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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


factor (or Q10 ), as expected for ectothermic animals.
In the chicken embryo, during a large portion of Differently, in the last phases of incubation, when con-
development, oxygen consumption (V̇O2 ) varies with fronted by a drop in T, the embryo attempts to increase
V̇O2 as a means of controlling body temperature, in
∗ Corresponding author. Tel.: +1 514 398 4335; what represents the early manifestation of endothermy.
fax: +1 514 398 7452. Hence, the embryo gradually shifts from the poikilo-
E-mail address: jacopo.mortola@mcgill.ca (J.P. Mortola). 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 The primary purpose of this study was to examine
be fully manifest post-natally (Whittow and Tazawa, the effect of hypoxia on the V̇O2 response to changes in
1991; Nichelmann and Tzschentke, 2003). T in the chicken embryo at various stages of its devel-
As for T, changes in O2 availability also cause cor- opment. Further, the possibility that the hypometabolic
responding changes in the embryo’s V̇O2 (Bartels and response to hypoxia is a regulated, rather than passive
Baumann, 1972; Stock et al., 1985; Bjønnes et al., (i.e., O2 supply-limited), response has been addressed
1987; Ar et al., 1991; Tazawa et al., 1992). The in- by examining the effect of changes in T on V̇O2 at var-
teraction between T and oxygenation in setting the ious levels of hypoxia.
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 2. Methods
ectothermic or the endothermic pattern is prevailing. At
low T, during the ectothermic phase of the embryonic Experiments were conducted on freshly laid fertil-
development, because V̇O2 is small, changes in oxy- ized eggs of White Leghorn chickens, obtained from
genation should have less impact on V̇O2 than at high T. a local supplier. On day 0, the eggs weighed between
Conversely, during the late phases of development, the 60 and 65 g. They were placed in a still air incubator
embryo is likely to be more O2 dependent at lower, than (Hova-bator Model, 1602), at 38 ◦ C and 60% relative
at higher T, because the low T promotes a thermogenic humidity, with automatic rotation of the eggs four times
effort with the rise in V̇O2 . Therefore, one may hypothe- per day. Measurements were performed on embryos at
size that, in the growing chicken embryo, hypoxia may days 11, 16, 20 (during the star fracture of the exter-
pose a limit to V̇O2 at high T during the earlier phases nal pipping phase), and on post-natal day 1 (within 24 h
and at low T during the late phases of development. from hatching). These groups will be referred to as E11,
The hypometabolic response during hypoxia could E16, E20 and H1, respectively.
result from the scarce O2 supply, like it would hap- The experiments consisted of measuring metabolic
pen in any reaction with substrate limitation. Hence, rate by indirect calorimetry (oxygen consumption, V̇O2
the V̇O2 level attained during the hypoxia-induced hy- and carbon dioxide production, V̇CO2 ) at several tem-
pometabolism would be the maximal possible for that peratures and levels of oxygenation, in various combi-
degree of oxygenation. In such a situation, a drop in T nations. After the measurements, the eggs were opened,
(during the endothermic phase of development) or an the embryos killed by cold (E11), exposure to CO2
increase in T (during the ectothermic phase) would not (E16, E20) or with an overdose of barbiturate anaesthe-
be able to raise hypoxic V̇O2 , since hypoxic V̇O2 is O2 - sia (H1), examined to exclude obvious gross anatomi-
limited. Conversely, the hypometabolic response could cal abnormalities and weighed with a digital scale ac-
reflect a regulated phenomenon, triggered by hypoxia curate to 10−3 g.
but not necessarily forced upon by the O2 limitation. If
this was the case, the level of hypometabolism would 2.1. Oxygen consumption
not be necessarily the maximal attainable and for a con-
stant level of hypoxia, the V̇O2 level could still change Measurements of oxygen consumption (V̇O2 ) were
with changes in T. A regulated hypometabolic response obtained by the open-flow method (Frappell et al.,
to hypoxia, or regulated O2 -conformism, seems to be 1992), with a setup adapted to the chicken embryo
a wide-spread phenomenon observed in invertebrates (Menna and Mortola, 2002, 2003; Mortola, 2004). The
(Paul et al., 1997; Wiggins and Frappell, 2000; Hicks eggs or the hatchlings, either in groups of two (E11 and
and McMahon, 2002; Alexander and McMahon, 2004), E16), or individually (E20 and H1), were placed in a
in reptiles (Hicks and Wang, 1999) and in adult and 300 ml plastic container maintained at the desired T by a
neonatal mammals (Saiki and Mortola, 1997; Rohlicek water bath. The flow of gases (air or hypoxic mixtures)
et al., 1998). During the embryonic period, the possi- through this respirometer was delivered by a pump.
