The Journal of Experimental Biology 207, 1543-1552 1543

Published by The Company of Biologists 2004

doi:10.1242/jeb.00909

Acclimation to hypothermic incubation in developing chicken embryos

(Gallus domesticus)
I. Developmental effects and chronic and acute metabolic adjustments
Juli L. Black and Warren W. Burggren*
Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA
*Author for correspondence (e-mail: burggren@unt.edu)

Accepted 22 January 2004

Summary
Chronic exposure to a low incubation temperature populations. Late stage (HH43–44) embryos incubated
clearly slows the development of poikilothemic chicken at 38°C could maintain VO∑ (approximately
embryos (or any other poikilotherms), but little is 27–33·µl·g–1·min–1) during an acute drop in Ta to
known about the more subtle developmental effects approximately 30°C. However, at the same stage 35°C
of temperature, especially on physiological regulatory embryos acutely measured at 38°C were unable to
systems. Consequently, two populations of chicken similarly maintain their VO∑, which fell as soon as Ta
embryos were incubated at 38°C and 35°C. When reached 36°C. Thus, while hypothermic incubation does
compared at the same development stage, incubation not affect gross development (other than would be
temperature had no significant impact on embryonic predicted from a simple effect of Q10), there is a significant
survival or growth. Moreover, the relative timing of major delay in the relative timing of the onset of
developmental landmarks (e.g. internal pipping), thermoregulatory ability induced by hypothermic
expressed as a percentage of development, was unaffected incubation.
by temperature. The ability to maintain the rate of oxygen
consumption (VO∑) during an acute drop in ambient
temperature (Ta) improved from Hamburger–Hamilton Key words: chicken embryo, Gallus domesticus, thermoregulation,
(HH) stages 39–40 to 43–44 in the 38°C but not the 35°C hypothermia, incubation, development, heterokairy.

