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Azzam et al., p. 1
Hypoxic incubation blunts the development of thermogenesis in chicken embryos and hatchlings
Milène A. Azzam, Kirsten Szdzuy and Jacopo P. Mortola *,
Department of Physiology, McGill University, 3655 Promenade Sir William Osler,
Montreal, Quebec, H3G 1Y6 Canada
Running title: Hypoxic incubation and thermogenesis
*Tel. : +1-514-3984335; fax: +1-514-3987452.
E-mail address: [email protected]
Page 1 of 30Articles in PresS. Am J Physiol Regul Integr Comp Physiol (March 8, 2007). doi:10.1152/ajpregu.00885.2006
Copyright © 2007 by the American Physiological Society.
Azzam et al., p. 2
Abstract
We asked to what extent sustained hypoxia during embryonic growth might interfere with the
normal development of thermogenesis. White Leghorn chicken eggs were incubated at 38oC
either in normoxia (Nx, 21% O2) or in hypoxia (Hx, 15% O2, from embryonic day E5 until
hatching). The Hx embryos had lower body weight (W) throughout incubation, and hatching was
delayed by about 10 hours. For both groups, all measurements were conducted in normoxia. At
embryonic day E11, the static temperature- oxygen consumption (ambient T-2OV& ) curve was
typically ectothermic (Q10 = 1.92-1.94), and similar between Nx and Hx. Toward the end of
incubation (E20), the Q10 averaged 1.41 (±0.06) in Nx and 1.79 (±0.08) in Hx (P<0.005),
indicating that the onset of the thermogenic response in Hx lagged behind Nx. In the 1-day old
hatchlings (H1) body weight did not significantly differ between Nx and Hx. At H1, the T-
2OV& curves were endothermic-type, and more so in the older (>8hrs old) than in the newly
hatched (<8 hrs old) chicks, whether examined statically or dynamically as function of time. In
either case, the thermogenic responses of Hx were lower than those of Nx. In a 43-31oC
thermocline, the preferred T of the Hx hatchlings was around 37.3oC, and similar to Nx,
suggesting a similar setpoint for thermoregulation. We conclude that hypoxic incubation blunted
the development of thermogenesis. This could be interpreted as an example of epigenetic
regulation, where an environmental perturbation during early development alters the phenotypic
expression of a regulatory system.
Key words. Embryonic development. Epigenetic adaptation. Hatching. Hypometabolism.
Hypoxia. Thermoregulation, development.
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Azzam et al., p. 3
Introduction
Mammals and birds are homeotherms, meaning that they regulate body temperature around a
set value, over a wide range of ambient temperatures. This characteristic develops during the late
prenatal period and begins to be functional at birth and hatching, when it manifests itself with a
combination of behavioural and autonomic mechanisms (4, 21, 43). One of these mechanisms is
heat production, or thermogenesis, a metabolic response to cold that, in young mammals, is
mostly contributed by the calorigenic properties of the brown adipose tissue and the activity of its
uncoupling protein (31). Hypoxia influences all aspects of thermoregulation. In particular, in
neonatal mammals, hypoxia depresses thermogenesis (27), decreases the brown fat mass and the
expression of its uncoupling protein (29, 30). In addition, as in the adults of many classes of
animals, hypoxia lowers the preferred ambient temperature (6, 7, 15, 25, 44). The depression of
heat production is one of the mechanisms to save on oxygen consumption (2OV& ); hence, it
represents an important strategy to survive hypoxia (22).
Like after birth, also in the prenatal period hypoxia decreases metabolic rate, but the energy
saving comes largely from the reduction in fetal growth (18, 21). In fact, during the embryonic
development of a bird or a mammal, thermogenesis is absent, and begins to be operational only
in the perinatal period (e.g., 14, 28). Because of this late development, it is possible that prenatal
hypoxia may have no effects on the formative process of thermogenesis. On the other hand, the
development of thermogenic mechanisms may be sensitive to the prenatal metabolic history, as
in the case of avian embryos incubated at below-normal temperatures. In these embryos, the
sustained hypometabolism provoked by cold-incubation not only stunted body growth but also
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Azzam et al., p. 4
blunted the normal developmental process of thermogenesis (3, 23). The eventuality that prenatal
hypoxia may interfere with the normal development of the mechanisms of heat production has
received little attention, and the few data available are open to different interpretations.
