3.5)

2.98

27

5

12.86

0.159

79.6

(0.27)

(0.006)

(3.2)

2.22

32

5

13.31

0.237

116.4

(0.32)

(0.003)

(2.3)

DAY 30

22

4

13.81

0.534

60.7

(0.40)

(0.021)

(1.9)

2.36

27

4

13.79

0.821

87.0

(0.18)

(0.043)

(1.7)

2.20

32

4

14.03

1.217

109.4

(0.31)

(0.028)

(5.0)

DAY 39

22

4

13.49

0.763

47.7

(0.15)

(0.008)

(3.3)

2.85

•

27

4

12.87

1.289

99.8

(0.38)

(0.039)

(10.0)

2.15

32

4

13.35

1.891

117.0

(0.26)

(0.011)

(6.6)

CONSUMPTION
(ml /h)

41

Figure 2-1 . Oxygen consumption (ml / h) of embryos at day 1 6 as a function of

oxygen tension at different temperatures. Values are means ± SE.

CONSUMPTION

42

Figure 2-2. Oxygen consumption (ml / h) of embryos at day 30 as a function of

oxygen tension at different temperatures. Values are means ± SE.

CONSUMPTION

43

1 1 1 1 1 1 1 1

0 40 80 120 160

P02 (mmHg)

Figure 2-3. Oxygen consumption (ml / h) of embryos at day 39 as a function of

oxygen tension at different temperatures. Values are means ± SE.

CONSUMPTION

44

Figure 2-4. Oxygen consumption (ml / h) of eggs as a function of temperature

at different incubation days. Values are means ± SE.

45

J 1 1 1 1 1 1 I I 1 1 I

21 24 27 30 33

rEMPERATURE ( °C)

Figure 2-5. Relationship of the critical oxygen tension (Pc) of embryos to

temperature at different incubation days. Values are means ± SE.

CONSUMPTION

46

Figure 2-6. Oxygen consumption (ml / h) of eggs as a function of incubation

days at different temperatures. Values are means ± SE.

47

1 1 . 1 1 1 1 1

1 0 20 30 40

DAYS

Figure 2-7. Relationship between the critical oxygen tension (Pc) of embryos

and incubation day at different temperatures. Values are means ±
SE.

48

02 CONSUMPTION (ml / h)

Figure 2-8. Relationship between critical oxygen tension (Pc) and oxygen

consumption of Florida red-bellied turtle embryos. Values are
means + SE.

0j-|^pyfip 3

INFLUENCES OF EGG HYDRATION ON METABOLISM, CRITICAL OXYGEN
TENSION, AND HATCHLINGS OF FLORIDA RED-BELLIED TURTLE

(PSEUDEMYS NELSONH

Introduction

Numerous studies have demonstrated that flexible-shelled reptilian eggs
take up water from their surroundings under favorable conditions, and the
amount of water uptake depends on the water potential and thermal
conductivity of the incubation media (Packard et al. 1977; Packard and Packard
1 984;1 989; Kam and Ackerman 1990). However, whether this water uptake
process affects gas exchange of egg is not clear. Even though water and
oxygen move across the eggshell-membrane complex by different pathways,
the oxygen conductance of eggs is still hydration-dependent (Ackerman and
Prange 1972; Lutz et al. 1980; Feder et al. 1982; Black et al. 1984). For
example, in flexible eggs of the snapping turtle (Chelvdra serpentina),
membranes contribute the majority of the resistance to oxygen diffusion.

Oxygen conductance of moist eggshell-membrane complexes falls 10-fold
when compared to that of dry eggshell-membrane complexes (Feder et al.
1982). Black et al. (1984) incubated Burmese python (Pvthon molurus
bioittatusl eggs in different moisture regimes, and they found that the oxygen
conductance of the eggshell-membrane complex is inversely correlated with the
amount of water uptake.

The reduction in oxygen conductance may not affect growth and
development of embryos if eggs are incubated under normoxic conditions, but it

49

50

could become a problem when eggs are incubated under hypoxic conditions.
Eggs incubated underground are potentially subjected to hypoxia when oxygen
concentration in the nest chambers is reduced. This reduction in oxygen can be
caused by a combination of a low gas conductance of the nest due to the
wetting of incubation medium by rainfall, flooding, and high tides (Chabreck
1975; Plummer 1976), microbial respiration (Seymour et al. 1986), and egg
metabolism (Ackerman 1977; Lutz and Dunbar-Cooper 1984).

Flexible-shelled eggs of the Florida red-bellied turtle, Pseudemys nelsoni
were used to investigate the effects of water relations of eggs on embryonic
metabolism and growth and respiration under hypoxic conditions. Critical
oxygen tension measurements were used to evaluate how well embryos
tolerate hypoxia.

Materials and Methods

A total of five clutches of Florida red-bellied turtle eggs was collected
from alligator nests in Florida during June and July, 1991 . One egg from each
clutch was opened to determine age. Eggs from each clutch were assigned to
both moisture regimes, 3 and 13 % gravimetric water content, to eliminate clutch
effects. A total of 38 eggs was assigned to each group, and they were half-
buried in sand and incubated at a constant 30 °C. Boxes were covered with
plastic sheets to reduce evaporative water loss, and water was added as
necessary.

Mass and critical oxygen tension were measured on six randomly
selected eggs from each group on day 16, 30, and 39. The critical oxygen
tension was determined by a method described elsewhere (see chapter 1).

51

Briefly, each egg was placed in a cup inside a metabolic chamber containing 5
ml of distilled water. The metabolic chambers were submerged in a water bath
at 32 0 C for 3 hours before measurements were taken. Chambers were
flushed and reflushed with a humidified gas mixture before stopcocks were
closed. A 30 ml gas sample was collected from each chamber at the end of
each measurement, and the final oxygen fraction was determined by injecting
the sample into an Applied Electro-chemistry S-3A O2 analyzer. Immediately
after the O2 concentration of all chambers was measured, eggs were subjected
to the next gas mixture, ranging from 90 to 10% air, and the oxygen
concentrations were determined by repeating the above protocol. Each new

gas mixture was 5 to 20 % lower in air content than the previous treatment.
Oxygen consumption, Vo2 (ml. STDP / h egg), was calculated using the

equation, Vo2 = V(Flo2-FEo2)/(1-FEo2)t, where V is the dry, C02-free air (ml),

FI02 and FE02 are the initial and final oxygen fraction (%) of a measurement,

and t is the duration (h) from start to end of a measurement (Vleck 1 987). The
critical oxygen tension, Pc (the P02 at which the oxygen consumption is first

reduced), was determined using a BASIC program that fits a two-segmented
straight line (Yeager and Ultsch 1 989). Data were analyzed individually and
averaged.

Wire partitions were installed when pipped eggs were found in any box.
Eggs were checked daily, and hatchlings were quickly frozen in air-tight
containers. They were later thawed at room temperature, and residual yolk and
carcass were separated, weighed, dried at 65 °C, and reweighed. Egg mass,
hatchling mass, and oxygen consumption were adjusted using analysis of
covariance (ANCOVA) and the initial mass was used as covariate. All means
were expressed as means (± standard error) unless otherwise noted. In most
cases, means were tested for significant difference using Student's t test.

52

Results

Eggs in both groups increased mass throughout incubation, and the
patterns of water uptake were similar (Fig. 3-1 ). Eggs incubated in 3 %
gravimetric water content increased mass slowly and gained 0.67 and 1 .59 g of
water by day 30 and 39 respectively (Fig. 3-1). In contrast, eggs incubated in 13
% gravimetric water content increased mass rapidly and gained 1 .83 and 3.47 g
of water by day 30 and 39, respectively (Fig. 3-1). Egg masses between two
groups were different at day 30 and 39 (P < 0.05 and P < 0.01 respectively).

Critical oxygen tension curves of embryos for both groups at day 16, 30,
and 39 are presented in Figures 3-2, 3-3, and 3-4 respectively. Oxygen
consumption of embryos were identical between two groups at day 16, 30, and
39. Figures 3-5 and 3-6, drawn from Figures 3-2 to 3-4, illustrate the changes of
oxygen consumptions and critical oxygen tensions during incubation. Oxygen
consumption measured in air were used in this comparison. The results show
embryos consumed more oxygen as they grew, and the rates of increase in the
two groups were almost identical (Fig. 3-5). The critical oxygen tensions
remained unchanged over the incubation period and were not different
between groups (Fig. 3-6). The dry masses of yolk-free hatchlings were the
same between groups. Eggs incubated in 13 % gravimetric water content,
however, had heavier wet mass of hatchlings and less yolk, wet and dry, than
eggs incubated in 3 % gravimetric water content (Table 3-1).