Introduction
The developing chicken embryo is a poikilotherm, and embryo as a unit (Bull, 1980; Temple et al., 2001; Gutzke and
consequently any decrease in ambient temperature precipitates Crews, 1988; Johnston et al., 1996; Spicer and Burggren,
a series of cascading events: embryo temperature follows the 2003). Susceptible to change in the chronically hypothermic
decline in ambient temperature (Ta), which in turn reduces embryo, then, are the absolute as well as the relative timings
metabolic rate and energy production, slowing embryonic of onset of different physiological process and the systems that
growth and maturation (Tazawa et al., 1988; Pelster, 1997). regulate them. This phenomenon has recently been termed
The most obvious consequence of experimental hypothermic ‘heterokairy’ to differentiate it from the rather broadly and
incubation is that it will take longer for a chronically loosely used ‘heterochrony’ (see Spicer and Burggren, 2003).
hypothermic embryo to reach the required level of maturity for The shift from poikilotherm to homeotherm in bird embryos
hatching. Not surprisingly, embryonic growth and the is an important physiological transition, beginning within the
development of physiological systems in chicken embryos last 20% of incubation. This thermoregulatory transition
incubated at 35°C are retarded compared to embryos incubated requires heat-producing metabolism and supporting oxygen
at the optimum temperature of 38°C, with an additional 4 days transport to mature as essential steps in the precise regulation
required for hatching (Tazawa et al., 1988). of body temperature. Such developmental events may be
Less obvious than the simple lengthening of the embryonic particularly susceptible to temperature-induced qualitative
period by chronic hypothermia, but potentially of considerable adjustments. In this and the following study on the late stages
physiological importance, is the impact on both the absolute of incubation of the chicken embryo (Gallus domesticus), we
and relative timing of the onset of different physiological examine both acute and chronic temperature influences on
processes and their regulation. Indeed, temperature changes metabolism, changes in blood-oxygen transport supporting
during development often have complex affects that go beyond metabolism, and thermoregulatory responses of chicken
simply accelerating or decelerating the development of the embryos. Our goal is to determine if chronic hypothermia
1544 J. L. Black and W. W. Burggren
(35°C) alters standard developmental patterns, providing pipping, external pipping and hatching was expressed by the
additional evidence for heterokairy. As a self-contained temperature quotient (Q10) calculated using the van’t Hoff
embryo that is very well characterized anatomically and equation:
reasonably well understood physiologically, the chicken
Q10=(k2/k1)10/(t1–t2) ,
embryo is an excellent model to examine how the changes in
the thermal environment can quantitatively and qualitatively where k1 and k2 are the timing of events expressed as a
influence the developmental timeline. percentage of incubation at temperatures t1=38°C and t2=35°C,
In this first study, we test the hypothesis that chronic respectively.
incubation in hypothermia (35°C) not only lengthens the
embryonic period but also alters the relative timing of hatching Static VO∑ measurements at 35°C and 38°C
events, the normal pattern of changes in metabolic activity, and Six eggs from each incubation temperature, at each stage,
the ability of the embryo to respond physiologically to acute were implanted with thermocouples. Thermocouple wires were
decreases in Ta. inserted immediately beneath the shell through a 0.5·mm hole,
and held in place with a 1·cm2 piece of tape. Preliminary
experiments revealed no detectable thermal gradient from
Materials and methods inside the embryo’s body to the shell exterior in incubating
Source and incubation of eggs eggs. Consequently, ‘surface temperature’ measured
Fertilized White Leghorn eggs (Gallus domesticus L.) were immediately under the shell is assumed to be embryo
obtained from Texas A&M University (College Station, Texas, temperature. The eggs were placed in an air-tight, water-tight
USA) and shipped to the University of North Texas (Denton, respirometer (240·ml) through which air was pumped
TX, USA) where they were incubated in commercial continuously at 70–75·ml·min–1. Water and carbon dioxide