bility of hypoxic hypometabolism being a regulated The flow was maintained steady at 50 ml/min (E11) or
phenomenon, rather than O2 limitation, has never been 100 ml/min (E16, E20 and H1), under the control of a
experimentally considered in any class of animals. precision flow meter. At the lower flow, the wash-out
J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106 99

time constant (time at 63% of complete wash-out) of protocol doubled the total number of experiments, it
the container (with the eggs inside it) was about 2 min was chosen to limit the possibility of carry-over effects
and about 8 min were required for 95% wash-out; at introduced by multiple exposures. The sequence of the
the higher flow, these times were halved. T exposures and the alternation between normoxia and
Gas mixtures were prepared by blending gases in the hypoxia varied among animals. By the end of the study,
appropriate proportions into gas-impermeable bags, values at each T and oxygenation were the average of
connected to the inflow port of the respirometer. The 10 sets (of two embryos each) at E11 and E16, of 10
inflowing and outflowing gases were sampled, passed embryos (at E20) and of eight hatchlings (at H1). One
through a drying column, and monitored by calibrated hour was allowed to elapse whenever we changed T and
O2 (OM-11, Beckman) and CO2 (CD-3A, Applied 30 min whenever we changed the oxygenation. Values
Electrochemistry) gas analyzers. The output of these were computed during the last 5 min of each condition.
analyzers was displayed on a computer monitor dur- The second protocol was performed on a total of 20
ing on-line acquisition. V̇O2 and V̇CO2 were computed E11 embryos, studied in sets of two. Their mean body
from the flow rate and the inflow–outflow gas con- weight was 3.9 g (±0.1). It consisted of exposures to
centration difference, averaged over the last 5 min of various levels of oxygenation, between 21 and 9% O2 ,
each condition. The values, calculated at standard tem- at both 33 and 39 ◦ C. The order of the O2 exposures
perature, pressure and dry conditions, are presented in was 21, 15, 9, 12 and 18%, alternating the sequence of
ml/min normalized by the body weight of the embryo the higher and lower T among embryos. As in protocol
expressed in kg (1 ml O2 STPD = 0.0446 mmol O2 ). 1, in this protocol also, 1 h was given after a T change
The ratio between V̇CO2 and V̇O2 represents the respi- and 30 min after a change in oxygenation.
ratory exchange ratio, RER. The third protocol was planned after the results of
protocols 1 and 2 became known. It was performed on
2.2. Protocols 20 E11 embryos, studied in sets of two. Their mean
body weight was 4.0 g (±0.1). It consisted of the fol-
The main experiment (Table 1) was performed on lowing sequence: 21% O2 at 36 ◦ C for 1 h, followed by
a total of 100 embryos (40 E11 and 40 E16, studied 16% O2 at 36 ◦ C for 30 min and concluded by 16% O2
in sets of two and 20 E20, studied individually) and at 39 ◦ C for 1 h. As in protocol 1, in protocols 2 and
on 16 hatchlings. Their mean body weights were, re- 3 also, the reported data refer to the last 5 min of each
spectively, 3.7 g (±0.1), 17.9 (±0.3), 44.1 (±0.8) and condition.