53

Discussion

The similarity of oxygen consumption and dry mass of yolk-free
hatchlings in the two groups suggests that growth and development are not
affected by the moisture levels of sand (Fig. 3-5; Table 3-1). A similar finding
has been reported for Burmese python eggs (Black et al. 1984). Eggs
incubated in different moisture regimes had similar oxygen consumption and
hatchling mass, while O2 conductance is inversely correlated to the moisture
levels (Black et al. 1984). Because metabolism and growth are not affected by
the moisture regimes, embryos incubated in moist substrates must have
maintained normal metabolism and growth by extracting more oxygen from
blood, which reduces the P02 beneath the eggshell (Black et al. 1984).

However, as mentioned earlier, the low P02 beneath the shell may have
detrimental effects on metabolism when eggs are exposed to hypoxia. The
transport of oxygen from atmospheric air to cells involves three major
compartments: the gas exchanger (the chorioallantoic membrane); the oxygen
carrier (blood); and the oxygen sink (cells) (Weibel 1984). These compartments
are in a series arrangement, and P02 drops as oxygen moves from one

compartment to the other. Note that two of the three compartments require
diffusion and are driven by a P02 gradient. Any factor that increases P02 at any
point could facilitate oxygen transport, whereas any factor that decreases P02 at

any point could impede oxygen transport. In theory, Burmese python eggs
incubated in the moist regimes should have a lower P02 under the eggshell

and are predicted to be less able to maintain normal oxygen consumption
under hypoxic conditions than eggs incubated in the dry regimes.

Results in this study disagree with the prediction, as indicated by the
similarity of critical oxygen tensions (day 30 and 39) even though eggs in 13 %

54

gravimetric water content gained significantly more water than those in 3 %
gravimetric water content. Two explanations could account for the differences.

1 ) Turtle eggs may have a higher O2 conductance and, therefore, the P02

gradient across the eggshell-membrane complex are smaller than in Burmese
python eggs. Consequently, the reduction in O2 conductance due to moisture
treatments in this study would not be severe enough to cripple the growth and
development of embryos. 2) Black et al. (1984) measured the P02 beneath the

shell when eggs were half-buried in vermiculite. Thus, the total resistance to
oxygen diffusion is the sum of the resistance of substrate and shell-membrane
complex. In contrast, critical oxygen tension of embryos was measured outside
the incubating substrates, and the total resistance to oxygen diffusion was
attributable only to the shell-membrane complex and presumably smaller than
measurements made by Black et al. (1984).

In conclusion, for reptilian eggs incubated underground in natural
environments, the incubating media may affect gas exchange of eggs directly
and indirectly. As the medium becomes wet, the resistance to oxygen diffusion
increases (Kam 1988; McGehee 1990). In addition, eggs take up more water
when incubated in moist conditions, and the increased water content in the
eggshell-membrane complex also increases the resistance to oxygen diffusion
(Black et al. 1984). However, the egg shell may crack as eggs take up water
and swell, which presumably increases O2 conductance (Lillywhite and
Ackerman 1984). Depending on the amount of water uptake, shell type, and the
severity of hypoxia inside nest chambers, the sum of these two resistances
could be high enough to impede oxygen diffusion and retard embryonic growth.

55

Table 3-1. Hatchling mass, residual yolk mass, and incubation period of

Florida red-bellied turtle eggs incubated in 3 and 13 % gravimetric
water content. Values are reported as means with standard error

in parenthesis.

P is significant

probability.

3 %

13 %

P

Sample size

14

14

Incubation time (days)

43 (0.3)

44 (0.4)

>.05

Wet hatchling mass (g)

7.37 (0.09)

7.92 (0.09)

< .001

Dry hatchling mass (g)

1.76 (0.05)

1 .88 (0.05)

> .05

Wet residual yolk mass (g)

1.65 (0.08)

1.41 (0.08)

< .05

Dry residual yolk mass (g)

0.82 (0.04)

0.66 (0.04)

< .01

56

r , i | « 1 « 1

io 20 30 40

AGE (days)

Figure 3-1 . Mean values (±SE) for changes in mass of eggs incubated in

sand with 3 (solid line) and 13 % (dashed line) gravimetric water
content. * and ** represent significant differences at 0.05 and 0.01
levels, respectively.

Oo CONSUMPTION

57

Po2 (mmHg)

Figure 3-2. Oxygen consumption (ml / h) of embryos at day 16 as a function of

oxygen tension. Eggs were incubated in sand with 3 (solid line)
and 1 3 % (dashed line) gravimetric water content. Values are
means + SE.

02 CONSUMPTION

58

• i 1 I 1 i ■ i

0 40 80 120 160

Po2 (mmHg)

Figure 3-3. Oxygen consumption (ml / h) of embryos at day 30 as a function of

oxygen tension. Eggs were incubated in sand with 3 (solid line)
and 1 3 % (dashed line) gravimetric water content. Values are
means ± SE.

59

o

I —
□l

CO

o

o

CM

O

2.1 -
1.8-
1.5-
1.2-
0.9-
0.6-
0.3-

0.0

0

40

80

• 3 % water

° 1 3 % water

120 160

P

o2

(mmHg)

Figure 3-4. Oxygen consumption (ml / h) of embryos at day 39 as a function of

oxygen tension. Eggs were incubated in sand with 3 (solid line)
and 13 % (dashed line) gravimetric water content. Values are
means ± SE.

CONSUMPTION

(ml / h)

60

Figure 3-5. Oxygen consumption of embryos incubated in sand with 3 (solid

line) and 13 % (dashed line) gravimetric water content. Values
are reported as means +SE.

\

Pc (mmHg)

61

AGE (days)

Figure 3-6. Relationship between the critical oxygen tension (Pc) of embiyos

and incubation day. Eggs were incubated in sand with 3 (solid
line) and 13 % (dashed line) gravimetric water content. Values
are means + SE.

CHAPTER 4

PHYSIOLOGICAL RESPONSES OF FLORIDA RED-BELLIED TURTLE
(PSEUDEMYS NELSONh EMBRYOS TO CHRONIC HYPOXIA

Introduction

Reptilian eggs incubated underground are potentially subjected to
hypoxia when oxygen concentration in nest chambers is reduced. Reduction in
oxygen can be caused by a combination of a low gas conductance of the nest
due to the wetting of incubation medium by rainfall, flooding, or high tides
(Chabreck 1975; Plummer 1976), microbial respiration (Seymour et al. 1986),
and egg metabolism (Ackerman 1977; Lutz and Dunbar-Cooper 1984).

Hypoxic effects on embryonic metabolism and growth of avian eggs have
received far more attention than in reptilian eggs. Avian embryos generally
tolerate moderate hypoxia (P02 = 125 mmHg) where development and growth

are not affected (Black and Snyder 1980; Synder et al. 1982; Carey et al. 1982).
Severe hypoxia (P02 = 95 mmHg or lower) retards development and growth

and prolongs the incubation period in chicken and montane coot (Fulica
americanal embryos (Wangesteen et al. 1974; Carey et al. 1989). In most
cases, the development and growth of high altitude species are not affected by
hypoxia encountered in natural habitats, as found in bar-headed geese (Anser
indicusl. horned larks fEremophila alpestrisl. and red-winged blackbirds
fAoelaius phoeniceusl (Carey et al. 1982; Snyder et al. 1982; Verbeek 1967;
Conry 1978). These studies and others have also shown that exposure to
hypoxia induces structural and physiological adjustments of embryos in

62

63

respiratory, circulatory, and tissue levels (Black and Synder 1980; Carey et al.
1982; Baumann et al. 1983; Dusseau and Hutchins 1988; Strick et al. 1991)

Hypoxic effects on embryonic growth and development of reptiles have
been studied only in sea turtle (Ackerman 1981a) and snapping turtle (Chelydra
serpentina) embryos (Kam 1988). Green turtle (Chelonia mvdas) and
loggerhead (Caretta carettal eggs were incubated in artificial nests where the
gas conductance of the sand could be manipulated. Ackerman (1981a) found
that eggs incubated in nests under a long sand column grow slower and hatch
later than those incubated in nests under a short sand column. Kam (1 988)
reported that embryos incubated in sand with 12 % gravimetric water content
grew more slowly than those with 4 % gravimetric water content. McGehee
(1990) incubated loggerhead eggs in sand with 0, 25, 50, 75, and 100 percent
moistures, and found that the hatching success is strongly related to the
moisture levels. These findings suggest that retarded metabolism and growth of
embryos are due to oxygen depletion and possibly carbon dioxide
accumulation in nests, which are caused by a low gas conductance in the
incubation medium, attributable to either sand thickness or moisture. Oxygen
and carbon dioxide tensions in nest chambers during incubation were not
reported or measured. Therefore, it is not known whether embryos could
tolerate hypoxia or could compensate for hypoxia and hypercapnia.