incubators. All experimental procedures were approved by the were removed from the outflow air by passing it through
University of North Texas’ Institutional Animal Care and Use Drierite™ and soda lime, respectively. Analysis of O2 content
Committee. of the air and calculation of oxygen consumption was carried
Populations of eggs were incubated at 38.0°C (control), or out using an oxygen analyzer (model FC-1B, Sable Systems
35.0°C (hypothermic), all at a relative humidity of 60%. To Inc., Henderson, NV, USA). The ventilated chamber was
determine the gross effects of incubation temperature on partially submerged in a programmable water bath (ISOTEMP
development, nine or more embryos incubated at each 1028P, Fisher Scientific, Hampton, NH, USA) and allowed to
temperature were staged for developmental maturity on days equilibrate to incubation temperature for a minimum of 30·min
13–14, 15–16, 17–18 and 19–20 (Hamburger and Hamilton, before measurements were started. Measurements of oxygen
1951). Hypothermic embryos have a slower rate of consumption were recorded simultaneously with egg
development than embryos incubated in control conditions, so temperature and ambient temperature (Chart software and
staging was completed through hatching to determine the Powerlab data acquisition system, ADInstruments, Colorado
length of incubation required for the 35°C embryos to reach Springs, CO, USA). All values of VO∑ (µl·O2·g–1·min–1) were
developmental stages equivalent to the 38°C embryos. expressed on an embryo mass-specific rather than egg mass-
All subsequent metabolic experiments were conducted on specific basis, unless otherwise indicated.
embryos at the following stages: stage 39–40, reached on days Basal VO∑ measurements made at constant Ta were
13–14 for 38°C and days 15–16 for 35°C; stage 41–42, designated as ‘static’ VO∑ measurements (in contrast to
reached on days 15–16 for 38°C and days 17–18 for 35°C; measurements during gradual cooling or warming, described
stage 43–44, reached on days 17–18 for 38°C and days 19–20 below). Static measurements were always recorded first at the
for 35°C. chronic incubation temperature of that particular egg. After this
initial measurement (for approximately 30·min), the water bath
Rates of survival and timing of hatching temperature was changed at a rate of 3°C·h–1 to expose the egg
Fertilized eggs (N=40 for 38°C and N=32 for 35°C) were to the other treatment incubation temperature (e.g. 35°C if the
incubated as described above. Eggs were candled every 2 days incubation temperature was 38°C) for a minimum of 2·h. VO∑
(D) from D4 to D18 of incubation, to determine survival. From was then determined for the same egg at the other treatment
D19 to D25 of incubation eggs were candled daily to determine temperature as described above.
survival at the pre-pip stage, internally pipped stage, externally
pipped stage and hatching success. Survival rates were VO∑ measurements during gradual cooling
calculated as number of eggs alive compared to total number VO∑ was also measured during gradual cooling, because the
of eggs incubated at that temperature. Counts of eggs on each Ta at which basal VO∑ (and the accompanying heat production)
day were converted to relative frequencies and plotted for each can no longer be maintained during cooling – the Tcrit – is an
day of incubation. Egg counts also allowed the calculation of indication of the maturity of thermoregulatory ability. Hence,
percentage survival and the timing of pipping and hatching eggs (N≥6 for each incubation temperature) with implanted
events. thermocouple wires were placed in metabolic chambers.
The effect of temperature on length of incubation to internal Chamber and egg surface temperature were simultaneously
 Metabolism in hypothermic chicken embryos 1545
recorded as described above. Static basal VO∑ A
measurements were determined at the egg’s chronic 100
38°C Infertile
incubation temperature before the start of the gradual 90 Dead
cooling protocol. For those eggs incubated at 38°C, Pre-IP
80
measurement of VO∑ at 38°C was followed by IP
continuous VO∑ measurements during gradual cooling 70 EP
Hatch
(3°C·h–1) of the water bath and metabolic chamber to a 60
final egg temperature of 30°C. For those eggs incubated
50
in hypothermia (35°C), a static VO∑ measurement was
determined at 35°C and the temperature of the water 40
bath was then increased to 38°C. Eggs were allowed a 30