In human infants born at high altitude, acute cold exposure caused a thermogenic response
significantly lower than in infants at sea level (11). This result could mean that the hypoxic
gestation of the high-altitude infants depressed the developmental processes of their thermogenic
mechanisms. However, it could also reflect the fact that the measurements were conducted at
high-altitude, and hence in a hypoxic condition that, as mentioned above, by itself reduces 2OV&
and thermogenesis.
Two-day old rats born from dams exposed to hypobaric hypoxia since conception had lower
body weight and 2OV& than controls, their brown fat was hypoplastic with decreased uncoupling
protein; yet, when subjected to cold, their thermogenic response was almost as in normoxic-
gestated animals (12, 30). These seemingly conflicting results were attributed to the stimulating
effects on thermogenesis caused by stress-related mechanisms, possibly of maternal origin. In
fact, during pregnancy, the maternal adaptation to hypoxia influences birth weight (19) and
causes numerous hormonal responses, including thyroid and catecholamine release, known to
have an impact on fetal development (9, 13, 41). After birth, maternal stress can diminish
lactation and the care for the litter, causing complex effects on the pup’s metabolism and the bio-
molecular machinery involved in heat production (2, 20, 34, 36). Avian embryos, in which the
chorioallantoic membrane provides gas exchange, offer an opportunity to study the effects of
prenatal hypoxia on the development of thermogenesis without the issue of the maternal
Page 4 of 30
Azzam et al., p. 5
interference on the embryo’s response. Therefore, for this study we have chosen the chicken
embryo, an animal model with a well characterised developmental pattern of heat control, and for
which the hypometabolic response to acute hypoxia is also well documented (1, 5, 24, 28, 37, 39,
40).
Materials and Methods
Experiments were performed on fertilized White Leghorn chicken eggs, obtained from a local
supplier. Eggs were stored at 15oC until the start of incubation, and for no longer than 7 days.
The eggs were weighed and placed in incubators (Hova-Bator, Savannah, GA) around midday
(day 0). The incubators maintained a steady temperature (T) of about 38oC and 60% relative
humidity, and provided a 45-degree egg rotation four times a day. At first, all eggs were
incubated in normoxic conditions. Then, at embryonic day E5, they were separated into two
groups. Some continued in normoxia (21% O2, Normoxic, Nx), others were transferred into a
hypoxic incubator kept at 15-16% O2 (on average, including the periods of incubator opening for
egg transfer and cleaning, 15.48±0.01) (Hypoxic, Hx). The desired level of hypoxia was obtained
by leaking a small stream of warmed and humidified N2 into the incubator from a pressurised
tank, under the control of a flowmeter. The O2 concentration within the incubator was
continuously sampled by a calibrated fuel cell gas analyser (Foxbox, Sable Systems Int., Las
Vegas, NV) and displayed on a computer monitor. A T-data logger and a hygrometer were inside
each incubator; the former collected the T value every 10 min, while humidity was read daily.
Body growth was measured from embryonic day E8 to hatching, at 3-day intervals. The
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Azzam et al., p. 6
metabolic responses to changes in T were measured in normoxia, in the Nx and Hx embryos at
the same chronological day (E11, E20), and in the hatchlings on the day of hatching (H1), either
within the first eight hours (<8 hrs, on average at 4.2±0.4 and 3.9±0.4 hrs in Nx and Hx,
respectively) or later during the day (8-24 hrs, on average 14.7±0.8 and 14.3±0.8 hrs in Nx and
Hx, respectively).
Egg weight at the start of incubation, embryo weight, and daily water loss (equal to the egg
weight loss) were measured for each egg. At the end of the experiment, the embryo was killed by
exposure to CO2 and cold, the egg was opened, the embryo examined to exclude gross
anatomical abnormalities, and its body weight (W) measured on a digital scale.