The hypoxic effects on the embryonic growth and development of Florida
red-bellied turtle (Pseudemvs nelsonil embryos were studied. Many Florida
red-bellied turtles found in wetlands of north-central Florida lay their eggs in
nest mounds of the American alligator (Alligator mississippiensis) (Goodwin
and Marion 1977). Oxygen content within a nest cavity is dependent upon the
nest location and rainfall during the nesting season. In this study, hypoxic
tolerance was determined by measuring the critical oxygen tension of embryos.

64

Morphometries of blood vessels in the chorioallantoic membrane, heart mass,
and hematocrit were measured to reveal possible responses to hypoxia.

Materials and Methods

A total of ten clutches of Florida red-bellied turtle eggs were collected
from alligator nests in Alachua county, Florida during June and July, 1991 . One
egg from each clutch was opened to determine age. Eggs were randomly and
equally assigned to a control and a hypoxic treatment. Both group of 77 eggs
initially were half-buried in sand with 4 % gravimetric water content and
incubated at a constant 30 °C. The boxes were covered with plastic sheets to
reduce evaporative water loss, and water was added as necessary.

At 21 days of incubation, eggs were transferred to sealed incubators
(dimension 45 x 30 x 25 cm) and half-buried in sand with 4 % gravimetric water
content. Two outlets, one for incurrent and one for outcurrent gas, were
installed on the top cover. Gas was humidified by bubbling through a gas-
sealed flask containing distilled water before being delivered into the
incubators. One incubator was perfused with air delivered by an air pump
located upstream, and the other incubator was perfused with a mixture of 90 %
N2 and 10 % O2 from a compressed gas tank. Flow rates were set at about 4-5
L / h. The oxygen tensions in both incubators were checked daily by taking a
gas sample and measured with an Applied Electro-chemistry S-3A O2 analyzer.
They averaged 150 ± 1 and 72 ± 5 mmHg for the normoxic and hypoxic
incubators, respectively. Relative humidity inside the incubators was high
throughout incubation, as indicated by the condensation of water vapor on the

65

walls. Eggs in both incubators had positive water balance throughout
incubation, which increased 5-10 % of their intial mass.

Mass and oxygen consumption were measured from 6-12 randomly
selected eggs from each incubator on day 21 , 26, 33, and 39. Eggs were then
opened. Yolk and embryo were separated, weighed, dried at 65 °C, and
reweighed.

Wire partitions were installed when pipped eggs were found in any
incubator. Eggs were checked daily, and hatchlings were killed with halothane.
Blood was collected from a carotid artery to determine hematocrit, and
hatchlings were frozen in air-tight containers. Later, residual yolk, ventricle, and
carcass were separated, weighed, dried at 65 °C, and reweighed.

The oxygen consumption of eggs was measured using a closed-system,
and Glass jars (Kerr Glass Inc.) fitted with two rubber stoppers and three-way
stopcocks were used as metabolic chambers. These chambers contained 5 ml
of distilled water and a piece of wet filter paper to keep the chambers at 1 00 %
relative humidity. Each chamber was flushed with humid air before stopcocks
were closed. The interval for gas sampling was dependent on the
developmental stage of embryos such that no more than 1 -2 % of total oxygen
was consumed. A 40 ml gas sample was taken at the end of incubation, and

the fractional O2 concentration was determined with an Applied Electro-
chemistry S-3A O2 analyzer. Oxygen consumption, Vo2 (ml O2. STDP / h egg),

was calculated using the equation, Vo2 =V(Flo2-FEo2)/(1-FEo2)t. where V is the

dry, C02*free air, Flo2 and FEo2 are the initial and final oxygen fractions (%) of

a measurement, and t is the duration (h) from start to end of a measurement

(Vleck 1987).

On day 21 and 33 of incubation, six eggs from each group were selected
to measure critical oxygen tensions. Methods used to measure the oxygen

66

consumption of embryos at different gas tensions and to determine the critical
oxygen tension are described earlier (Chapter 1 ). After the experiments, eggs
were fixed in 10 % buffered formalin for 1-3 days. Eggs were later opened, the
chorioallantoic membrane (CAM) was removed and fixed, and embryo and yolk
were separated, dried at 65 °C, and weighed.

The morphometry of CAM (surface area and length of blood vessels) was
quantified using a digitizer adjacent to a Nikon Labophot microscope. Raw data
were stored in an Apple lie microcomputer interfaced with the digitizer. A
vascular density index was also determined by counting the number of vessels
that intersected with the lines of a grid. Measurements were done at 40 X
magnification, and all counting was done in triplicate and averaged.

Hatchling mass and oxygen consumption were adjusted using analysis
of covariance (ANCOVA) and the initial mass was used as covariate. The mass
and oxygen consumption of embryos from the critical oxygen tension
experiments were not different from that of the other experiment, and therefore,
the data were pooled. All means were expressed as means (SE) unless
otherwise noted.

Results

Eggs in control and hypoxic groups increased oxygen consumption
during incubation and reached 1.38 and 1.36 ml O2 / h, respectively, at day 39
(Fig. 4-1). Oxygen consumption of eggs in the normoxic conditions reached a
plateau at day 33, whereas that of eggs in hypoxic conditions did not reach the
plateau until day 39. The oxygen consumption of eggs in both groups was
different at day 33.

67

Embryos in normoxic and hypoxic conditions grew at an increasing rate
(Fig. 4-2). Dry embryonic masses were not different between groups until day
39 (control group with 1 .18 ± 0.04 g and hypoxic group with 0.88 ± 0.05 g, P <
0.01 ). Hypoxic embryos hatched at the same time but with smaller masses than
normoxic embryos (Table 4-1). In addition, the dry mass of ventricle was
similar, but relative ventricular mass and hematocrit was higher, for the
hatchlings in hypoxic conditions (Table 4-1).

The critical oxygen tension and oxygen consumption of embryos at day
21 were not different between the control and hypoxic group (P > 0.05, Table 4-
2). At day 33, embryos that were exposed to normoxic conditions increased
their critical oxygen tensions by 9 %, whereas embryos that were exposed to
hypoxic conditions reduced their critical oxygen tensions by 24 %. The
difference in the critical oxygen tensions of embryos between the two groups
was different (P < 0.001). Measurements of blood vessels in CAM, including the
number of intersections, surface area, and length per unit area, were statistically
the same at day 21 for both groups (Table 4-3). By day 33, only the blood
vessel length was different (P < 0.01) (Table 4-3).

Discussion

Oxygen consumption and growth

The metabolic pattern of normoxic embryos resembles patterns

previously reported for Chrvsemvs and Kinosternon (Lynn and von Brand 1 945)
in that Vo2 increases during incubation, then reaches a plateau. The

metabolism of normoxic embryos reached a plateau at day 33, suggesting that
embryonic growth has reached a maximum level. In contrast, hypoxic embryos

68

did not grow at the same rate as normoxic embryos, as indicated by lower
metabolic levels (Figs. 4-1 , 4-2). As a result, metabolism of hypoxic embryos
did not reach the plateau at day 33 as normoxic embryos did; consequently,
they continued to consume oxygen at an increasing rate and took six more days
to reach the maximum level.

Embryos incubated in hypoxic conditions were smaller at day 39 than
those in normoxic conditions, indicating that hypoxia retards embryonic growth
(Fig. 4-2). Similar findings have been reported for eggs of the green sea turtle,
the loggerhead, and the snapping turtle (Ackerman 1981a; Kam 1988). In avian
eggs, depression of metabolism and growth by hypoxia has been interpreted
as either an inability of embryos to take up enough oxygen from surroundings
(Carey et al. 1982; Snyder et al. 1982; Carey et al. 1989), or an adaptive
adjustment to maintain air-cell P02 adequate for tissue oxygenation

(Wangensteen et al. 1974; Beattie and Smith 1975).

Much information is available on the effects of hypoxia on growth and
development of avian embryos. Chicken embryos incubated at moderate
hypoxia (at 1500 m, P02 = 125 mmHg) maintain normal oxygen consumption

and embryonic growth, and hatch with the same mass as embryos in normoxic
condtions (Snyder and Black 1980). Because hypoxic embryos probably have
lower air-cell P02. and, consequently, lower arterial Po2> adjustments must

have compensated for the hypoxia. Exposure to severe hypoxia (at 3800 m,

P02 = 93 mmHg) results in depressed metabolic rate and growth, prolonged

incubation period, reduced hatchling mass, and reduced hatchability (Smith et
al. 1969; Wangensteen et al. 1974).