Survival, as relative frequency (%)

minimum of 2·h at 38°C before entering the same
20
gradual cooling protocol described for the control eggs
incubated at 38°C. Preliminary measurements indicated 10
that the temperature in the deep interior of the egg was 0
always identical to surface temperatures at the cooling 0 2 4 6 8 10 12 14 16 18 20 22 42 26
rates used in these experiments.
B
100
Stage and mass determination
90 35°C
Upon completion of VO∑ measurements, embryos
were killed by placing eggs in a container containing 80
halothane vapor. Each embryo was then removed from 70
the egg, blotted dry with towels and its wet mass
determined using a microbalance (Denver Instrument 60
Company, Denver, CO, USA). The ventricle was 50
dissected from the embryo, blotted dry and weighed. 40
The embryo was staged to confirm that it was at the
30
expected developmental stage, weighed, and then dried
in a 40°C oven for 2 days for subsequent determination 20
of dry mass. 10

Statistical analysis of VO∑ and mass data 0

0 2 4 6 8 10 12 14 16 18 20 22 42 26
All data were tested for normality of distributions
Incubation time (days)
(Shapiro–Wilks normality test) and equality of
variances. SAS (Version 8.0) was used to conduct all Fig.·1. The length of incubation, survival through incubation, and timing of
statistical analyses. All statistical decisions were made hatching events (IP, internal pipping; EP, externally pipping) for embryos
with a 0.05 level of significance and all values are chronically incubated at 38°C (A) and 35°C (B). Sample sizes are indicated
presented as means ± S.E.M. in Table·1.
Significance of differences between static oxygen
consumption measurements at 35°C and 38°C for
embryos incubated at the same temperature was determined Differences in embryo wet mass, dry mass, and heart mass
using a paired t-test. The same oxygen consumption data were were determined using ANOVA and SNK multiple range test.
tested for significance of differences between incubation
temperatures at a particular measurement temperature using
independent t-tests. Results
The rate of oxygen consumption during gradual cooling at Survival and timing of hatching events
each stage and each incubation temperature were tested with The length of incubation, survival, and timing of hatching
repeated-measures ANOVA (block design) to determine Tcrit, events for embryos incubated in normothermal conditions
the temperature at which a significant decrease in rate of (38°C) and hypothermal conditions (35°C) are summarized in
oxygen consumption from control values occurred during the Figs·1 and 2. In the early stages of incubation there was little
cooling protocol. Comparisons of oxygen consumption rates difference in survival rates between the two incubation
during gradual cooling between stages and incubation temperatures. At both temperatures there was an initial loss due
temperatures were performed using a one-way ANOVA. to the presence of infertile eggs between D0 and D4 (12.5% at
Student–Newman–Keuls (SNK) multiple range tests were 38°C and 18.7% at 35°C, respectively). Embryos incubated at
performed following each ANOVA for post-hoc detection of 38°C had a slightly higher rate of survival (75.0%) between
specific differences between means. D4 and internal pipping than 35°C embryos (68.8%), and a
1546 J. L. Black and W. W. Burggren
HH 39– 40 HH 41– 42 HH 43– 44 difference (P>0.05) in wet mass or dry mass between
35°C embryos incubated at 35°C (7.60±1.03·g and
0.79±0.13·g, respectively) and 38°C (5.54±0.29·g and
0.51±0.03·g, respectively). All embryos experienced a
38°C
significant increase in both wet and dry mass from
stage 39–40 to stage 41–42 (15.71±0.81·g and
14 15 16 17 18 19 20 2.38±0.17·g for 35°C embryos and 13.75±0.40·g and
Incubation time (days) 2.20±0.11·g for 38°C embryos, F=62.29, P<0.01), but
there was still no differences attributable to incubation
Fig.·2. Developmental stage and corresponding day of incubation (mean ± 1
temperature. By stage 43–44 the 35°C embryos had a
S.E.M.) for embryos incubated at 35°C and 38°C. Sample sizes are indicated in
significantly higher wet mass (22.26±0.98·g) than 38°C
Table·1.
embryos (19.42±0.51·g) (F=62.29, P<0.01), but there
was no significant difference in dry embryo mass
higher overall survival (48% and 31.3% for 38°C and 35°C, (4.16±0.25·g and 4.62±0.36·g, respectively).
respectively). No apparent differences in embryo body mass were detected
Hypothermic embryos required an incubation time of 23.7 between incubation temperatures at stage 41–42; nonetheless,
days, an average of 3.1 days longer than 38°C embryos the wet ventricle mass of 35°C embryos (0.22±0.04·g) was
(Table·1). Internal pipping in 35°C embryos occurred an significantly larger than that of the 38°C embryos at stage
average of 2.9 days later than in normothermal conditions, 41–42 (0.10±0.01·g) (F=5.72, P=0.0016), and the ratio of
where it occurred at 22.2 days. Similarly, external pipping ventricle mass to embryo mass was significantly larger in 35°C
occurred at 22.8 days, 3.0 days later than in warmer embryos. embryos at all stages examined (F=30.66, P<0.0001) (Fig.·4).
The effect of hypothermic incubation on the relative timing Between stages 41–42 and 43–44 there was no significant
of each hatching event was determined by converting the change in ventricle mass in embryos at either incubation
length of each event interval to a percentage of total temperature. Because embryonic wet mass significantly
incubation length, and then determining Q10 (Table·1). increased as development progressed, the ratio of ventricle
Incubation in hypothermic conditions had no effect on the mass to embryo mass decreased significantly in all embryos
relative timing of each hatching event, as indicated by (F=30.66, P<0.0001).
Q10 values of 1.00 for each event interval. Thus, internal
pipping occurred at 93% of development and external Static VO∑ at 35°C and 38°C
pipping at 96% of development, regardless of incubation There was a general decrease in mass-specific VO∑ with
temperature. increasing stage (Fig.·5), with stage 43–44 embryos from both
incubation temperatures having a significantly lower VO∑ than
Growth and developmental progress at stage 39–40. Mean mass-specific VO∑ for 35°C embryos
Embryos incubated at 35°C reached HH 39–40 on D15–D16 ranged from 25±1.7·µl·g–1·min–1 at stage 39–40 to
of incubation (Fig.·2), and by D17–D18 they were at HH 13±0.35·µl·g–1·min–1 at stage 43–44 (Table·2). The mass-
41–42. After 19–20 days 35°C embryos were at HH 43–44. specific VO∑ of 38°C embryos ranged from
Developmental progress of hypothermic embryos in late stages 33±0.82·µl·g–1·min–1 at stage 39–40 to 27±2.2·µl·g–1·min–1
of incubation generally lagged behind 38°C embryos by 2–3 at stage 43–44 (Table·2). Mass-specific VO∑ of 35°C and
days. 38°C embryos at the same developmental stage were not
Growth of embryos at each incubation temperature was significantly different.
determined by measuring both wet and dry embryo mass Each group of embryos, regardless of incubation
(Fig.·3). At the earliest stage (39–40) there was no significant temperature or stage, experienced a significant change in