Metabolic rate was measured in normoxic conditions by indirect calorimetry (oxygen
consumption, 2OV& , and carbon dioxide production,
2COV& ), with an open-flow methodology (10)
adapted to the chicken embryo (16, 17, 28). Measurements were performed either in sets of two
(E11, E20) or individually (H1) in a respirometer, which consisted of a 300-ml plastic container
maintained at the desired T by a water bath. A steady gas flow of either 100 (E11) or 200 ml/min
(E20 and H1) was continuously delivered through the respirometer, under the control of a
precision flowmeter. The inflow and outflow O2 and CO2 concentrations were monitored by gas
analysers (OM-11, Beckman and CD-3A, Applied Electrochemistry) arranged in series, after the
gas had passed through a drying column. The outputs of the analysers were displayed on a
computer monitor during on-line acquisition. 2OV& and
2COV& were computed from the flow rate
and the inflow-outflow concentration difference. The values, calculated at standard temperature,
pressure, and dry conditions, are presented in ml/min.
Page 6 of 30
Azzam et al., p. 7
Protocol 1: 2OV& at steady ambient temperatures. These measurements were performed on day
11 (E11) and day 20 (E20) embryos, and in H1 during the first (H1<8hrs) or late hours
(H1>8hrs). Each age group consisted of different animals. Embryos were studied in sets of two,
and the hatchlings were studied individually. Measurements of gaseous metabolism were
collected at T=39-36-33-30oC. Each T was maintained for one hour, and data refer to the last 5
minutes of each exposure. T was changed either in ascending or descending sequence, alternating
the order among animals. Because of the thermal inertia of the water bath, any change in T
required about 15 min to reach complete equilibrium.
Protocol 2: time-course of response to cold. These measurements were performed at H1. After
at least a half-hour of acclimatisation to the respirometer, gaseous metabolism was measured
continuously for 30 min at 39oC and for 1 hour following an abrupt drop in water bath
temperature to 30oC.
Protocol 3: preferred temperature. These measurements were performed on Nx and Hx
hatchlings at < 8hrs and > 8hrs, studied individually in a thermocline. This consisted of a plastic
tube 70 cm long with an inner diameter of 10 cm, closed by lids at either end, with steady airflow
of 400 ml/min. An internal platform about 7 cm wide ran through the whole length. Two roller
pumps circulated the water from separate water baths through long polyethylene tubing coiled
around the thermocline, each coil a few cm apart. By regulating the temperature of the baths, the
water flows and the relative distance of the coils, it was possible to generate a temperature
difference between the two ends of the thermocline, from about 43 to 31 oC, with an internal
linear gradient of 5 cm/oC. Four tungsten-constantan thermocouples were permanently placed at
Page 7 of 30
Azzam et al., p. 8
various locations, to make sure that the prefixed T gradient was stable throughout the recording;
the correlation coefficient of the distance-temperature regression line was never below 0.96, and
averaged 0.984±0.001.
The hatchling was placed in the middle of the thermocline, and was left undisturbed for at
least 1 hour. Then, the chick’s position was noted (± 1.5 cm) continuously every min for 30 min.
The average min-by-min difference in position represented the average distance travelled over
the 30 min of the measurements. Position data were transformed into thermocline T from the
slope and intercept of the position-T linear regression.
Number of animals, data normalisation and statistics. For each experiment, numbers of
animals are given in the corresponding sections of Results, Table and Figure legends. All group
data are presented as means ± 1 S.E.M. Linear regression through the data points was employed
to compute the Q10 values of E11 and E20, according to the van’t Hoff equation Q10 = (2OV& ’/
2OV& ”) 10 / (T’-T”), where 2OV& ’ and
2OV& ” are the values of 2OV& at ambient temperature 39oC (T’)
and 30oC (T”). Statistically significant differences between two groups of data (e.g., body
weights at any given age, Q10 values, 2OV& at a given T) were evaluated by two-tailed t test.
Comparisons of the responses to the thermocline were done by two-way ANOVA (one grouping
factor being age, the second being the normoxic or hypoxic incubation), with post hoc
Bonferroni’s limitations for the four comparisons. In all cases, a difference was considered
statistically significant at P<0.05.
Results
Page 8 of 30
Azzam et al., p. 9
Number of animals, egg weights, age and postnatal hours of the experiments, body weights of
Table 1 embryos and hatchlings are presented in Table 1.