In bar-headed geese (Anser indicus) and Canada geese (Bran.13
canadensis), oxygen consumption, incubation period, and hatchling mass of
embryos show no differences between moderate and severe hypoxic groups

69

(P02 = 125 and 94 mmHg respectively) (Snyder et al. 1982). Similar results are

found in the incubation period and hatchability of eggs among populations of
horned larks (Eremophila aipestrisl breeding at 2400 m and 3800 m (Verbeek
1967; Conry 1978). A study on red-winged blackbirds (Agelaius phoeniceus)
over 2900 m altitudinal gradient found no differences in embryonic metabolism,
incubation period, and hatchling mass (Carey et al. 1982). In contrast, eggs of
montane coots (Fulica americanal laid at 4150 m have depressed metabolic
rate and prolonged incubation period, but hatch with the same mass as that of
lowland coots (Carey et al. 1989).

Incubation period, hatching success, and hatchling mass.

Exposure to chronic hypoxia resulted in retarded growth, depressed
metabolism, comparable incubation period and hatchability, and reduced
hatchling mass in red-bellied turtle embryos. Different patterns have been
observed in the green and loggerhead turtles in that embryos exposed to more
hypoxic and hypercapnic environments grow slowly and hatch later with higher
mortality than embryos exposed to less hypoxic and hypercapnic environments
(Ackerman 1981a). Furthermore, the size of hatchlings varies among
populations. Eggs from Costa Rica hatch with the same size regardless of the
treatments, whereas eggs from Ascension Island hatch smaller in the more
hypoxic treatments. Another study on loggerhead turtle eggs has shown that
incubation period increases but hatchability and hatchling sizes decrease as
moisture levels of the incubating media increase (McGehee 1990). Snapping
turtle embryos incubated in wet media grow more slowly and hatch later with
similar mass and mortality when compared to embryos that are incubated in dry
media (Kam 1988).

70

Embryos incubated in hypoxic conditions could have at least four
different outcomes in reptilian and avian embryos. First, embryonic growth is
not affected by hypoxia, and embryos have comparable incubation period and
hatchling mass as embryos in normoxic conditions. This pattern has been
reported in chicken eggs at 1500 m (P02 = 133 mmHg) (Black and Synder
1980); horned lark eggs at 2400 and 3800 m (P02 = 117 and 93 mmHg

respectively) (Drury 1961; Verbeek 1967; Conry 1978); red-winged blackbird
eggs at 1600, 2400, 2900 m (P02 = 131, 117, 109 mmHg respectively) (Carey

et al. 1982). Second, embryonic growth is retarded by hypoxia, and embryos
take longer time to grow but hatch with a mass similar to eggs in normoxia. This
pattern has been reported in montane coot eggs at 4150 m (P02 = 87 mmHg);

green and loggerhead turtle eggs from Costa Rico (oxygen tensions were not
reported) (Ackerman 1981a) and snapping turtle eggs (oxygen tensions were
not measured) (Kam 1988). Third is a pattern similar to the previous one except
that embryos hatch with smaller sizes and mortality is increased. This has been
reported in chicken eggs at 3800 m (P02 = 93 mmHg) (Smith et al. 1969;

Wangensteen et al. 1974); green turtle eggs from Ascension Island (oxygen
tensions were not reported) (Ackerman 1981a) and loggerhead turtle eggs
(oxygen tensions were not measured) (McGehee 1990). Fourth, embryonic
growth is retarded by hypoxia, and embryos have incubation period and
hatchability that are similar to normoxic-raised embryos, but hatchling mass is
smaller (P02 = 72 mmHg) (present study).

It is clear that when embryonic growth is retarded by hypoxia, embryos
could prolong the incubation period and hatch either with similar mass or
smaller mass than normoxic embryos, as found in patterns 2 and 3. In contrast,
some embryos do not prolong incubation time and simply hatch smaller, as
found in pattern 4. The difference between pattern 2 and 3 can be attributed to

71

the different severity of hypoxic effects on embryonic growth and development.
In pattern 2, hypoxic exposure is not severe, and the physiological functions are
limited but not affected. Embryos simply take longer time to grow and hatch
later with the same hatchling mass, as found in pattern 2. In contrast, embryos
in pattern 3 are exposed to severe hypoxia, and the physiological functions are
not only limited but also affected; therefore, embryos would not only grow more
slowly but also hatch later with smaller mass and relatively high mortality.

Florida red-bellied turtle embryos in hypoxic conditions hatched at the
same time but with smaller mass when compared to normoxic embryos,
suggesting that this is a hypoxia-induced hatching event. Previous studies
have shown that hypoxia or anoxia induces hatching in the fish, Fundulus
heteroclitus. the frog, Pseudophrvne bibroni. and the turtle, Carettochelvs
insculpta (DiMichele and Taylor 1980; Webb et al. 1986; Bradford and Seymour
1988). Hypoxic-induced hatching can only succeed when a size is attained
sufficient to break the eggshell and there is a sufficient level of maturity of organ
systems. In this study, embryos were subjected to hypoxia only during the
second half of the incubation period, which coincides with the end of the
differentiation phase and beginning of the growing phase. Thus, embryos were
subjected to hypoxia after differentiation had been completed, and hypoxia
presumably affects embryonic growth but not differentiation. Similar findings
have been reported for chicken embryos in that hypoxia has a greater effect
upon differentiation than upon growth at middle stages (Smith et al. 1969).

The mechanism by which hypoxia could initiate hatching in reptilian eggs
is poorly understood. In bird eggs, glucocorticoid has been reported to play a
major role in hatching of turkey embryos, as indicated by an increase of
hatchability after exogenous administrations of glucocorticoid (corticosterone)
(Wentworth and Hussein 1985). Glucocorticoid is also important in synthesis of

72

surfactant in the embryonic lung of chickens right before hatching (Hylka and
Doneen 1 983). However, it is not known if the glucocorticoid plays the same
role in inducing early hatching during hypoxia.

The described patterns may not be the only ways hypoxia influences
embryonic growth and development. More patterns will probably emerge as
additional studies are done. These patterns may also be confounded by some
differences in the experimental protocols. For example, snapping turtle, green
turtle and loggerhead eggs are subjected not only to hypoxia but also
hypercapnia, and gas concentrations were not constant during incubation
(Ackerman 1981a; Kam 1988). Bird eggs are usually exposed to hypoxia at
lower barometric pressures, but reptilian eggs are usually exposed to hypoxia
at normal barometric pressures. Timing of hypoxic treatment may result in
different results. In this study hypoxia was introduced at about 50 % of the
incubation period, which is different from previous studies.

Embryonic adjustments to hvpoxia

Embryos exposed to chronic hypoxia reduced critical oxygen tension by
24 %, indicating that embryos can increase oxygen transport in response to
hypoxia. The increase of oxygen transport is probably the result of adjustments
of circulatory (i.e., relative ventricular mass, hematocrit) and respiratory (CAM
morphometry) systems. However, embryonic growth in the hypoxic group was
lower than in the control group, suggesting that the increase of oxygen transport
could only partially compensate for the hypoxic effects. Snyder et al. (1982)
incubated eggs of bar-headed and Canada goose at a moderate (P02 = 125
mmHg) and severe (P02 = 95 mmHg) hypoxic conditions. They found that the
critical oxygen tensions are lower in the former species. In addition, the critical
oxygen tension of bar-headed geese is not affected by severe hypoxia, but is

73

reduced in Canada geese. Eggs of the bar-headed goose, laid at high altitude
(as high as 5500 m), possess adaptations to maintain normal oxygen
consumption in moderate and severe hypoxia. In contrast, eggs of the Canada
goose, laid at near sea level, do not tolerate hypoxia well; however, they can
increase the oxygen transport in response to a severe hypoxic treatment.