Table·1. Effects of chronic incubation at 35°C and 38°C on the length of incubation and the relative amount of development
each day
Survival to Days to Time to event
Event interval Sample size event end (%) end event (% of total)
Temperature
Start → End 35°C 38°C 35°C 38°C 35°C 38°C 35°C 38°C effect (Q10)
0 → IP 13 16 40.6 40.0 22.2 19.3 93.5 93.6 1.0
0 → EP 9 9 28.1 22.5 22.8 19.8 96.1 96.2 1.0
0→H 9 9 28.1 21.8 23.7 20.6 100 100 1.0
IP → EP 9 9 69.0 56.2 0.6 0.5 2.6 2.6 1.0
EP → H 9 9 100.0 77.7 0.9 0.8 3.8 3.8 1.0

Incubation is divided into intervals based on hatching events from day 0 (0), internal pipping (IP), external pipping (EP) and hatching (H).
 Metabolism in hypothermic chicken embryos 1547

A 0.30 A
25
38°C
* (15) 35°C
38°C 0.25
20 * (17)
35°C
(22)
Embryo wet mass (g)

Ventricle wet mass (g)

(22) * 0.20
15 (15)
* (13)
0.15
10
(11) (17)
0.10 (13)
5 (11)

0.05
0
39–40 41–42 43–44
0
41–42 43–44
6 B
0.014 B
5 38°C
38°C * (17) 35°C
Embryo dry mass (g)

35°C * (15) 0.012 (22)

4

Ventricle mass/body mass ratio 0.010

3
(22)
* 0.008
* (15)
2 * (13) (13)
0.006
1 (11) * (17)
(11) 0.004
0
39–40 41–42 43–44 0.002
Developmental stage (HH)
0
Fig.·3. Wet (A) and dry (B) mass of embryos incubated at 35°C and
41–42 43–44
38°C. A box surrounds points that are not significantly different from
each other (P>0.05). Asterisks indicate a significant increase in mass Developmental stage (HH)
from the previous stage, at the same incubation temperature
Fig.·4. Wet ventricle mass (A) and ratio of ventricle mass to embryo
(P<0.05). Values are means ± 1 S.E.M. N values are in parentheses.
mass (B) for embryos incubated in 35°C and 38°C. Plotting
convention as described for Fig.·3.