Fig. 1 Embryo’s growth and hatching. The body weights (W) of the embryos growing in hypoxia,
collected every three days from day 5 to hatching, were lower than in Nx (Fig. 1). At E20, all the
hypoxic embryos had the yolk separated, while more than half of the Nx embryos studied had the
yolk already incorporated in the abdomen, increasing their W by about 12 g. The hatching age of
Hx occurred was 21.04 ± 0.07 days, an age slightly, yet significantly, longer than that of Nx
(20.59 ± 0.07; P<0.001). During the first post-hatching day (H1) the W of the Hx hatchlings
(39.7 ± 0.5 g, N=53) was close to that of Nx (40.5 ± 0.5 g, N=52; P>0.05). Specifics of body
weight, days of hatching and post-hatch hours for the H1 studied in the various protocols are
given in Table 1.
Sustained changes in ambient temperature. The W of the E11 embryos used for these
measurements averaged 4.2 ± 0.1 g, and 3.6 ± 0.1, in Nx and Hx, respectively (P<0.001). At any
given T, the 2OV& values of the Hx were lower than in Nx (Fig.2, top panel). This was due to the
differences in embryo’s W; in fact, after W-normalisation the curves overlapped. The shape of
the individual curves could be compared readily after expressing the T-2OV& relationships in
percent of the values at 39oC (bottom panel); they did not differ significantly from each other.
Fig. 2 Also the Q10 values of the two groups did not differ significantly (Nx, 1.92 ± 0.075; Hx, 1.94 ±
0.10).
The E20 of the Hx group used for these measurements weighed 27.2 g (± 1.0), significantly
less than the Nx embryos (40.5 ± 1.6 g). In either group of embryos the decrease in T lowered
Page 9 of 30
Azzam et al., p. 10
2OV& . At any given T, the 2OV& values of Hx were lower than in Nx (Fig. 3, top left panel). After
Fig. 3 expressing the T-2OV& relationships in percent of the values at 39oC (Fig. 3, bottom left panel),
the Hx values were significantly lower than in Nx at T=33oC (P<0.05) and 30oC (P<0.01). The
steeper T-2OV& curve in Hx than in Nx was reflected by the differences in Q10 values, which
averaged 1.41 (± 0.06) in Nx and 1.79 (± 0.08) in Hx (P<0.005).
The W of the hatchlings (H1) used for these measurements did not differ significantly between
Nx and Hx, neither for the groups studied in the first hours (<8 hrs) nor for the groups studied in
the later hours (8-24 hrs) (Table 1). In Nx, 2OV& at 33 and 36oC was higher than at 39oC. With
further cooling to 30oC this thermogenic response was not maintained in the younger hatchlings,
while it was well maintained in the older group (Fig. 3, middle and right panels). This general
pattern was also apparent in the Hx hatchlings, with two main differences. At the warmer
temperatures, the absolute values (ml/min) of 2OV& of the Hx hatchlings exceeded the Nx values
(Fig. 3, top panels, at center and right), the difference reaching statistical significance in the
younger group. After expressing each curve in percent of the corresponding 39oC value (Fig. 3,
bottom panels, at center and right), the T-2OV& functions of the Hx were below Nx. At each T, this
difference was statistically significant at 33 and 36oC (younger group) and at 30oC (older group).
A statistical analysis of all the data points of the thermogenic response (i.e, the 30-36oC range)
indicated a significantly lower response in Hx than in Nx, both for the < 8hrs group (Hx: 107% ±
5; Nx: 140% ± 8, P<0.001) and the > 8hrs group (Hx: 131% ± 7; Nx: 158% ± 10, P<0.05).
For all the age groups examined, the changes in 2COV& with T, and the differences among
Page 10 of 30
Azzam et al., p. 11
groups, were qualitatively similar to those described for 2OV& .