42.2 (±0.9). The experiment consisted in measuring In an additional group of 12 E16 embryos (studied
gaseous metabolism at various T (39, 36, 33 and 30 ◦ C) in sets of two, mean body weight 17.7 g (±0.7)), V̇O2
during normoxia (21% O2 ) and hypoxia (11% O2 ). was monitored continuously for 40 min in air, 40 min in
Each animal was studied at only two of the four tem- 11% O2 and 40 min in air at T = 36 ◦ C. This was done
peratures, both in normoxia and hypoxia. Although this to assess to what extent anaerobic sources may have

Table 1
Experimental protocols
Conditions Age and number of embryos (E) and hatchlings (H)
1.Hypoxia and response to changes in temperature: various T E11: N = 40, in sets of two
(39, 36, 33 and 30 ◦ C), in random order, in 21 and 10% O2
E16: N = 40, in sets of two
E20: N = 20, individually
H1: N = 16, individually
2.Temperature and hypoxic response: various O2 concentrations E11: N = 20, in sets of two
(21, 15, 9, 12 and 18%) at T = 33 and 39 ◦ C
3.Temperature and hypoxic hypometabolism: sequence of 21% O2 E11: N = 20, in sets of two
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 E16: N = 12, in sets of two
constant T = 36 ◦ C
100 J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106

compensated the energetic deficit during the period in 3.2. Changes in T during hypoxia
hypoxia. In fact, upon return to normoxia, V̇O2 is ex-
pected to exceed the pre-hypoxic level by a magnitude In both E11 and E16, hypoxia significantly lowered
equivalent to the O2 debt contracted during the hypoxia V̇O2 at 39, 36 and 33 ◦ C, whereas it had no statistically
(Fahey and Lister, 1989; Frappell et al., 1991). significant effects at 30 ◦ C. None of the hypometabolic
values (i.e., at 39, 36 and 33 ◦ C) differed signifi-
2.3. Statistical analysis cantly from each other (Fig. 1, left panels). Hence, for
T > 33 ◦ C, hypoxic V̇O2 was independent of T.
Data are presented as group means ±1 S.E.M. Q10 In the external pippers (E20) hypoxia lowered V̇O2
values were calculated by linear regression through at all values of T (Fig. 1, right top panel) and, as in
the normoxic data points. For the analysis of protocol normoxia, V̇O2 was independent of T. After hatching
1, two-way repeated-measures ANOVA (the O2 level (Fig. 1, right bottom panel), hypoxic V̇O2 was less than
being the one-factor repetition) was performed, with in normoxia. Nevertheless, hypoxic V̇O2 at 33 ◦ C was
post hoc Bonferroni’s limitations for all the possible significantly higher than that at 39 ◦ C, indicating that
comparisons, i.e. the four comparisons between the hypoxia did not abolish the thermogenic response.
two O2 levels within a given T and the 12 comparisons
among the four T within each O2 level. Also the results 3.3. V̇CO2 and RER
of protocol 2 were treated statistically with a two-way
repeated-measures ANOVA, although, in this case, the The changes in V̇CO2 with T and oxygenation were
protocol permitted to adopt two-factor repetitions (the qualitatively similar to those described above for V̇O2
O2 level and T), followed by post hoc Bonferroni’s (Sections 3.1 and 3.2). In normoxia, the RER (respira-
limitations for all the possible comparisons within each tory exchange ratio = V̇CO2 /V̇O2 ) was around 0.75–0.8
factor. Results of protocol 3 were analyzed by one-way at all ages and temperatures (Fig. 2, top). In hypoxia,
repeated-measures ANOVA, with post hoc Bonfer- RER values were slightly higher than in normoxia
roni’s limitations for the two comparisons of interest, (Fig. 2, bottom). Furthermore, in E11 and in E16, the
i.e., that between 21% O2 , 36 ◦ C and 16% O2 , 36 ◦ C hypoxic RER values at 36–39 ◦ C were higher than at
and that between 16% O2 , 36 ◦ C and 16% O2 , 39 ◦ C. 30–33 ◦ C, even exceeding unity. This was the case also
Similarly, we compared statistically the data during for E20 at 39 ◦ C. Differently, in H1 the hypoxic RER
hypoxia with those during post-hypoxia recovery in air values were close to 0.8 and did not change signifi-
by one-way repeated-measures ANOVA. In all cases, cantly with T.
a significant difference was considered at P < 0.05.
3.4. E11: various levels of hypoxia at 33 and 39 ◦ C
3. Results
At 39 ◦ C, exposure to progressively lower O2 con-
3.1. Changes in T during normoxia centrations decreased the embryo’s V̇O2 , once past the
threshold of 18% (Fig. 3, open symbols).
