Respiratory Physiology & Neurobiology 146 (2005) 97–106

Oxygen consumption of the chicken embryo: interaction

between temperature and oxygenation
Jacopo P. Mortola∗ , Katherine Labbè
Department of Physiology, McGill University, 3655 Sir William Osler Promenade, Montreal, Que., Canada H3G 1Y6

Accepted 28 October 2004

Abstract

We measured the effects of hypoxia and changes in ambient temperature (T) on the oxygen consumption (V̇O2 ) of chicken
embryos at embryonic days 11, 16 and 20 (E11, E16 and E20, respectively), and post-hatching day 1 (H1). Between 30 and
39 ◦ C, at E11 and E16, V̇O2 changed linearly with T, as in ectothermic animals, with a Q10 of about 2.1. At E20, V̇O2 did not
significantly change with T, indicating the onset of endothermy. At H1, a drop in T increased V̇O2 , a clear thermogenic response.
Hypoxia (11% O2 for 30 min) decreased V̇O2 , by an amount that varied with T and age. At H1, hypoxia lowered V̇O2 especially
at low T. At E20, hypoxic hypometabolism was similar at all T. At E11 and E16, hypoxia lowered V̇O2 only at the higher T. In
fact, at E11, with T = 39 ◦ C even a modest hypoxia (15–18% O2 ) decreased V̇O2 . Upon return to normoxia after 40 min of 11%
O2 , V̇O2 did not rise above the pre-hypoxic level, indicating that the hypometabolism during hypoxia did not generate an O2
debt. At E11, during modest hypoxia (16% O2 ) at 36 ◦ C, the drop in V̇O2 was lifted by raising the T to 39 ◦ C, suggesting that
the hypoxic hypometabolism at 36 ◦ C was not due to O2 -supply limitation. In conclusion, the hypometabolic effects of hypoxia
on the chicken embryo’s V̇O2 depend on the development of the thermogenic ability, occurring predominantly at high T during
the early (ectothermic phase) and at low T during the late (endothermic) phase. At E11, both low T and low oxygen force V̇O2
to drop. However, at a near-normal T, modest hypoxia promotes a hypometabolic response with the characteristics of regulated
O2 conformism.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Birds; Hatching; Embryos; Development; Oxygen; Hypoxic hypometabolism; Gaseous metabolism; Thermoregulation; Hypoxia

1. Introduction ambient temperature (T) according to the Arrhenius

In the chicken embryo, during a large portion of
development, oxygen consumption (V̇O2 ) varies with
∗ Corresponding author. Tel.: +1 514 398 4335;
fax: +1 514 398 7452.
E-mail address: jacopo.mortola@mcgill.ca (J.P. Mortola).
factor (or Q10 ), as expected for ectothermic animals.
Differently, in the last phases of incubation, when con-
fronted by a drop in T, the embryo attempts to increase
V̇O2 as a means of controlling body temperature, in
what represents the early manifestation of endothermy.
Hence, the embryo gradually shifts from the poikilo-
thermic pattern that characterizes a large portion of its