Time-course of the metabolic response to acute cold. As it was the case for the previous tests,
also the Hx hatchlings used for these measurements, compared to Nx, had very similar W (Table
1) and higher 2OV& at 39oC (0.85 ± 0.04 ml/min versus 0.68 ± 0.03, P<0.001). Upon exposure to
30oC, 2OV& of Nx increased by about 40% and 65%, in the young and older group, respectively
(Fig. 4). Also the Hypoxic hatchlings increased 2OV& , but the increase was short-lasting at either
Fig. 4 age, and, from 30 min on, their metabolic response was significantly less than in Nx. To quantify
the ability to maintain a sustained metabolism in the cold, the 2OV& values at 60 min were
expressed in percent of the values attained at 15 min. In the Nx of the younger age, 2OV& at 60
min was 95% ± 10 of that attained at 15 min, a percentage significantly higher than in the same-
age Hx hatchlings (66% ± 2; P<0.02). At the older ages, the same ratios were 110% ± 11 (Nx)
and 83% ± 3 (Hx) (P<0.05).
Response to thermocline. During the 30-min recording in the thermocline, on average,
hatchlings moved at 4.0 ± 0.5 cm/min, with no significant differences between groups or age
periods. In Nx, the most frequently chosen T was 37.6 oC ± 0.4 and 37.4 oC ± 0.4 in the younger
Fig. 5 and older group, respectively. The preferred T of Hx did not differ significantly from Nx at either
age (Fig. 5, bar graph at left; two-way ANOVA, P>0.05). When the same statistical analysis was
applied to the 30 min data points of all animals combined (Fig. 5, panels at right), a small
significant difference emerged between the young Hx and Nx groups, and between the younger
and the older Hx groups (two-way ANOVA, post-hoc Bonferroni’s limitations, P<0.05).
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Azzam et al., p. 12
However, as it is obvious from the inspection of the histogram distributions, such differences
were quite small.
Discussion
The main information obtained from this study is that sustained embryonic hypoxia blunts the
development of the thermogenic mechanisms. Previously, it was noticed that high-altitude infants
had a lower thermogenic capacity than sea-level gestated infants (11). However, because the
measurements were performed at high altitude, it was unclear to what extent the infant’s blunted
thermogenesis was a consequence of the hypoxic gestation or was part of the acute response to
hypoxia, which, in neonates, is characterised by hypometabolism (27). Measurements of the
metabolic response to cooling temperatures in neonatal rats studied in normoxia after hypoxic
gestation circumvented this problem (30). However, they could not eliminate the confounding
issue of the maternal hypoxia, a stress-evoking condition that has implications on fetal and
newborn development, including temperature control (2, 20, 34, 36). The current results in an
avian model, therefore, give substantial weight to the possibility that prenatal hypoxia by itself,
and independently of maternal responses, disturbs the normal development of thermogenesis.
Prenatal hypoxia may interfere with the normal development of thermogenesis through a
variety of mechanisms. Chronic prenatal hypoxia blunts embryonic growth (Fig. 1) (5, 8), and,
during cold exposure, a small-size animal decreases metabolic rate (through the Q10 effect) more
readily than a large-size animal because of its smaller heat capacitance and larger surface-to-mass
ratio. However, the possibility that the smaller body size may be the main reason for the
Page 12 of 30
Azzam et al., p. 13
differences in thermogenesis between Hx and Nx cannot apply to the current results, either at
E20 or at H1. At E20, egg mass, not embryo mass, is the pertinent factor determining heat
capacitance, and the Hx eggs were not smaller than the Nx eggs. The two groups of hatchlings
did not differ in body size; in fact, the slightly longer incubation time allowed the Hx embryos to
incorporate their yolk, so that, by hatching time, they had reached a body weight comparable to
Nx, meaning that they also had a comparable surface-to-mass ratio. A similar phenomenon was
seen in embryos incubated in the cold, in which the lower embryo’s weight was compensated by
a longer incubation time (23).
Another possibility is the existence of cardiopulmonary malformations in the hatchlings of the
Hx group causing hypoxemia, a situation that would decrease their metabolic rate and impede a
calorigenic response (28). However, although embryonic hypoxia can modify the growth of
various organs, neither the lungs nor the heart are affected by the level of hypoxia (15% O2)
adopted in this study (5, also for review). In addition, the resting 2OV& (at 39oC) of the Hx
hatchlings was not lower than in Nx. On the contrary, in the younger group it was slightly higher
than in Nx, presumably to pay back some lactic acid accumulation and O2 debt contracted during
the hatching efforts in hypoxia or for the compensatory (catch-up) growth upon return to
normoxia. Hence, it does not seem probable that the Hx chicks had hypoxemia, and that this was
the cause of their poor thermogenesis.