oxygen consumption after 2·h at the opposite incubation

temperature (Matched-pairs t-tests with the probability for 41–42 and 43–44 for either incubation temperature, except for
each group; P<0.018, Fig.·5). the initial static measurement at 35°C for the 35°C embryos.
Although there are no significant differences in gradual
VO∑ during acute cooling cooling VO∑ between the 35°C and 38°C embryos compared at
Some embryos from each stage and each incubation each stage, there are important differences in the temperature
temperature were able to increase VO∑ briefly during the (Tcrit) at which they first experienced a significant decline in
earliest stages of the cooling protocol. However, all embryos VO∑ from the baseline rate at 38°C (Fig.·7). Fig.·7A shows that
eventually experienced a significant decline in VO∑ at some the youngest embryos (stage 39–40) experienced a significant
point during the gradual 8°C drop in Ta (Fig.·6). The youngest decrease in VO∑ when Ta reached 34°C. By stage 41–42
embryos of each incubation temperature had significantly (Fig.·7B) the 38°C embryos had a Tcrit of 34°C, while 35°C
higher VO∑ values than the older embryos (Fig.·7). This was embryos had a higher Tcrit of 36°C. Just prior to hatching at
evident for the entire duration of the cooling protocol in the stage 43–44 (Fig.·7C), 38°C embryos can endure a drop in Ta
35°C embryos (Fig.·7A), and until Ta fell to 36°C in the 38°C- of 8°C (Tcrit of 30°C) before suffering a significant decrease in
incubated embryos (Fig.·7B). There were no significant VO∑, but the stage 43–44 35°C embryos were not as efficient,
differences in VO∑ during gradual cooling between stages showing a significant decline at a Tcrit of just 36°C – 1/4 the
1548 J. L. Black and W. W. Burggren
Table·2. Rate of oxygen consumption by chicken embryos as a 40 A
function of incubation and measurement temperatures and 35°C incubation
developmental age 35 HH 43–44
HH 41–42
Temperature HH 39–40
incubation/ 30 (6)
*
measurement Day of VO∑ *

Rate of oxygen consumption (µl O2 g–1 min–1)

(°C) incubation (µl·min–1·egg–1) · Reference 25 (9)
38/38 16 382 Tazawa, 1973
38/38 16 417 Tazawa et al., 20
1992
38/38 16 350 Pearson et al., *
15
1996 (12)
38/38 16 333 Howe et al.,
1996
38/38 16 333±7 (9)* Present study 35 36 37 38 39
38/38 18 400 Tazawa, 1973 40 B
38/38 18 433 Tazawa et al., 38°C incubation
1992 HH 43–44
35
38/38 18 383 Pearson et al., HH 41–42
1996 HH 39–40 (8)
38/38 18 420 Dzialowski et al., 30
2002
38/38 18 450±38 (5)* Present study 25
*
(9)
*
35/35 16 417±30 (6)* Present study
35/35 18 383±20 (10)* Present study 20
35/35 20 317±7 (12)* Present study (7)
15
*
*Values are means ± 1 S.E.M. (N = number of eggs).

temperature decrease at which the 38°C began to show a VO∑ 35 36 37 38 39

decline. VO∑ values for each of these groups at 38°C and at Tcrit Measurement temperature (°C)
are summarized in Table·3.
Fig.·5. Mass-specific static rate of oxygen consumption VO∑ for
embryos incubated at 35°C (A) and 38°C (B). The arrow direction
Discussion indicates that the VO∑ for each embryo was first determined at that
individual’s incubation temperature, and then again after 2·h at the
Survival, timing of hatchling events and extension of other incubation temperature. Plotting convention as described for
developmental timeline Fig.·3. N values for each 35/38°C pair are indicated in parentheses.
Bird embryos are poikilothermic throughout incubation,
making them susceptible to fluctuations in body temperature
dictated by the incubation environment (Tazawa et al., 1988, not only lengthen the developmental timeline, but would also
1989; Whittow and Tazawa, 1991). One of the primary change development qualitatively. Yet our data show that
and most obvious impacts of chronic hypothermia on incubation temperature had no effect on the relative timing of
poikilotherms is a slowing of physiological rates and internal and external pipping in the chicken embryo, two
processes, including metabolism and development. Incubation critical landmarks in avian development. There was a slight
at 38°C has long been considered the optimum temperature for difference in the overall rates of survival between embryos
rearing chicken embryos (Whittow, 2000), clearly based on incubated at 35°C and 38°C (31% and 48%, respectively) but,
imitating the incubation environment created by the hen. Yet, overall, temperature had little effect on survival of the chicken
there is little evidence to suggest that incubation at a embryos throughout incubation. Moreover, small numbers of
temperature other than 38°C has consequences for survival or embryos at each temperature died at approximately the same
fitness of the embryo. The results of this study confirm that, as points in development (Fig.·1A,B). These identical mortality
expected, chicken embryos incubated at 35°C developed more rates during early incubation suggest that the embyros
slowly than those embryos incubated at 38°C, spending an experienced a common failure of an organ or a system at a key
average of 3.1 extra days in the egg before hatching. Similar point in development.
studies reported that 35°C incubation is near the lower end of
the viable incubation temperatures, and results in an incubation Incubation temperature and embryonic growth
period of up to 25 days in the chicken embryo (Tazawa, 1973). Embryonic wet mass for 38°C embryos at HH 39–40 and
Importantly, we hypothesized that incubation at 35°C would HH 41–42 closely resembled previously reported values
 Metabolism in hypothermic chicken embryos 1549