1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.resp.2004.10.011
98 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106
development to the homeothermic behavior, which will
be fully manifest post-natally (Whittow and Tazawa,
1991; Nichelmann and Tzschentke, 2003).
As for T, changes in O2 availability also cause cor-
responding changes in the embryo’s V̇O2 (Bartels and
Baumann, 1972; Stock et al., 1985; Bjønnes et al.,
1987; Ar et al., 1991; Tazawa et al., 1992). The in-
teraction between T and oxygenation in setting the
embryo’s metabolic rate has not been studied with
specific experiments. One may expect that the effects of
the interaction should vary depending on whether the
ectothermic or the endothermic pattern is prevailing. At
low T, during the ectothermic phase of the embryonic
development, because V̇O2 is small, changes in oxy-
genation should have less impact on V̇O2 than at high T.
Conversely, during the late phases of development, the
embryo is likely to be more O2 dependent at lower, than
at higher T, because the low T promotes a thermogenic
effort with the rise in V̇O2 . Therefore, one may hypothe-
size that, in the growing chicken embryo, hypoxia may
pose a limit to V̇O2 at high T during the earlier phases
and at low T during the late phases of development.
The hypometabolic response during hypoxia could
result from the scarce O2 supply, like it would hap-
pen in any reaction with substrate limitation. Hence,
the V̇O2 level attained during the hypoxia-induced hy-
pometabolism would be the maximal possible for that
degree of oxygenation. In such a situation, a drop in T
(during the endothermic phase of development) or an
increase in T (during the ectothermic phase) would not
be able to raise hypoxic V̇O2 , since hypoxic V̇O2 is O2 -
limited. Conversely, the hypometabolic response could
reflect a regulated phenomenon, triggered by hypoxia
but not necessarily forced upon by the O2 limitation. If
this was the case, the level of hypometabolism would
not be necessarily the maximal attainable and for a con-
stant level of hypoxia, the V̇O2 level could still change
with changes in T. A regulated hypometabolic response
to hypoxia, or regulated O2 -conformism, seems to be
a wide-spread phenomenon observed in invertebrates
(Paul et al., 1997; Wiggins and Frappell, 2000; Hicks
and McMahon, 2002; Alexander and McMahon, 2004),
in reptiles (Hicks and Wang, 1999) and in adult and
neonatal mammals (Saiki and Mortola, 1997; Rohlicek
et al., 1998). During the embryonic period, the possi-
bility of hypoxic hypometabolism being a regulated
phenomenon, rather than O2 limitation, has never been
experimentally considered in any class of animals.
The primary purpose of this study was to examine
the effect of hypoxia on the V̇O2 response to changes in
T in the chicken embryo at various stages of its devel-
opment. Further, the possibility that the hypometabolic
response to hypoxia is a regulated, rather than passive
(i.e., O2 supply-limited), response has been addressed
by examining the effect of changes in T on V̇O2 at var-
ious levels of hypoxia.
2. Methods
Experiments were conducted on freshly laid fertil-
ized eggs of White Leghorn chickens, obtained from
a local supplier. On day 0, the eggs weighed between
60 and 65 g. They were placed in a still air incubator
(Hova-bator Model, 1602), at 38 ◦ C and 60% relative
humidity, with automatic rotation of the eggs four times
per day. Measurements were performed on embryos at
days 11, 16, 20 (during the star fracture of the exter-
nal pipping phase), and on post-natal day 1 (within 24 h
from hatching). These groups will be referred to as E11,
E16, E20 and H1, respectively.
The experiments consisted of measuring metabolic
rate by indirect calorimetry (oxygen consumption, V̇O2
and carbon dioxide production, V̇CO2 ) at several tem-
peratures and levels of oxygenation, in various combi-
nations. After the measurements, the eggs were opened,
the embryos killed by cold (E11), exposure to CO2
(E16, E20) or with an overdose of barbiturate anaesthe-
sia (H1), examined to exclude obvious gross anatomi-
cal abnormalities and weighed with a digital scale ac-
curate to 10−3 g.
2.1. Oxygen consumption
Measurements of oxygen consumption (V̇O2 ) were
obtained by the open-flow method (Frappell et al.,
1992), with a setup adapted to the chicken embryo
(Menna and Mortola, 2002, 2003; Mortola, 2004). The