At E11, i.e. at an age when the embryo is purely ectothermic, the lower metabolic rates of Hx
were undoubtedly a consequence of their blunted body growth. In fact, the differences in 2OV&
from Nx could be entirely corrected after normalisation by weight, or after expressing the curves
Page 13 of 30
Azzam et al., p. 14
in percent of the value attained at 39oC (Fig. 1). Also the Q10 values were similar between the
two groups, and close to 2, which is the normal value for ectothermic embryos (23, 28, 39, 43).
Because thermogenic mechanisms begin to function toward the last days of incubation, as
apparent by a decrease in Q10, a developmentally retarded embryo retains the ectothermic pattern
for a longer time during incubation, with higher Q10 values than normal. This is what was
observed previously in normoxic embryos growth-retarded by a low incubation temperature (23),
and could explain the differences between Hx and Nx at E20 and H1. Hatching is a biological
event, not linked to a specific chronological age, and hypoxia delayed its occurrence by about 11
hours. Therefore, the lower thermogenesis in Hx than Nx may reflect a generalised blunting
effect of embryonic hypoxia on growth and a delay on the timing of hatching. Alternatively, the
similar body weight at a time when thermogenesis was decreased may be interpreted as
indicating that embryonic hypoxia did not affect body growth and the processes surrounding
hatching as much as it affected thermogenesis. A disturbance in the relative timing of the onset of
different physiological processes has been termed heterokairy (38), which is a special case of
epigenetic adaptation. This latter expression refers to the long-term effects of early
environmental perturbations on gene expression and the development of physiological
mechanisms. Cold exposure during prenatal development has been quoted in the past as an
example of epigenetic adaptation for the postnatal manifestation of thermogenesis (23, 32, 33,
42). The current results may suggest that embryonic hypoxia is another example of epigenetic
adaptation of thermogenesis. Because both hypoxia and cold during most of embryonic
development cause hypometabolism, it would be of interest to study other interventions causing
Page 14 of 30
Azzam et al., p. 15
sustained metabolic changes, such as caloric restriction or pharmacological interventions, to test
the possibility that embryonic hypometabolism represents the common event for the blunting in
the development of thermoregulation. Equally, it would be informative to know whether the
effects of hypoxia on the development of thermogenesis have specific time-windows during
embryonic growth, as it is the case for body and organ development (5, 8).
The preferred ambient T of the Hx hatchlings essentially coincided with that of Nx. Only the
younger Hx group showed a small tendency for lower temperatures, but the difference from Nx
was minute and, presumably, was dictated by their higher resting 2OV& . Our interpretation of these
results, therefore, is that prenatal Hx did not cause a long-term modification of the normoxic
setpoint of thermoregulation. This is different from the drop in setpoint known to occur in the
presence of hypoxia, and demonstrated in many classes of animals (6, 7, 15, 25, 44).
In conclusion, embryonic hypoxia delayed the establishment of thermogenic mechanisms. The
blunted thermogenesis was manifest also after hatching, at a time when the hypoxia-incubated
animals had reached normal body weight. It is possible that the blunted thermogenesis is one
aspect of a generalised hypoxia-mediated slowing of all embryonic developmental trajectories.
Alternatively, it may be indicate that prenatal hypoxia has differential impact on growth, the
timing of hatching, and thermoregulation. If these data were extrapolated to humans, they would
suggest that fetal hypoxia, as with maternal smoking or high altitude pregnancy, could endanger
the heat control mechanisms of the neonate, not only because of the infant’ small size (26, 35),
but also because of the epigenetic effects on neonatal thermogenesis.
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Azzam et al., p. 16
Grants
Funds for this research came from a Canadian Institute of Health Research grant.
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Azzam et al., p. 17
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Figure legends
Fig. 1. Growth curves for the normoxia- (open symbols) and the hypoxia-incubated embryos
(filled symbols). Symbols are group means, bars represent 1 SEM. When not indicated, SEM
were within the symbol size. Numbers indicate the number of animals studied. At E20, 9 of
the Nx embryos had the yolk already incorporated in the abdomen. Hatching time was slightly,
yet significantly, delayed in the hypoxic-incubated embryos, by about 11 hours (§, P<0.005).