At days E11 and E16, changes in T were accompa- In normoxia, V̇O2 at 33 ◦ C averaged 75% of the value
nied by corresponding changes in V̇O2 . The relation- at 39 ◦ C. This value changed little and not significantly,
ship between T and V̇O2 was linear for E11 (r2 = 1.0, with a decrease in O2 concentration, until [O2 ] was
Q10 = 2.16) and very close to linear for E16 (r2 = 0.96, 12% or lower (Fig. 3, filled symbols). Hence, at 33 ◦ C
Q10 = 2.13) (Fig. 1, left panels, open symbols). During and for [O2 ] > 12%, V̇O2 was independent of oxygen
the external pipping phase (E20) V̇O2 /kg was less than availability.
at the earlier ages and almost unaffected by changes in
T (Fig. 1, top right panel, open symbols). Finally, in the 3.5. E11: increase in T during hypoxic
hatchlings (H1), a drop in T from 39 to 33 ◦ C caused hypometabolism
a large increase in V̇O2 (thermogenic response), which
eventually decreased with a further drop in T (Fig. 1, The previous results in E11 (Sections 3.2 and 3.4)
right bottom panel, open symbols). 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% rose upon recovery in air. In none of the embryos the
(Fig. 3). Therefore, to test the possibility that the post-hypoxic values exceeded the pre-hypoxic V̇O2 val-
value of V̇O2 during hypoxic hypometabolism was ues.
not limited by O2 availability, we have chosen a V̇CO2 in hypoxia dropped, but much less than V̇O2
T > 33 ◦ C (specifically, 36 ◦ C) and [O2 ] > 12% (specif- did; hence, as in the previous experiment (Section
ically, 16%). Exposure to 16% O2 at 36 ◦ C signifi- 3.3), RER during hypoxia increased, even exceeding
cantly lowered V̇O2 to 89.6% (±2.2) of the normoxic unity (filled circles). Upon return to normoxia, V̇CO2
value. While the hypoxia was maintained, raising T remained at values significantly lower than the pre-
to 39 ◦ C significantly increased V̇O2 to 100.1% (±3.5) hypoxic values (open triangles).
(Fig. 4).

3.6. E16: recovery after hypoxic hypometabolism 4. Discussion

The time course of V̇O2 , measured in 5 min bins, The main results of this study indicated that: (1) the
during normoxia (30 min), hypoxia (11% O2 , 40 min) interaction between hypoxia and T in setting V̇O2 varied
and return in normoxia (40 min), was studied on E16 with the embryo’s development and (2) the V̇O2 level
embryos at 36 ◦ C (Fig. 5, open circles). After the hy- in hypoxia did not result necessarily from a limitation
poxic drop from about 0.3–0.2 ml/min, V̇O2 promptly 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- Fig. 4. Oxygen consumption in 11-day-old chicken embryos during
mogenic effort during cooling (H1) reflects the embry- normoxia (21% O2 ) at 36 ◦ C, hypoxia (16% O2 ) at 36 ◦ C and hypoxia
onic development of endothermy, previously studied (16% O2 ) at 39 ◦ C, tested in this order. At left, values are expressed
by several investigators (Tazawa et al., 1988; Whittow 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
and Tazawa, 1991; Nichelmann and Tzschentke, 2003).
means of 10 sets of two embryos each; bars are 1 S.E.M. Arrows
At E16, the lack of a statistical difference in normoxic indicate the experimental sequence. a, b significant difference from
V̇O2 between 33 and 36 ◦ C is a sign that thermal con- the previous conditions (one-way repeated-measures ANOVA, with
trol is already beginning at this early embryonic age. 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 about 0.75 to about 0.8, Fig. 2) probably reflects the
by the egg-shell (Tazawa et al., 1988). In this experi- higher use of carbohydrates as metabolic substrate and
ment, however, we observed a marked increase in V̇O2 the hypoxic hyperventilation, similar to what occurs in
between E20 and H1. It is difficult to explain such an in- neonatal mammals during hypoxia (Mortola and Dotta,
crease as a removal of O2 -diffusion limitation, because 1992). In eggs, ambient hypoxia lowers the partial pres-
the E20 embryos were in the external pipping phase, sure gradient for O2 and the difference with CO2 is en-
a period with well-established pulmonary ventilation hanced at high T, when gas diffusion increases. This
(Menna and Mortola, 2002). Hence, the mechanisms explains why, during hypoxia at high temperatures (36
responsible for the rapid improvement in heat produc- and 39 ◦ C), the RER increased in E11 and E16 em-
tion with hatching remain unclear. bryos. Upon return to normoxia there was a tendency
to reverse the process (Fig. 5), to replenish the egg
4.2. Effects of hypoxia on gaseous metabolism CO2 stores. At E20, a stage when gas exchange oc-
curs through both pulmonary convection and chorio-
The hypoxic depression of V̇O2 is a phenomenon allantoic diffusion (Menna and Mortola, 2002), RER
widespread in all classes of animals (Boutilier, 2001; increased significantly only at the highest T.