eggs or the hatchlings, either in groups of two (E11 and
E16), or individually (E20 and H1), were placed in a
300 ml plastic container maintained at the desired T by a
water bath. The flow of gases (air or hypoxic mixtures)
through this respirometer was delivered by a pump.
The flow was maintained steady at 50 ml/min (E11) or
100 ml/min (E16, E20 and H1), under the control of a
precision flow meter. At the lower flow, the wash-out
 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106
time constant (time at 63% of complete wash-out) of
the container (with the eggs inside it) was about 2 min
and about 8 min were required for 95% wash-out; at
the higher flow, these times were halved.
Gas mixtures were prepared by blending gases in the
appropriate proportions into gas-impermeable bags,
connected to the inflow port of the respirometer. The
inflowing and outflowing gases were sampled, passed
through a drying column, and monitored by calibrated
O2 (OM-11, Beckman) and CO2 (CD-3A, Applied
Electrochemistry) gas analyzers. The output of these
analyzers was displayed on a computer monitor dur-
ing on-line acquisition. V̇O2 and V̇CO2 were computed
from the flow rate and the inflow–outflow gas con-
centration difference, averaged over the last 5 min of
each condition. The values, calculated at standard tem-
perature, pressure and dry conditions, are presented in
ml/min normalized by the body weight of the embryo
expressed in kg (1 ml O2 STPD = 0.0446 mmol O2 ).
The ratio between V̇CO2 and V̇O2 represents the respi-
ratory exchange ratio, RER.
2.2. Protocols
The main experiment (Table 1) was performed on
a total of 100 embryos (40 E11 and 40 E16, studied
in sets of two and 20 E20, studied individually) and
on 16 hatchlings. Their mean body weights were, re-
spectively, 3.7 g (±0.1), 17.9 (±0.3), 44.1 (±0.8) and
42.2 (±0.9). The experiment consisted in measuring
gaseous metabolism at various T (39, 36, 33 and 30 ◦ C)
during normoxia (21% O2 ) and hypoxia (11% O2 ).
Each animal was studied at only two of the four tem-
peratures, both in normoxia and hypoxia. Although this
Table 1
Experimental protocols
Conditions
1.Hypoxia and response to changes in temperature: various T
(39, 36, 33 and 30 ◦ C), in random order, in 21 and 10% O2
2.Temperature and hypoxic response: various O2 concentrations
(21, 15, 9, 12 and 18%) at T = 33 and 39 ◦ C
3.Temperature and hypoxic hypometabolism: sequence of 21% O2
at 36 ◦ C, 16% O2 at 36 ◦ C and 16% O2 at 39 ◦ C
4.Post-hypoxic recovery: sequence of 21, 16 and 21% O2 , at
constant T = 36 ◦ C
99
protocol doubled the total number of experiments, it
was chosen to limit the possibility of carry-over effects
introduced by multiple exposures. The sequence of the
T exposures and the alternation between normoxia and
hypoxia varied among animals. By the end of the study,
values at each T and oxygenation were the average of
10 sets (of two embryos each) at E11 and E16, of 10
embryos (at E20) and of eight hatchlings (at H1). One
hour was allowed to elapse whenever we changed T and
30 min whenever we changed the oxygenation. Values
were computed during the last 5 min of each condition.
The second protocol was performed on a total of 20
E11 embryos, studied in sets of two. Their mean body
weight was 3.9 g (±0.1). It consisted of exposures to
various levels of oxygenation, between 21 and 9% O2 ,
at both 33 and 39 ◦ C. The order of the O2 exposures
was 21, 15, 9, 12 and 18%, alternating the sequence of
the higher and lower T among embryos. As in protocol
1, in this protocol also, 1 h was given after a T change
and 30 min after a change in oxygenation.
The third protocol was planned after the results of
protocols 1 and 2 became known. It was performed on
20 E11 embryos, studied in sets of two. Their mean
body weight was 4.0 g (±0.1). It consisted of the fol-
lowing sequence: 21% O2 at 36 ◦ C for 1 h, followed by
16% O2 at 36 ◦ C for 30 min and concluded by 16% O2
at 39 ◦ C for 1 h. As in protocol 1, in protocols 2 and
3 also, the reported data refer to the last 5 min of each
condition.
In an additional group of 12 E16 embryos (studied
in sets of two, mean body weight 17.7 g (±0.7)), V̇O2
was monitored continuously for 40 min in air, 40 min in
11% O2 and 40 min in air at T = 36 ◦ C. This was done
to assess to what extent anaerobic sources may have
Age and number of embryos (E) and hatchlings (H)
E11: N = 40, in sets of two
E16: N = 40, in sets of two
E20: N = 20, individually