Adjustments in response to hypoxia have been reported for avian
embryos at the respiratory, circulatory, and tissue levels. Chronic hypoxia
stimulates angiogenesis in the CAM of chicken eggs (Dusseau and Hutchins
1988; Strick et al. 1991). Exposure to hypoxia increases hemoglobin-oxygen
affinity in chicken and other domestic fowl embryos (Black and Snyder 1980;
Baumann et al. 1983) and heart mass of domestic chicken and Canada goose
hatchlings (Black and Snyder 1980; Snyder et al. 1982). Hypoxia probably
induces a redistribution of blood flow to embryonic tissue such that blood is
preferentially distributed to areas with a high priority (e.g. heart) than to areas
with a low priority (e.g. limb muscle). Chicks hatched in hypoxic conditions
have significantly heavier heart and lighter gastrocnemius muscle when
compared to chicks that hatched in the normoxic conditions, even though body
masses are the same (Black and Snyder 1980)

The other possible adaptive adjustment of embryos is to modify the
resistance of the eggshell-membranes to oxygen diffusion. Eggs laid at high
elevations develop in hypoxic conditions and encounter greater water iosses
due to the increase of the diffusion coefficient of gases at lower barometric
pressures. Increased O2 conductance of the eggshell promotes O2 uptake but
suffers a much greater water loss. The opposite is also true. Some eggs are
built to maintain water balance at the expense of maximizing oxygen uptake
(Wangensteen et al. 1974, Carey et al. 1982). Consequently, the oxygen
requirement for normal development is met by modifying the resistance of the

74

inner barrier to oxygen diffusion in red-winged blackbirds (Carey et al. 1982).
However, coot eggs (Fulica americanal laid at 4150 m have increased O2 and
CO2 conductance of eggshells, which favor gasous diffusion and increased
water loss (Carey et al. 1 989).

At lower elevations, hypoxia occurs only when eggs are buried
underground, such as in the case of the Brush Turkey (Alectura lathami) and
Mallee Fowl (Leipoa ocellatal (Seymour et al. 1986). Here, the selective
pressure for eggshell conductance to maintain water balance is relaxed
because of the high humidity inside the nest. Thus, a high gaseous
conductance of the eggshell is adaptive to facilitate exchange of respiratory
gases (Seymour 1985; Seymour et al. 1986).

In conclusion, low critical oxygen tensions at day 21 and day 33 indicate
that Florida red-bellied turtle embryos tolerate hypoxia better than bird embryos
(Visschedijk et al. 1980; Synder et al. 1982; Seymour 1985). This is probably
attributed to the high O2 conductance of reptilian eggs (Ackerman and Prange
1972; Lutz et al. 1980; Feder et al. 1982) and the low metabolic rate of embryos
over long incubation periods (Ackerman 1980). However, embryonic growth
was retarded when Florida red-bellied turtle eggs were incubated in hypoxic
conditions at P02 = 72 mmHg, indicating that embryos are no longer able to

maintain normal metabolism at this hypoxic level. Furthermore, hypoxic
embryos grew slower but hatched at the same time as normoxic embryos,
suggesting that hypoxia only influences embryonic growth but not development
during the latter half of incubation.

The decrease of critical oxygen tensions by 24 % in response to hypoxia
indicates that Florida red-bellied turtle embryos are flexible, structurally and
physiologically, in enhancing oxygen transport to compensate for hypoxia.
These findings are in agreement with previous studies in chicken and Canada

75

goose embryos (Dusseau and Hutchins 1988; Strick et al. 1991 ; Baumann et al.
1983; Snyder et al. 1982).

76

Table 4-1. Hatchling, residual yolk, and ventricular mass, incubation period,

and hematocrit of Florida red-bellied turtle hatchlings in normoxic
and hypoxic conditions. Values are reported as means with
standard error in parenthesis. P is significant probability.

Normoxic

Hypoxic

P

Sample size

20

23

Incubation time (days)

43

42

>.05

(1.2)

(1.6)

Wet hatchling mass (g)

6.899

6.184

< .05

(0.257)

(0.225)

Dry hatchling mass (g)

1.608

1.323

< .01

(0.083)

(0.059)

Wet yolk mass (g)

1.352

1.872

< .001

(0.095)

(0.070)

Dry yolk mass (g)

0.696

0.9871

< .001

(0.051)

(0.045)

Wet ventricular mass (mg)

15.2

15.1

> .05

(0.73)

(0.94)

Dry ventricular mass (mg)

2.8

3.2

> .05

(0.16)

(0.25)

Relative ventricular mass (%)

0.18

0.25

<.01

(0.11)

(0.19)

Hematocrit (%)

27

34

< .001

(0.9)

(13)

77

Table 4-2. Oxygen consumption (V02) and critical oxygen tension (Pc) of

embryos at day 21 and 33. Values are reported as means with
standard error in parenthesis. Sample size is 6 for each group. P
is significant probability.

Normoxic

Hypoxic

P

Dav 21

V02 (ml / h)

0.588

(0.041)

0.626

(0.032)

> .05

Pc (mmHg)

94.4

(3.8)

94.5

(3.8)

> .05

Dav 33

V02 (ml / h)

1.393

(0.032)

1.271

(0.062)

> .05

Pc (mmHg)

103.7

(4.0)

72.4

m

< .001

78

Table 4-3. Morphometry measurements of blood vessels in the

chorioallantoic membrane of embryos incubated in normoxic and
hypoxic conditions. Values are reported as means with standard
error in the parenthesis. Sample size is 6 for each group. P is
significant probability.

Normoxic

Hypoxic

P

No. of intersection

Day 21

40.4

41.5

> .05

(2.3)

(2.9)

Day 33

45.8

54.2

> .05

(3.6)

(3.2)

Surface area Imm2)

Day 21

0.664

0.674

> .05

(0.020)

(0.026)

Day 33

0.895

0.980

> .05

(0.053)

(0.076)

Lenath (mm)

Day 21

8.41

8.65

> .05

(0.58)

(0.23)

Day 33

9.17

11.29

< .01

(0.32)

(0.42)

NOIldlAinSNOO 20

79

Figure 4-1 . Oxygen consumption (ml / h) of embryos incubated in normoxic

and hypoxic conditions as a function of incubation days. Values
are reported as means ± SE. * represents a significant difference
at a 0.05 level.

DRY MASS (g)

80

Figure 4-2. The increase of embryonic mass with incubation time. Values are

reported as mean + SE. ** and *** represent significant
differences at 0.01 and 0.001 levels, respectively. "H" symbols
indicate hatchlings.

02 CONSUMPTION

81

0.8-

0.6-

0.4-

0.2-

0.0 --
0

O HYPOXIC

j i i i | * i

40 80 120 160

Pq, (mmHg)

Figure 4-3. Oxygen consumption (ml / h) of embryos at day 21 as a function of

oxygen tension. Values are means + SE.

CONSUMPTION

82

Figure 4-4. Oxygen consumption (ml / h) of embryos at day 33 as a function of

oxygen tension. Values are means + SE.

CHAPTER 5

EFFECTS OF SIMULATED FLOODING ON METABOLISM, WATER BALANCE,

AND HATCHING SUCCESS OF FLORIDA RED-BELLIED TURTLE

(PSEUDEMYS NELSONh EMBRYOS

Introduction

Reptilian eggs incubated underground are potentially subjected to
flooding when water levels are raised by rainfall, high tides, or other natural
phenomena. Laboratory or field studies have demonstrated flooding is a major
cause of egg mortality in Crocodvlus porosus (Webb et al. 1977), Alligator
mississipiensis (Hines et al. 1968, Joanen 1969, Kushlan and Kushlan 1980),
and Trionvx m. muticus (Plummer 1 976). Egg mortality is dependent on the
duration of submergence.

Although studies have been directed at the effect of flooding on egg
mortality, no attempt has been made to investigate its influence on water
balance and embryonic metabolism and development. When eggs are
inundated, the oxygen availability drops, and the eggshells are saturated with
water, which favors water exchange but not gas exchange. It is not known how
embryos respond to a sudden change from an aerial environment to an
aqueous environment. It is unclear if flooding has any effect on embryonic
development and metabolism in those embryos that survive.

Many Florida red-bellied turtles (Pseudemvs nelsoni) found in Florida
wetlands lay their eggs in nest mounds of the American alligator. Eggs are
usually located either level with or below alligator nest cavities, which are about
25 cm above the water level (Goodwin and Marion 1977). The nest mounds

83

84

have horizontal bases of about 150-160 cm in diameter and 50 cm in height,
and in close proximity to permanent water (Goodwin and Marion 1978). An
annual survey found that about 20-30 % of nests are flooded during at least
some part of the incubation period (G. Masson, personal communication). The
purpose of this study was to assess the physiological effects of flooding on
water balance, metabolic rate, growth, survivorship, and hatchlings of
parchment-shelled eggs and embryos of P. nelsoni.

Materials and Methods

»

A total of seven clutches of red-bellied turtle eggs were collected from
alligator nests in Alachua county, Florida during June and July, 1991. One egg
from each clutch was opened to estimate age. Eggs were randomly and
equally assigned to a control and three flooding treatments: 1 , 3, and 6 days of
submersion. Each group of 27 eggs were half-buried in sand with 4 %
gravimetric water content and incubated at a constant 30°C. The boxes were
covered with plastic sheets to reduce evaporative water loss, and water was
added as necessary.