McMahon, 2001; Hicks and Wang, 2004; Jackson, In neonatal mammals, during moderate levels of
2004). In homeotherms, hypoxia blunts predominantly hypoxia, the accumulation of lactic acid is small or
temperature-control (Mortola and Gautier, 1995). The absent, with minimal indication of an O2 debt (Mortola
current results in H1 and E20 closely resemble those and Gautier, 1995). Also in the embryos that we tested
observed in neonatal mammals, in which the magni- (E16), upon return to normoxia, there was no evidence
tude of the hypoxic inhibition on V̇O2 depends on the of a payment back of the O2 debt, indicating that
degree of the normoxic thermogenesis (Mortola, 2001). the drop in V̇O2 during hypoxia reflected the drop
The effects of hypoxia on gaseous V̇CO2 are close to, in metabolic rate. This agrees with the view that
but not necessarily the same as, those on V̇O2 for sev- anaerobic pathways play a minor role in the energetics
eral reasons. In H1, the slight increase in RER (from 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 O2 -limited, changes in T had no further effects on V̇O2 .
curtailed by hypoxia. In neonatal mammals, acute hy- Hence, at a first look, the responses of the E11 embryos
poxia mainly depresses thermoregulation (Mortola and seemed to comply fully with a pattern of strict T- and
Gautier, 1995) and this could be the primary mech- O2 -conformism. On the other hand, a closer inspec-
anism also during the last phases of the chicken tion suggested that this was not an accurate interpreta-
embryonic development. However, at day 11, when tion of the data. In fact, at the lowest [O2 ], the 33 ◦ C
thermogenesis is absent, hypometabolism may re- [O2 ]–V̇O2 relationship (Fig. 3, filled symbols) crossed
sult primarily from inhibitions of cell excitability over the 39 ◦ C curve. This was due to the higher O2
(Hochachka, 1986). Additional O2 saving could be solubility at the lower T. Hence, had we plotted V̇O2
achieved by decreasing cell repair and tissue growth, as function of [O2 ] in the embryo’s body fluids, rather
with the result of a blunted embryonic growth (Stock than as function of [O2 ] delivered to the egg, the 33 ◦ C
and Metcalfe, 1987; Xu and Mortola, 1989). curve would have been shifted to the right, in com-
parison to that shown in Fig. 3. The magnitude of the
4.3. Hypoxic hypometabolism in the chicken shift should correspond approximately to the ratio of
embryo: O2 conformism or regulation? the O2 solubilities at the two temperatures, or about
11%. The effect of this correction is that, at the same
Some experiments in newborn and adult mammals [O2 ] of 12%, the data points of the two curves would
have considered the possibility that the hypometabolic present a difference larger than currently apparent from
response to hypoxia represented a controlled down- the figure and the mere presence of such a difference
regulation of O2 usage, rather than a response forced is not compatible with strict O2 -conformism.
by the limitation of O2 supply (Saiki and Mortola, It was because of this reasoning that we planned the
1997; Rohlicek et al., 1998). It was found that, experiments of protocol 3 (Fig. 4), in which the values
upon pharmacological or physiological stimulation, of hypoxia and T were chosen ad hoc for best testing
hypoxic V̇O2 increased, indicating that the hypoxic the possibility of regulated conformism. The low V̇O2
level of V̇O2 was not limited by the availability of O2 . during 16% O2 at 36 ◦ C was significantly increased
Similarly, in our hatchlings, hypoxic V̇O2 at 36 ◦ C by raising the T to 39 ◦ C (Fig. 4) and the magnitude
significantly exceeded the value at 39 ◦ C. Therefore, of the increase is underestimated because the lower
the hypometabolism at 39 ◦ C could not be interpreted O2 solubility at the higher T was not taken into ac-
as a limitation in O2 availability; rather, it suggested count. Hence, the hypoxic hypometabolism observed
a phenomenon of regulated O2 -conformism. Also at 36 ◦ C cannot be attributed to O2 limitation. In fact,
the responses of hypoxic ectothermic vertebrates and had this been the case, it would not have been pos-
invertebrates to an increase in T or to pharmacological sible to raise V̇O2 by increasing T, nor by any other
stimulation agree with a regulated control of hypoxic intervention. In conclusion, like newborn and adult an-
hypometabolism (Paul et al., 1997; Hicks and Wang, imals, also chicken embryos respond to hypoxia with
1999; Wiggins and Frappell, 2000; Hicks and regulated O2 -conformism, provided that neither T nor
McMahon, 2002; Alexander and McMahon, 2004). It oxygenation are too low.