H1: N = 16, individually
E11: N = 20, in sets of two
E11: N = 20, in sets of two
E16: N = 12, in sets of two
100 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106
compensated the energetic deficit during the period in
hypoxia. In fact, upon return to normoxia, V̇O2 is ex-
pected to exceed the pre-hypoxic level by a magnitude
equivalent to the O2 debt contracted during the hypoxia
(Fahey and Lister, 1989; Frappell et al., 1991).
2.3. Statistical analysis
Data are presented as group means ±1 S.E.M. Q10
values were calculated by linear regression through
the normoxic data points. For the analysis of protocol
1, two-way repeated-measures ANOVA (the O2 level
being the one-factor repetition) was performed, with
post hoc Bonferroni’s limitations for all the possible
comparisons, i.e. the four comparisons between the
two O2 levels within a given T and the 12 comparisons
among the four T within each O2 level. Also the results
of protocol 2 were treated statistically with a two-way
repeated-measures ANOVA, although, in this case, the
protocol permitted to adopt two-factor repetitions (the
O2 level and T), followed by post hoc Bonferroni’s
limitations for all the possible comparisons within each
factor. Results of protocol 3 were analyzed by one-way
repeated-measures ANOVA, with post hoc Bonfer-
roni’s limitations for the two comparisons of interest,
i.e., that between 21% O2 , 36 ◦ C and 16% O2 , 36 ◦ C
and that between 16% O2 , 36 ◦ C and 16% O2 , 39 ◦ C.
Similarly, we compared statistically the data during
hypoxia with those during post-hypoxia recovery in air
by one-way repeated-measures ANOVA. In all cases,
a significant difference was considered at P < 0.05.
3. Results
3.1. Changes in T during normoxia
At days E11 and E16, changes in T were accompa-
nied by corresponding changes in V̇O2 . The relation-
ship between T and V̇O2 was linear for E11 (r2 = 1.0,
Q10 = 2.16) and very close to linear for E16 (r2 = 0.96,
Q10 = 2.13) (Fig. 1, left panels, open symbols). During
the external pipping phase (E20) V̇O2 /kg was less than
at the earlier ages and almost unaffected by changes in
T (Fig. 1, top right panel, open symbols). Finally, in the
hatchlings (H1), a drop in T from 39 to 33 ◦ C caused
a large increase in V̇O2 (thermogenic response), which
eventually decreased with a further drop in T (Fig. 1,
right bottom panel, open symbols).
3.2. Changes in T during hypoxia
In both E11 and E16, hypoxia significantly lowered
V̇O2 at 39, 36 and 33 ◦ C, whereas it had no statistically
significant effects at 30 ◦ C. None of the hypometabolic
values (i.e., at 39, 36 and 33 ◦ C) differed signifi-
cantly from each other (Fig. 1, left panels). Hence, for
T > 33 ◦ C, hypoxic V̇O2 was independent of T.
In the external pippers (E20) hypoxia lowered V̇O2
at all values of T (Fig. 1, right top panel) and, as in
normoxia, V̇O2 was independent of T. After hatching
(Fig. 1, right bottom panel), hypoxic V̇O2 was less than
in normoxia. Nevertheless, hypoxic V̇O2 at 33 ◦ C was
significantly higher than that at 39 ◦ C, indicating that
hypoxia did not abolish the thermogenic response.
3.3. V̇CO2 and RER
The changes in V̇CO2 with T and oxygenation were
qualitatively similar to those described above for V̇O2
(Sections 3.1 and 3.2). In normoxia, the RER (respira-
tory exchange ratio = V̇CO2 /V̇O2 ) was around 0.75–0.8
at all ages and temperatures (Fig. 2, top). In hypoxia,
RER values were slightly higher than in normoxia
(Fig. 2, bottom). Furthermore, in E11 and in E16, the
hypoxic RER values at 36–39 ◦ C were higher than at
30–33 ◦ C, even exceeding unity. This was the case also
for E20 at 39 ◦ C. Differently, in H1 the hypoxic RER
values were close to 0.8 and did not change signifi-
cantly with T.
3.4. E11: various levels of hypoxia at 33 and 39 ◦ C
At 39 ◦ C, exposure to progressively lower O2 con-
centrations decreased the embryo’s V̇O2 , once past the
threshold of 18% (Fig. 3, open symbols).
In normoxia, V̇O2 at 33 ◦ C averaged 75% of the value
at 39 ◦ C. This value changed little and not significantly,
with a decrease in O2 concentration, until [O2 ] was
12% or lower (Fig. 3, filled symbols). Hence, at 33 ◦ C
and for [O2 ] > 12%, V̇O2 was independent of oxygen
availability.
3.5. E11: increase in T during hypoxic
hypometabolism
The previous results in E11 (Sections 3.2 and 3.4)
indicated that [O2 ] = 11% limited V̇O2 at T > 33 ◦ C
 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106 101