Egg mass and oxygen consumption of 9 randomly selected eggs from
each group were measured periodically throughout the incubation starting at
day 19. For the control and 1-day submergence treatment, measurements were
also made at 1 , 3, 6, 9, 16, and 24 days after submersion. For the 3- and 6-day
submergence treatments, measurements were made at 3 and 6 days,
respectively, after submersion.

Eggs of the submergence groups were incubated in sand throughout
incubation except during the flooding experiments. The flooding experiments

85

were carried out in the same walk-in environmental chamber where eggs were
incubated. Eggs were submerged in distilled water (30°C) at 19 days of
incubation and removed from water after 1 , 3, and 6 days of submersion. Each
egg was weighed immediately after removal from water, and oxygen
consumption was determined. Because the relative humidity in the metabolic
chambers was saturated and the duration for measuring oxygen consumption
was short (no more than 45 minutes), it was assumed that the oxygen
consumption was measured when the eggshell was still near, if not fully
saturated.

The oxygen consumption of eggs was measured using a closed-system,
and Glass jars (Kerr Glass Inc.) fitted with two rubber stoppers and three-way
stopcocks were used as metabolic chambers. These chambers contained 5 ml
of distilled water and a piece of wet filter paper to keep the chambers at 1 00 %
relative humidity. Each chamber was flushed with humid air before stopcocks
were closed. The interval for gas sampling was dependent on the
developmental stage of embryos such that no more than 1-2 % of total oxygen
was consumed. A 40 ml of gas sample was taken at the end of incubation, and

the fractional O2 concentration was determined with an Applied Electro-
chemistry S-3A O2 analyzer. Oxygen consumption, Vo2 (ml STPD / h), was

calculated using the equation, V(Flo2-FEo2)/(1‘FEo2)t. where V is the dry, CO2-
free air, FI02 and FE02 are the initial and final oxygen fraction (%) of a
measurement, and t is the duration (h) from start to end of a measurement
(Vleck 1987).

Wire partitions were installed in every box when pipped eggs were found
in any box. Boxes were checked everyday, and hatchlings were killed by
freezing. Later, hatchlings were thawed at room temperature, and wet mass

86

was weighed. Residual yolk and carcass were separated, weighed, dried at 65
0 C, and reweighed.

Egg mass, hatchling mass, and oxygen consumption were adjusted
using analysis of covariance (ANCOVA) and the initial mass was used as
covariate. All means were expressed as mean (± standard error) unless
otherwise mentioned. Dry hatchling and residual yolk mass were tested for
significant difference using ANOVA.

Results

Embryonic metabolism

The oxygen consumption of control eggs increased throughout
incubation and reached 1.63 ml 02 / h at day 43 (Fig. 5-1). All submerged eggs
reduced their oxygen consumption to low levels at around 0.07-0.09 ml O2 / h.
Eggs of thel-day submergence treatment resumed oxygen consumption quickly
and almost reached the same level as eggs in the control group at 24 days after
the flooding experiment was started (i.e., day 43) (1 .56 ml O2 / h, P > 0.05) (Fig.
5-1). In contrast, the oxygen consumption of eggs of the 3- and 6-day
submergence treatments decreased and did not recover to the levels at day 19
(Fig. 5-2).

Hatching success, incubation period, and hatchlings

The hatching success and incubation time of red-bellied turtle eggs were
significantly affected by the duration of submergence (Table 5-1). The percent
hatching was highest for eggs in the control group and progressively lowered
for eggs in the 1- and 3-day submergence treatments, while no eggs in the 6-

87

day submergence treatment survived. Eggs in the control required significantly
less time to hatch than eggs in the 1- and 3-day submergence treatments (P <
0.001, P < 0.01 respectively).

The dry masses of carcass and residual yolk and water content of
hatchlings were not different among groups (Table 5-2). In the 1-day
submergence treatment, the dry mass of hatchlings (1.788± 0.07 g, n=7) was
significantly heavier than that of dead embryos (1 .384 ± 0.07 g, n=1 4) (P <
0.01). The dry mass of dead embryos of the 1-day submergence treatment was
heavier than that of the 3- and 6-day submergence treatments (0.04 ± 0.04 g, n
= 20 and 0.05 ± 0.04 g, n=27, P < 0.001 , P < 0.001 respectively), but the latter
two were not significantly different from each other (P > 0.05).

Egg mass during incubation

Eggs in the control and 1-day submergence treatment increased mass
throughout incubation, and the patterns of water uptake were similar (Fig. 5-3).
They gained 1 .43 and 1 .83 g of water, respectively, at 24 days after the flooding
experiment started, and the difference was not significantly different (P > 0.05).
In contrast, eggs in the 3- and 6-day submergence treatments lost water
throughout incubation except during submergence (Fig. 5-4). They lost 0.59
and 0.79 g of water respectively at 24 days after the flooding experiments were
started, and the difference was not significantly different (P > 0.05).

Simulated flooding had significant effects on the water uptake of eggs
(Table 5-3). Eggs in the 1- and 3-day submergence treatments gained 84 and
42 % respectively (P < 0.001 and P < 0.05) more water than eggs in the control
within the same duration. However, eggs in the control gained 322 % more
water than eggs in the 6-day submergence treatment within the same duration
(P<0.01).

88

Discussion

Patterns of metabolism

The metabolic pattern of control eggs resembles patterns reported for
Chrvsemvs and Kinosternon (Lynn and von Brand 1945) in that Vo2 increases

during incubation, then reaches a plateau. A similar metabolic pattern is
exhibited by embryos of precocial birds (Vleck et al. 1980). The Vo2 of red-

bellied turtle embryos reached a plateau at day 35, which was about 78 % of

the incubation period (Incubation period = 45 days).

In contrast, the Vo2 pattern of embryos of the 1-day submergence

treatment differed from that of the control group in that Vo2 increased during

incubation but did not reach a plateau (Fig. 5-1). This difference is attributed to
the effect of flooding. The embryonic metabolism of the control group reached a
plateau at day 35, suggesting that embryonic growth had reached a maximum
level. In contrast, the embryonic metabolism of the 1-day submergence

treatment was suppressed during submersion and resumed after eggs removal
from water. However, Vo2 was still somewhat lower than the control group,

suggesting that embryos grow at slower rates. Embryonic metabolism did not
reach the plateau at day 35 as in the control; consequently, embryos continued

to grow at an increasing rate and took 8 more days to reach the maximum level.
Embryos in the 3- and 6-day submergence treatments had \Zo2 patterns

which differed from those of controls. Metabolism was suppressed during
submersion and did not return to the initial values after eggs were removed from
water (Fig. 5-2). The low levels of metabolism suggest that physiological
processes are permanently damaged during submersion. This hypothesis is

89

supported by the findings that only one embryo out of 54 eggs survived
submersion, and the dry mass of carcasses was not different from that of
embryos measured at the day of experiment.

Hatching success

Hatching success was lowest for eggs that were submerged in water for
6 days, indicating that a long duration of submergence is harmful to the
developing embryos. Earlier laboratory studies have shown that duration of
submergence reduces the hatching success of eggs of Trionvx m. muticus
(Plummer 1976), Alligator mississippiensis ( Joanen et al. 1977), and
Crocodvlus porosus (Magnusson 1982). The interaction between duration of
submergence and age is important in determining hatching success. In
mississipiensis. a long duration of inundation (48 h) kills embryos at all ages.
Short durations of inundation (2-6 h) yields a high hatching success of eggs at
all ages. However, an intermediate duration of inundation (12 h) kills more old
embryos than young embryos (Joanen et al. 1977). It appears that in A*
mississiooiensis. embryos at all ages could tolerate severe hypoxia for at least
6 h, but no more than 48 h. Between 6-48 h, the degree of tolerance is
dependent upon the age of embryos, which, in turn, depends largely on the
metabolic levels of embryos. Young embryos have comparatively low
metabolic rates; therefore, they could tolerate hypoxia longer than older
embryos. Kraemer and Bell (1980) reported that loggerhead (Caretta caretta)
embryos at all developmental stages are equally vulnerable to the flooding rain,
suggesting that the duration of inundation must have well passed the upper limit
of the tolerance of embryos.