A D
120 120
35°C, HH 39–40 38°C, HH 39–40
110 110
100 100
90 90
80 80
70 70
60 60
50 50

35 38 36 34 32 30 38 36 34 32 30
Normalized oxygen consumption (% of VO2 at 38°C)

B E
120 120
.
110 35°C, HH 41–42 110 38°C, HH 41–42

100 100
90 90
80 80
70 70
60 60
50 50

35 38 36 34 32 30 38 36 34 32 30

C F
120 120 38°C, HH 43–44
35°C, HH 43–44 110
110
100 100
90 90
80 80
70 70
60 60
50 50

35 38 36 34 32 30 38 36 34 32 30
Measurement temperature (°C)
Fig.·6. VO∑ during gradual cooling for individual embryos incubated at 35°C and 38°C to stages 39–40 (A and D, respectively), stages 41–42 (B
and E, respectively), and stages 43–44 (C and F, respectively). Data are normalized to the measured VO∑ value at 38°C; cross hatched bars
represent 5% variation from the measurement at 38°C.

(Tazawa et al., 1971; Dzialowski et al., 2002). The difference total body water in D18 (HH 44) embryos at 81.8%. Their
in wet body mass at HH 43–44 between incubation study also determined that D12 embryos (HH 38) contained
temperatures in the present study is accounted for by the higher 91.7% body water, a value comparable to that in our study for
total body water content in the 35°C embryos. Calculations of HH 39–40 embryos incubated at both 35°C (89.6%) and 38°C
% total body water from the wet and dry mass data confirmed (90.8%).
that at stage 43–44 the 35°C the wet mass of the embryo Overall, these data reveal that embryo growth is proportional
consisted of 81% water, compared to only 76% in the 38°C to developmental stage, irrespective of how long it takes to
embryos. Dzialowski et al. (2002) reported similar values of reach that stage. This emphasizes the importance of making
1550 J. L. Black and W. W. Burggren

A Fig.·7. Gradual cooling mass-specific rate of oxygen consumption for

embryos at stages 39–40 (A), 41–42 (B) and 43–44 (C). A box
35
surrounding points indicates no significant difference between stages
* * HH 39–40 (P>0.05). The asterisks indicate the temperature at which the oxygen
30
consumption first became significantly lower than the initial oxygen
25 consumption at 38°C. The subsequent transition to broken lines after
the asterisk indicates that all subsequent oxygen consumptions
20 (6) remain significantly different during further cooling. Numbers of
15 * embryos used in each cooling run are indicated in parentheses.
(6) Values are means ± 1 S.E.M.
10 38°C
35°C developmental time (see Pritchard et al., 1996; Weltzien et al.,
5
1999). However, as Spicer and Burggren (2003) and Burggren
0 and Crossley (2002) emphasize, temperature effects on
35 38 37 36 35 34 33 32 31 30
development are complex, selective and not easily unraveled.
In contrast to whole embryo mass, ventricular mass showed
Rate of oxygen consumption (µl O2 g–1 min–1)

B
evidence of complex temperature effects. The ventricle mass
35
of stage 43–44 embryos incubated at 38°C (0.10g±0.01)
30 HH 41–42 closely resembled the heart mass data for D18 chicken
embryos (0.12g±0.01) obtained by Dzialowski et al. (2002).
25 Our study revealed that both ventricle mass and the size of the
* ventricle relative to embryo mass were significantly higher in
20 *
the 35°C embryos (Fig.·4) at stage 41–42. Previous studies
15 examining the effects of hypoxic stress on the development of
(6)
* (5) the chicken embryo saw increases in heart mass of D12
10
38°C embryos that were exposed to hypoxia between days 6 and 12
5 35°C of incubation (Dzialowski et al., 2002). That study, however,
was unable to show a similar response in D18 embryos or in
0 hatchlings. Future experiments to examine the differential
35 38 37 36 35 34 33 32 31 30
effects of incubation temperature on heart mass will help to
unravel why hypothermic incubation leads to cardiac
C hypertrophy.
35