Fig. 1. Oxygen consumption (normalized by embryo’s weight, V̇O2 /kg) at different ambient temperatures (T) in chicken embryos of various ages
and in newly hatched chicks, during normoxia (21% O2 , open symbols) and hypoxia (11% O2 , filled symbols). At each T, values are means of
10 sets of two embryos each (days 11 and 16), or 10 embryos (day 20), or eight hatchlings. Bars are 1 S.E.M. Different letters indicate significant
differences in V̇O2 (two-way ANOVA, repeated-measures for O2 level, with post hoc Bonferroni’s limitations, P < 0.05).

(Fig. 1) and that T = 33 ◦ C limited V̇O2 at [O2 ] > 12%
(Fig. 3). Therefore, to test the possibility that the
value of V̇O2 during hypoxic hypometabolism was
not limited by O2 availability, we have chosen a
T > 33 ◦ C (specifically, 36 ◦ C) and [O2 ] > 12% (specif-
ically, 16%). Exposure to 16% O2 at 36 ◦ C signifi-
cantly lowered V̇O2 to 89.6% (±2.2) of the normoxic
value. While the hypoxia was maintained, raising T
to 39 ◦ C significantly increased V̇O2 to 100.1% (±3.5)
(Fig. 4).
rose upon recovery in air. In none of the embryos the
post-hypoxic values exceeded the pre-hypoxic V̇O2 val-
ues.
V̇CO2 in hypoxia dropped, but much less than V̇O2
did; hence, as in the previous experiment (Section
3.3), RER during hypoxia increased, even exceeding
unity (filled circles). Upon return to normoxia, V̇CO2
remained at values significantly lower than the pre-
hypoxic values (open triangles).