Whether hatching success is affected by the oxygen content in water is
debatable. Theoretically, when eggs are submerged, the air-filled pathways in

90

the eggshell fill with water, and continuous liquid columns are formed.
Consequently, eggs could obtain oxygen via water-filled pathways by diffusion.
However, Kutchai and Steen (1971) demonstrated that the O2 permeability of
the compound membranes in a chicken egg is about 30 times higher than that
of a layer of water of the same thickness. If this is true for reptilian eggs, then
the limiting factor would be the O2 permeability of the eggshell and not the
amount of oxygen in the water.

The duration of anoxia tolerance may be species-specific, but more
measurements from other species and a standard experimental protocol are
needed before such a generalization can be reached. For example, eggs do
not survive in water for 2 days in Al. mississippiensis (Joanen et al. 1 977), 1 3 h
in C porosus (Magnusson 1982), 15 days in T. m. muticus (Plummer 1976), and
6 days in P. nelsoni (present study). These differences can largely be
explained by the difference in oxygen consumption. The longer duration of
tolerance in water for turtle eggs when compared to crocodilian eggs is
attributed to the difference in egg sizes and metabolic rates. Furthermore, the
difference in tolerance between T. m. muticus and P. nelsoni is because the
former was subjected to submersion at a younger age than was the latter (day
12 and 19 with incubation periods of 60 and 44 days at 30 0 C respectively).

Causes of mortality

Flooding kills embryos in two different ways. First, it kills embryos during
submergence by limiting oxygen availability, i.e., suffocation. In the 3- and 6-
day submergence treatments, metabolism was suppressed during submersion
and did not return to the initial values after eggs were taken out of water. All
except one embryo in the 3- and 6-day submergence treatments were dead and

91

dry carcass mass was not different from that of embryos measured at the day of
the experiment. Both measurements indicate that physiological processes are
permanently damaged during submersion. A heavy rainfall kills embryos of £*
caretta by suffocation (Kraemer and Bell 1980). This is inferred because the
developmental stages of dead embryos correspond with their ages at the time
of being flooded by rainfall.

Second, flooding kills embryos during the late incubation period by some
unknown mechanism(s). Based on patterns of Vo2. embryonic growth in the 1-

day submergence treatment was not affected by flooding except during

submersion. Submersion experiments were started when embryos were at 19
days of incubation, and the Vo2 of embryos until 24 days later were periodically

measured . The normal Vo2 pattern suggests that embryos were alive at least

up to day 43, 4 days before hatching. Because only 25 % of eggs in this
treatment hatched, the remaining 75 % of eggs must have died a few days
before hatching.

McGehee (1990) incubated UcaESlla eggs in sand with 0, 25, 50, 75,
and 100 % moisture and found that the hatching success is largest at 25 %
moisture and significantly less at higher and lower moisture levels. This
suggests the cause of mortality is due to oxygen depletion and possibly carbon
dioxide accumulation in nests. Kam (1988) found gas conductance of the
incubating media is correlated to moisture levels such that snapping turtle
embryos incubated in sand with 12 % gravimetric water content, which have
lower gas conductance, grow slower than those with 4 % gravimetric water

content.

92

incubation period and hatchling size

Incubation time increased as duration of submergence increased. The
long incubation period is attributed to suppressed metabolism during
submersion (Fig. 5-1). The similarity of dry mass in hatchling and residual yolk
of control and treatments suggest flooding does not affect overall embryonic
development and growth. Although embryos suppressed metabolism and
delayed development and growth during submersion, they attained normal
hatching size by taking a longer time to grow.

Water balance

During submersion, the daily water uptake by eggs in the 1- and 3-day
submergence treatments was higher than in controls (Table 5-3). When
submersed, air-filled pathways are presumably flooded with water. The amount
and direction of water exchange between embryos and surroundings depend
on the water potential gradient and water conductivity of the eggshell and its
membranes. Distilled water has a water potential of zero, whereas the water
potential of turtle eggs is about - 700 to - 800 kPa, depending on the osmotic
pressure in embryos (Tracey et al. 1978). Thus, water moves from the
surroundings to eggs in liquid water. In contrast, eggs of the control were
incubated in sand with 4 % gravimetric water content and a water potential of
about -4 to -7 kPa (Kam and Ackerman 1990), which does not differ much from
that in water. Eggs in the control exchange water mainly via vapor phase, and
the water vapor conductivity is much lower than the saturated hydraulic
conductivity of eggs in water (Hillel 1982). Consequently, water uptake by eggs
in the control group was less than those in 1- and 3-day submergence
treatments. It is surprising to find that eggs in the 6-day submergence treatment

93

gained less water than control eggs even though eggs in the water apparently
have higher water conductivity than eggs buried in the sand.

After being removed from water, eggs either continued to increase in
mass (control and the 1 -day submergence treatment, Figs. 5-1 , 5-3), or they
started to lose mass (3- and 6-day submergence treatmentsm Figs. 5-2, 5-4).
This difference in water balance apparently is related to whether embryos are
alive after removed from water. Eggs in the 1-day submergence treatment
consumed oxygen at the same rate as eggs in the control, indicating that
embryos are still alive. In contrast, eggs in the 3- and 6-day submergence
treatments did not resume oxygen consumption at the previous levels,
indicating that embryos may have died due to permanent damage to
physiological processes of the embryos. It is unclear why the viability of
embryos could reverse the direction of the water exchange process, even
though the water potential gradient clearly favors water moving from the
surroundings into eggs.

Ecological considerations

The hatching success of embryos was related to the duration of
submergence, suggesting that red-bellied turtle embryos do not have particular
physiological adaptations to flooding. This is probably also true for eggs of A,.
mississipiensis. porosus. and L EL muticus (Plummer 1 976; Joanen et al.
1977; Magnusson 1982). Obviously, flooding is an important factor in
determining the nesting success of these species. Trionvx m. muticus
experienced 3 % nesting success in a unusually wet year when compared to a
65 % nesting success in a dry year (Plummer 1 976). All porosus eggs laid
beside rivers are killed by flooding (Magnusson 1982) . Numerous field studies
have reported that all or nearly all C. caretta eggs are killed by excessive

94

rainfall (Ragotzkie 1959; Dean and Talbert 1975; Kraemer and Bell 1980),
suggesting that C. caretta eoas also do not possess physiological adjustments

to flooding.

For those species that bury eggs in habitats where flooding regularly
occurs, flooding presumably acts as a strong selective force in the evolution of
physiological adaptation of eggs. One possible adaptation available only to
well-developed embryos is to hatch when flooding occurs. Treatments such as
submergence or perfusion with nitrogen gas induce hatching of Carettochelvs
insculota embryos (Webb et al. 1986). Anoxia-induced hatching not only
prevents embryos from suffocation, but also supplies more water for hatchlings
to disperse.

The other possible adaptation is developmental arrest, which is
characterized by low metabolic rates and allows embryos to tolerate adverse
conditions (Ewert 1985). The developmental arrest has been documented in
eggs of some winter-nesting turtles when ambient temperatures are below
optimum. They go through the winter with extremely slow growth and
development. Such arrested development ends upon the arrival of warm
temperatures, and embryos begin to develop and grow again. As a result, the
incubation periods are much longer those of summer breeders (Goff and Goff
1932; Goode and Russell 1968). Embryonic diapause prevents embryos from
developing under less optimal conditions, which may lead to gross
abnormalities and even death.

There is no evidence at the present time that reptilian embryos exhibit
developmental arrests in response to flooding; however, it is possible that
developmental arrest evolves as a preadaption (exaptation). For species that
exhibit embryonic diapause in response to cold temperatures, the diapause

95

could be a preadaptation for flooding. In other words, embryos that are in
diapause during winter would not be affected by flooding.

For those eggs that are vulnerable to flooding, nest site selection of
females becomes important in determining the nesting success and recruitment
of young to a population. First, females could nest in different kinds of habitat
such that if eggs in a particular habitat are killed by flooding, hatchlings from
non-flooded habitats could disperse and replenish the population. Female
porosus lay eggs not only along the river banks but also in swamp habitats
(Magnusson 1982). No swamp nests are flooded, and predation by animals
and humans are also lower when compared to nests on river bank habitats.
Second, if females choose to nest in a particular habitat, then microhabitats that
are least susceptible to flooding are preferred. Caretta carett? nests on high
sloping beaches backed by rounded dunes or vegetation, and are usually
above the high tide line (Baldwin and Lofton 1959; McGehee 1990). In flatback
turtles, Chelonia depressa. 94 % of nests are located on the top of the dunes or
on the steep seaward slope, and the remaining 6 % are located between the
foot of dunes and the high tide mark (Limpus 1 971 ). Nests of Graptemvs
oculifera and G. pulchra are clumped at the highest points of open sand bars
along the edge of woods (Anderson 1 958). The females of 1. el. muticus
preferably nest on open sandbars free of vegetation, weeds, and sediment
markings, but the nest elevation varies among geographical populations. In
Kansas, nests are clumped at the highest points of sandbars (Fitch and
Plummer 1975: Plummer 1976), whereas, in Iowa, Louisiana, and Mississippi,
they are scattered along level sandbars (Goldsmith 1944; Anderson 1958).
Goldsmith (1944) reported that during low water level, females nest on the
sandy shores between the low and high water marks. These nests are
inundated when water level rises.