*, statistically significant difference in body weight between the two groups (P<0.05).
Fig. 2. Ambient temperature - oxygen consumption (2OV& ) relationships in embryos at day 11,
with 2OV& expressed in absolute values (top panel) and in percent of the values attained at 39oC
(bottom panel). Open symbols, normoxia-incubated embryos. Filled symbols, hypoxia-
incubated embryos. Symbols are the average of 10 sets of two embryos each. *, statistically
significant differences between the two groups (P<0.05).
Fig. 3. Ambient temperature - oxygen consumption (2OV& ) relationships in 20-day old embryos
(left panels) and in hatchlings, during the first 8 hours from hatching (middle panels), or
during the remaining hours of the day (right panels). 2OV& is expressed in absolute values (top)
and in percent of the values attained at 39oC (bottom). Open symbols, normoxia-incubated
animals. Filled symbols, hypoxia-incubated animals. Symbols are the averages of 10 sets of
two embryos each, or of 10 hatchlings. Bars represent 1 SEM. *, statistically significant
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differences between the two groups (P<0.05).
Fig. 4. Changes in oxygen consumption (2OV& ) during 1 hour of exposure to 30oC ambient
temperature, expressed in percent of the value at 39oC. Data refer to normoxia-incubated
(open symbols) and hypoxia-incubated hatchlings (filled symbols), during the first 8 hours
from hatching (left panel), or during the remaining hours of the day (right panel). For each
curve, symbols are the averages of 7-10 animals; bars represent 1 SEM. *, statistically
significant differences between the two groups (P<0.05).
Fig. 5. At left: average preferred ambient temperature in normoxia-incubated (open columns) and
hypoxia-incubated hatchlings (filled columns), during the first 8 hours from hatching and
during the remaining hours of the day. Columns are averages of 10 animals, bars are SEM. At
right: histogram distributions of the preferred ambient temperatures during the 30 min of
recording, for all the animals combined (total of 300 min per group).
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Table 1- Number of animals, egg weight, age and body weight
N egg wt Day of Post-hatch Body
N at E0, g hatching hours weight, g
Protocol 1 - Steady ambient temperatures (30-33-36-39oC):
E11 Nx 10 sets a 57.0±0.6 - - 4.2±0.1
Hx 10 sets a 60.3±1.0(<0.001) - - 3.6±0.1(<0.001)
E20 Nx 10 sets a 56.2±0.8 - - 40.5±1.6
Hx 10 sets a 60.9±0.7(<0.001) - - 27.2±1.0(<0.0001)
H1<8hrs Nx 7 54.1±1.7 20.7±0.3 3±1 39.1±1.5
Hx 7 58.8±0.7(<0.05) 21.0±0.0 3±1 39.9±1.6
H1>8hrs Nx 7 59.8±1.8 20.9±0.2 13±3 40.5±1.2
Hx 11 58.2±1.8 21.1±0.1 13±1 38.7±1.0
Protocol 2 - Time-course of response to 30oC:
H1<8hrs Nx 8 60.8±1.5 20.8±0.2 4±1 42.4±1.7
Hx 8 59.9±1.6 20.9±0.1 4±1 42.7±1.5
H1>8hrs Nx 10 59.5±1.2 20.5±0.2 16±1 42.4±0.7
Hx 7 66.7±1.8 20.6±0.2 15±1 41.3±1.1
Protocol 3 - Preferred temperature in thermocline:
H1<8hrs Nx 10 53.6±1.1 20.4±0.1 5±1 39.6±0.9
Hx 10 52.7±0.9 21.3±0.1(<0.001) 4±0.4 38.6±0.7
H1>8hrs Nx 10 55.5±1.2 20.4±0.1 15±1 39.0±0.8
Hx 10 54.2±1.5 21.2±0.2 (<0.001) 16±1 38.2±1.1
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Legend of Table 1
Values are mean ±1 SEM. Nx, normoxia-incubated; Hx, hypoxia-incubated. E11, E20, embryos at
age 11 and 20; H1, first day post-hatching. In brackets, P value of the statistical comparison between
Hx and Nx. a Each set comprises two embryos. Post-hatch hours indicate the hours following
hatching at the time of the measurements.
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