3.6. E16: recovery after hypoxic hypometabolism 4. Discussion

The time course of V̇O2 , measured in 5 min bins,
during normoxia (30 min), hypoxia (11% O2 , 40 min)
and return in normoxia (40 min), was studied on E16
embryos at 36 ◦ C (Fig. 5, open circles). After the hy-
poxic drop from about 0.3–0.2 ml/min, V̇O2 promptly
The main results of this study indicated that: (1) the
interaction between hypoxia and T in setting V̇O2 varied
with the embryo’s development and (2) the V̇O2 level
in hypoxia did not result necessarily from a limitation
in O2 supply.
102 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106

Fig. 3. Oxygen consumption (normalized by embryo’s weight,

V̇O2 /kg) in 11-day-old chicken embryos during exposures to various
levels of oxygen concentrations, at ambient temperatures of 33 ◦ C
(filled symbols) or 39 ◦ C (open symbols). Values are means of 10
sets of two embryos each; bars are 1 S.E.M. Different letters indicate
significant differences in V̇O2 (two-way repeated-measures ANOVA,
with post hoc Bonferroni’s limitations, P < 0.05).

Mass-specific metabolic rates are typically low in the

avian embryo and rapidly rise after hatching, similarly
to what observed in the mammalian fetus at birth (Rahn,
1982). The higher post-hatching values have been at-

Fig. 2. Respiratory exchange ratio (ratio between carbon dioxide

production and oxygen consumption) of chicken embryos at days
11, 16 and 20 (E11, E16, E20) and 1-day-old hatchlings (H1) at
various ambient temperatures (T) during normoxia (top panel) and
hypoxia (bottom panel). At each T, values are means of 10 sets of
two embryos each (days 11 and 16), or 10 embryos (day 20), or
eight hatchlings. Bars are 1 S.E.M. In general, hypoxic values were
higher than the normoxic ones. Also, in E11, E16 and E20, but not
in H1, RER values during hypoxia at 36–39 ◦ C were higher than at
30–33 ◦ C (two-way repeated-measures ANOVA for O2 level, with
post hoc Bonferroni’s limitations, P < 0.05).

4.1. T and normoxic V̇O2

The progression from a Q10 of about 2.1 (in E11 and

E16), to the maintenance of V̇O2 (E20) and a clear ther-
mogenic effort during cooling (H1) reflects the embry-
onic development of endothermy, previously studied
by several investigators (Tazawa et al., 1988; Whittow
and Tazawa, 1991; Nichelmann and Tzschentke, 2003).
At E16, the lack of a statistical difference in normoxic
V̇O2 between 33 and 36 ◦ C is a sign that thermal con-
trol is already beginning at this early embryonic age.
Fig. 4. Oxygen consumption in 11-day-old chicken embryos during
normoxia (21% O2 ) at 36 ◦ C, hypoxia (16% O2 ) at 36 ◦ C and hypoxia
(16% O2 ) at 39 ◦ C, tested in this order. At left, values are expressed
normalized by embryo’s weight (ml O2 × kg−1 × min−1 ), at right,
in percent of the value measured in normoxia at 36 ◦ C. Values are
means of 10 sets of two embryos each; bars are 1 S.E.M. Arrows
indicate the experimental sequence. a, b significant difference from
the previous conditions (one-way repeated-measures ANOVA, with
post hoc Bonferroni’s limitations, P < 0.05).
 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106 103

Fig. 5. Oxygen consumption (V̇O2 , open circles) and carbon dioxide production (V̇CO2 , open triangles) in 16-day-old chicken embryos at 36 ◦ C,
during normoxia (21% O2 ), hypoxia (11% O2 ) and recovery in normoxia. The corresponding respiratory exchange ratios (RER = V̇CO2 /V̇O2 ,
Y-axis at right) are indicated by the filled symbols. Values are means of seven sets of two embryos each; bars are 1 S.E.M. Data are averages of
5 min bins. The first values in hypoxia and after return to normoxia were collected at 7 min, to allow for complete chamber wash-out (see Section
2). Statistical analysis was performed on the last 10 min of each condition. For each parameter, different letters indicate significant differences
(one-way repeated-measures ANOVA, with post hoc Bonferroni’s limitations, P < 0.05).