96

Table 5-1 . Survivorship and incubation time from eggs of the Florida red-

bellied turtle subjected to various durations of submersion.

Control

1 day

3 days

6 days

No. eggs

27

27

27

27

No. live hatchlings

24

7

1

0

% hatched

69

26

4

0

Incubation time (days)

43.5

(0.2)

46.7

(0.8)

47

97

Table 5-2. Hatchling and residual yolk mass of Florida red-bellied turtle eggs

subjected to various durations of submersion. Values are reported
as mean ± SE.

Control

1 day

3 days

P

Sample size

13

7

1

Dry hatchling mass (g)

1.87

(0.05)

1.79

(0.07)

1.79

> .05

Dry yolk mass (g)

0.65

(0.02)

0.65

(0.03)

0.59

> .05

98

Table 5-3. Water uptake by eggs at various durations in sand and water.

Values are reported as mean + SE. Sample size = 9.

Dailv water uptake (a / dav)

Half-buried in

Submerged in

P

sand

water

1 day

0.119

0.220

< .001

(0.005)

(0.027)

3 days

0.287

0.407

< .05

(0.009)

(0.054)

6 days

0.520

0.123

< .01

(0.028)

(0.144)

CONSUMPTION

99

Figure 5-1 . Oxygen consumption (ml / h) of eggs of the control (dashed line)

and 1-day submergence treatment (solid line) as a function of
incubation days. Values are reported as means ± SE.

CONSUMPTION

100

Figure 5-2. Oxygen consumption ( ml / h) of eggs of 3- (dashed line) and 6-day

submergence (solid line) treatments as a function of incubation
days. Values are reported as means ± SE.

EGG MASS (g)

101

1 5 1

14-

13

• CXDNTROL
° 1 DAY

12

1 0

20 30 40

AGE (DAYS)

50

Figure 5-3. Changes in mass of eggs in the control (dashed line) and 1 -day

submergence treatment (solid line). Values are reported as
means ± SE.

EGG MASS (g)

102

Figure 5-4. Changes in mass of eggs in 3- (dashed line) and 6-day

submergence (solid line) treatments. Valus are reported as
means ± SE.

GENERAL DISCUSSION

This study focused on gas exchange and physiological responses of
reptilian embryos to acute and chronic hypoxia. I determined critical oxygen
tensions of turtle, squamate, and alligator embryos in to evaluate their tolerance
of hypoxia. I chose the Florida red-bellied turtle, P. nelsoni, as a model
organism to further examine environmental influences on gas exchange and
physiological responses of embryos to chronic hypoxia and flooding.

Embryos of the American alligator, Alligator mississippiensis. the
loggerhead sea turtle, Caretta caretta. the Florida red-bellied turtle, Pseudemys
nelsoni. the yellow rat snake, Elaphe obsoleta quadrivittata. and the southern
fence lizard, Sceloporus undulatus undulatus maintained a constant Vo2 to

some degree of hypoxia. This indicates that they are metabolic regulators. A
low Vo2 and a high eggshell conductance for 02 explain how reptilian embryos

are able to maintain their metabolism in hypoxic conditions. Hypoxic tolerance

decreased over incubation, as indicated by an increase in Pc. Embryonic
development characterized by high Vo2 increases the Pc, whereas the

development of circulatory and respiratory functions that promote the Gc reduce

the Pc. The Pc increased presumably because increments of Gc, a measure of
02 transport capacity, did not offset the increase of Vo2 during development.

The similarity of Pc of embryos between species during late incubation
suggests that hypoxic tolerance of reptilian embryos is independent of shell
type and egg size. The ability of embryos to tolerate hypoxia does not correlate

103

104

with their habitat. For example, C. caretta embryos are the least hypoxic
tolerant species even though they clearly experience hypoxia when the P02 of

nest chambers decreases rapidly during the second half of incubation
(Ackerman 1 977). In contrast, JL &. quadrivittata and ll undulatus embryos

tolerate hypoxia best, yet these species are shallow nesters and most likely do
not encounter hypoxia conditions. Eggs are laid in hollow, rotting logs and
vegetation, under railroad ties, beneath rocks, at the base of grass clumps and
sawdust piles (Ashton and Ashton 1985; Ashton and Ashton 1988; Ernest and
Barbour 1989).

I used Florida red-bellied turtle eggs as model organisms to study effects
of temperature and hydration on critical O2 tension curves. Results show that
temperature but not hydration has significant effects on the critical oxygen
tension of embryos. Oxygen consumption and Pc of embryos were significantly
affected by temperature. The Q10 values of 2-3 at different incubation days
indicate thermal sensitivity of metabolism remains the same during
development. At each incubation day, Pc increased directly proportional to
temperature, indicating it is temperature dependent. Oxygen consumption and
Pc of the turtle embryos were not affected by egg hydration. Eggs incubated in
13 % gravimetric water content gained mass faster than at 3 % gravimetric
water content. However, oxygen consumption, critical oxygen tension, and
hatchling mass did not differ between groups.

Critical oxygen tensions at different ages, temperatures, and hydrations
were measured when embryos were exposed to acute hypoxia.

Generalizations about the ecological adaptation of reptilian embryos to hypoxic
conditions in the field may be premature because information concerning the
responses of embryos to chronic hypoxia is not available. Thus, the
physiological responses of turtle embryos to chronic hypoxia was subsequently

105

studied. Exposure to chronic hypoxia (10 % air) resulted in retarded growth,
depressed metabolism, comparable incubation period and hatchability, and
reduced hatchling mass. However, embryos are flexible, structurally and
physiologically, in enhancing oxygen transport to compensate hypoxic effects.
Hypoxic embryos reduced critical oxygen tensions by 22 mmHg from day 21 to
33, whereas normoxic embryos increased critical oxygen tensions by 9 mmHg.
The reduction in the critical oxygen tension of hypoxic embryos indicates that
they are capable of increasing oxygen transport in response to hypoxia, which
is the result of adjustments of circulatory (i.e., relative ventricular mass,
hematocrit) and respiratory (CAM morphometry) systems.

Anoxia is an extreme case of hypoxia, and it could occur when eggs are
flooded. About 20-30 % of alligator nests are flooded during at least some part
of the incubation periold. Turtle eggs were studied to assess the physiological
effects of flooding on water balance, metabolic rate, growth, survivorship, and
hatchlings of parchment-shelled eggs and embryos. Results show that
simulated flooding has dramatic effects on water balance, embryonic
metabolism and development of embryos. The 1-day submergence treatment
did not affect water balance and oxygen consumption of embryos, but did
increase egg mortality. The 3- and 6- day submergence treatments affected
water balance, oxygen consumption, and hatching success of eggs. Flooding
kills embryos either during submergence by suffocation, as found in the 3- and
6-day submergence treatments, or during late incubation by some unknown
mechanism(s), as found in the 1-day submergence treatment.

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BIOGRAPHICAL SKETCH

Yeong-Choy Kam was born in Negeri Sembilan, Malaysia on October, 7,
1959. He lived in Malaysia, Taiwan, Milwaukee, and Ames before moving to
Florida. He graduated from Chung Hwa High School in Kluang, Johore, in
1974. He received a Bachelor of Science degree from the National Taiwan
University in 1982. He graduated from the Iowa State University in 1988 with a
Master of Science in zoology. He married Chiung-Fen, Iris, Yen on January 1 ,
1985, and has a son, Michael, and will have a daughter. He is a member of the
American Society of Zoologists and the American Society of Ichthyologists and
Herpetologists.

114

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.

Q>

-e

iltL-

Harvey B. Lillywhite, Chair
Professor of Zoology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.

in F. Anderson
[ssociate Professor of Zoology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.

Associate Professor of Zoology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the

Associate Professor of Zoology

degree of Doctor of Philosophy.

ichele G. Wheatly

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is ful y adequate, in scope
and quality, as a dissertation for the degree..of DoctorJiLElTilosophy.

IddhjL —

Charles E. Wood

Associate Professor of Physiology

This dissertation was submitted to the Graduate Faculty of the
Department of Zoology in the College of Liberal Arts and Sciences and to the
Graduate School and was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

May, 1 992

Dean, Graduate School