is also known that there is no correlation between the In mammals, the mechanisms behind the controlled
intracellular O2 and tissue V̇O2 , unless the intracellular drop in V̇O2 during hypoxia are not understood, al-
O2 pressure becomes extremely small (e.g., <3 mmHg, though it is likely that they are coordinated at the hy-
Marcinek et al., 2003). Hence, there is ample evidence pothalamic level (Mortola and Gautier, 1995). In the
that the occurrence of hypoxic hypometabolism is not young embryo, it is possible that cardiovascular adjust-
necessarily an indication of O2 -limitation, but can be a ments to T and oxygenation contribute to the regulated
form of regulated conformism. However, whether this conformism. For example, the rise in T during hypoxia
could be the case also during embryonic development may increase heart rate (Khandoker et al., 2004) and
had never been addressed experimentally in any class with it the perfusion of the chorio-allantoic membrane
of animals. (Ar et al., 1991), or change the regional vascular resis-
At E11, once the low T was limiting V̇O2 , changes tance, modifying the effects of hypoxia on the embryo’s
in O2 were inconsequential; similarly, once V̇O2 was distribution of blood flow (Crossley et al., 2003, Paul
J.P. Mortola, K. Labbè / Respiratory Physiology & Neurobiology 146 (2005) 97–106 105

et al., 1997). If, during hypoxia, the T-dependent re- Frappell, P.B., Lanthier, C., Baudinette, R.V., Mortola, J.P., 1992.
distribution of blood flow improved the O2 delivery to Metabolism and ventilation in acute hypoxia: a comparative
organs with higher V̇O2 , this would result in an increase analysis in small mammalian species. Am. J. Physiol. 262,
R1040–R1046.
of the embryo’s V̇O2 . Hicks, D.W., McMahon, R.F., 2002. Respiratory responses to tem-
perature and hypoxia in the nonindigenous Brown Mussel, Perna
perna (Bivalvia, Mytilidae), from the Gulf of Mexico. J. Exp.
Mar. Biol. Ecol. 277, 61–78.
5. Conclusions Hicks, J.W., Wang, T., 1999. Hypoxic hypometabolism in the anes-
thetized turtle, Trachemys scripta. Am. J. Physiol. 277, R18–
Of all the substrates required for the embryo’s de- R23.
velopment, heat and oxygen are the only two not stored Hicks, J.W., Wang, T., 2004. The hypoxic metabolic response in
reptiles: behavioural and physiological mechanisms that re-
within the egg. Both are crucial in setting the embryo’s duce aerobic demands. Respir. Physiol. Neurobiol. 141, 261–
metabolic rate and their relative importance changes 271.
with development. During the ectothermic phase, hy- Hochachka, P.W., 1986. Defense strategies against hypoxia and hy-
poxia lowers V̇O2 at high T, because the low T curtails pothermia. Science 231, 234–241.
the embryo’s V̇O2 to the point that a decrease in oxy- Jackson, D.C., 2004. Hypoxic hypometabolism and acid–base bal-
ance. Respir. Physiol. Neurobiol. 141, 273–283.
genation has no further effects. During the endothermic Khandoker, A.H., Fukazawa, K., Dzialowski, E.M., Burggren, W.W.,
phase of late development, hypoxia blunts the embryo’s Tazawa, H., 2004. Maturation of the homeothermic response of
thermogeic effort; therefore, the hypoxic effects on V̇O2 heart rate to altered ambient temperature in developing chick
are more apparent at low, rather than at high, T. In the hatchlings (Gallus gallus domesticus). Am. J. Physiol. 286,
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