tributed to the removal of the O2 limitation imposed
by the egg-shell (Tazawa et al., 1988). In this experi-
ment, however, we observed a marked increase in V̇O2
between E20 and H1. It is difficult to explain such an in-
crease as a removal of O2 -diffusion limitation, because
the E20 embryos were in the external pipping phase,
a period with well-established pulmonary ventilation
(Menna and Mortola, 2002). Hence, the mechanisms
responsible for the rapid improvement in heat produc-
tion with hatching remain unclear.
4.2. Effects of hypoxia on gaseous metabolism
The hypoxic depression of V̇O2 is a phenomenon
widespread in all classes of animals (Boutilier, 2001;
McMahon, 2001; Hicks and Wang, 2004; Jackson,
2004). In homeotherms, hypoxia blunts predominantly
temperature-control (Mortola and Gautier, 1995). The
current results in H1 and E20 closely resemble those
observed in neonatal mammals, in which the magni-
tude of the hypoxic inhibition on V̇O2 depends on the
degree of the normoxic thermogenesis (Mortola, 2001).
The effects of hypoxia on gaseous V̇CO2 are close to,
but not necessarily the same as, those on V̇O2 for sev-
eral reasons. In H1, the slight increase in RER (from
about 0.75 to about 0.8, Fig. 2) probably reflects the
higher use of carbohydrates as metabolic substrate and
the hypoxic hyperventilation, similar to what occurs in
neonatal mammals during hypoxia (Mortola and Dotta,
1992). In eggs, ambient hypoxia lowers the partial pres-
sure gradient for O2 and the difference with CO2 is en-
hanced at high T, when gas diffusion increases. This
explains why, during hypoxia at high temperatures (36
and 39 ◦ C), the RER increased in E11 and E16 em-
bryos. Upon return to normoxia there was a tendency
to reverse the process (Fig. 5), to replenish the egg
CO2 stores. At E20, a stage when gas exchange oc-
curs through both pulmonary convection and chorio-
allantoic diffusion (Menna and Mortola, 2002), RER
increased significantly only at the highest T.
In neonatal mammals, during moderate levels of
hypoxia, the accumulation of lactic acid is small or
absent, with minimal indication of an O2 debt (Mortola
and Gautier, 1995). Also in the embryos that we tested
(E16), upon return to normoxia, there was no evidence
of a payment back of the O2 debt, indicating that
the drop in V̇O2 during hypoxia reflected the drop
in metabolic rate. This agrees with the view that
anaerobic pathways play a minor role in the energetics
of the hypoxic embryos (Bjønnes et al., 1987). It is
104 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106
difficult to say what are the energy-requiring functions
curtailed by hypoxia. In neonatal mammals, acute hy-
poxia mainly depresses thermoregulation (Mortola and
Gautier, 1995) and this could be the primary mech-
anism also during the last phases of the chicken
embryonic development. However, at day 11, when
thermogenesis is absent, hypometabolism may re-
sult primarily from inhibitions of cell excitability
(Hochachka, 1986). Additional O2 saving could be
achieved by decreasing cell repair and tissue growth,
with the result of a blunted embryonic growth (Stock
and Metcalfe, 1987; Xu and Mortola, 1989).
4.3. Hypoxic hypometabolism in the chicken
embryo: O2 conformism or regulation?
Some experiments in newborn and adult mammals
have considered the possibility that the hypometabolic
response to hypoxia represented a controlled down-
regulation of O2 usage, rather than a response forced
by the limitation of O2 supply (Saiki and Mortola,
1997; Rohlicek et al., 1998). It was found that,
upon pharmacological or physiological stimulation,
hypoxic V̇O2 increased, indicating that the hypoxic
level of V̇O2 was not limited by the availability of O2 .
Similarly, in our hatchlings, hypoxic V̇O2 at 36 ◦ C
significantly exceeded the value at 39 ◦ C. Therefore,
the hypometabolism at 39 ◦ C could not be interpreted
as a limitation in O2 availability; rather, it suggested
a phenomenon of regulated O2 -conformism. Also
the responses of hypoxic ectothermic vertebrates and
invertebrates to an increase in T or to pharmacological
stimulation agree with a regulated control of hypoxic
hypometabolism (Paul et al., 1997; Hicks and Wang,
1999; Wiggins and Frappell, 2000; Hicks and
McMahon, 2002; Alexander and McMahon, 2004). It
is also known that there is no correlation between the
intracellular O2 and tissue V̇O2 , unless the intracellular
O2 pressure becomes extremely small (e.g., <3 mmHg,
Marcinek et al., 2003). Hence, there is ample evidence
that the occurrence of hypoxic hypometabolism is not
necessarily an indication of O2 -limitation, but can be a
form of regulated conformism. However, whether this
could be the case also during embryonic development
had never been addressed experimentally in any class
of animals.
At E11, once the low T was limiting V̇O2 , changes
in O2 were inconsequential; similarly, once V̇O2 was
O2 -limited, changes in T had no further effects on V̇O2 .
Hence, at a first look, the responses of the E11 embryos
seemed to comply fully with a pattern of strict T- and
O2 -conformism. On the other hand, a closer inspec-
tion suggested that this was not an accurate interpreta-
tion of the data. In fact, at the lowest [O2 ], the 33 ◦ C
[O2 ]–V̇O2 relationship (Fig. 3, filled symbols) crossed
over the 39 ◦ C curve. This was due to the higher O2
solubility at the lower T. Hence, had we plotted V̇O2
as function of [O2 ] in the embryo’s body fluids, rather
than as function of [O2 ] delivered to the egg, the 33 ◦ C
curve would have been shifted to the right, in com-
parison to that shown in Fig. 3. The magnitude of the
shift should correspond approximately to the ratio of
the O2 solubilities at the two temperatures, or about
11%. The effect of this correction is that, at the same
[O2 ] of 12%, the data points of the two curves would
present a difference larger than currently apparent from
the figure and the mere presence of such a difference
is not compatible with strict O2 -conformism.
It was because of this reasoning that we planned the
experiments of protocol 3 (Fig. 4), in which the values
of hypoxia and T were chosen ad hoc for best testing
the possibility of regulated conformism. The low V̇O2
during 16% O2 at 36 ◦ C was significantly increased
by raising the T to 39 ◦ C (Fig. 4) and the magnitude
of the increase is underestimated because the lower
O2 solubility at the higher T was not taken into ac-
count. Hence, the hypoxic hypometabolism observed
at 36 ◦ C cannot be attributed to O2 limitation. In fact,
had this been the case, it would not have been pos-
sible to raise V̇O2 by increasing T, nor by any other
intervention. In conclusion, like newborn and adult an-
imals, also chicken embryos respond to hypoxia with
regulated O2 -conformism, provided that neither T nor
oxygenation are too low.
In mammals, the mechanisms behind the controlled
drop in V̇O2 during hypoxia are not understood, al-
though it is likely that they are coordinated at the hy-
pothalamic level (Mortola and Gautier, 1995). In the
young embryo, it is possible that cardiovascular adjust-
ments to T and oxygenation contribute to the regulated
conformism. For example, the rise in T during hypoxia
may increase heart rate (Khandoker et al., 2004) and
with it the perfusion of the chorio-allantoic membrane
(Ar et al., 1991), or change the regional vascular resis-
tance, modifying the effects of hypoxia on the embryo’s
distribution of blood flow (Crossley et al., 2003, Paul
 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106
et al., 1997). If, during hypoxia, the T-dependent re-
distribution of blood flow improved the O2 delivery to
organs with higher V̇O2 , this would result in an increase
of the embryo’s V̇O2 .
5. Conclusions
Of all the substrates required for the embryo’s de-
velopment, heat and oxygen are the only two not stored
within the egg. Both are crucial in setting the embryo’s
metabolic rate and their relative importance changes
with development. During the ectothermic phase, hy-
poxia lowers V̇O2 at high T, because the low T curtails
the embryo’s V̇O2 to the point that a decrease in oxy-
genation has no further effects. During the endothermic
phase of late development, hypoxia blunts the embryo’s
thermogeic effort; therefore, the hypoxic effects on V̇O2
are more apparent at low, rather than at high, T. In the
ectothermic embryo, both low T and low O2 force V̇O2
to drop in what seems strict T- and O2 -conformism.
However, modest hypoxia at a close-to-normal T causes
a hypometabolic response that presents the character-
istics of regulated O2 -conformism.
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