30 HH 43–44 Temperature and VO∑

25 VO∑ measurements obtained in the present study agree with
previous measurements for D12, 14, 16 and 18 chicken
20 * embryos incubated at 38°C (Table·2). However, this is the first
* study to complete measurements of VO∑ in embryos chronically
15
(6) incubated at 35°C. The mass-specific VO∑ of stage 43–44
10 (9) embryos incubated at 35°C appears low in comparison with
38°C
* measurements from 38°C embryos at the same stage in this and
5 35°C
previous studies. The significantly larger embryo wet mass of
0 the 35°C embryos contributes to this difference, but this group
35 38 37 36 35 34 33 32 31 30 also demonstrated a large amount of variation in VO∑.
Observations made during incubation indicated that the
Measurement temperature (°C)
chorioallantoic membrane (CAM) in the 35°C embryos failed
to line the entire inner surface of the shell. Although CAM
comparisons at equivalent stages of development, rather surface area data was not collected in these experiments, a
than solely at arbitrary periods of development defined smaller surface area for gas exchange might have an impact on
chronologically. Because the development of embryos and the embryos with the largest metabolic demand, i.e. HH 43–44
organ systems is non-linear, absolute time is not a good embryos. The importance of CAM surface area is debated.
descriptor when comparing embryos incubated at different Okuda and Tazawa (1988) covered up to 50% of the chicken
temperatures. The concept of ‘degree days’ (days of egg with epoxy, effectively reducing the surface area of the
development × the temperature of development) has been used CAM able to exchange gases with the environment. They found
to normalize the effect of temperature on development and that the reduction in gas conductance reduced VO∑. In contrast,
thus address the discrepancies between absolute time and Wagner-Amos and Seymour (2002) reported that metabolic
 Metabolism in hypothermic chicken embryos 1551
Table·3. Rate of oxygen consumption during gradual cooling from 38°C
Incubation
temperature Baseline VO∑ VO∑ at Tcrit Probability
(oC) Stage (HH) N (µl·min–1·g–1) Tcrit (oC) (µl·min–1·g–1) (SNK)
38 39–40 6 24.3±2.9* 34 21.1±2.7 0.002
38 41–42 5 19.0±1.7 34 16.4±1.2 <0.05
38 43–44 9 16.8±1.0 30 11.3±1.7 0.017
35 39–40 6 30.0±5.1 34 26.1±4.4 <0.001
35 41–42 6 20.0±1.4 36 17.0±1.3 0.037
35 43–44 6 17.8±0.9 36 16.8±1.0 0.013

Baseline temperature = 38oC; Tcrit = temperature at which basal VO∑ can no longer be maintained.
*Values are means ± 1 S.E.M.
SNK, Student–Newman–Keuls test.

activity was not significantly correlated with reductions in gas suggesting the onset of thermoregulatory ability in late-stage
conductance, accomplished by applying wax to the shell. embryos: (1) the ability of some individual embryos actually
Incubation temperature did not profoundly affect basal VO∑ to increase briefly their VO∑ by 5–10% upon a 1°C drop in Ta
of chicken embryos in the final stages of development. The very and (2) the ability of the embryo generally to maintain VO∑
similar VO∑ exhibited by 35°C- and 38°C-incubated embryos at during an 8°C drop in Ta.
HH 43–44 and the general trend of a decrease in mass-specific Incubation temperature appears to modify the
oxygen consumption with development may both be explained developmental onset of the chicken embryo’s ability to trigger
by the internal O2 levels late in development. In such late stages, endogenous heat production as part of developing
VO∑ of the embryo is constrained by O2 diffusion rates across thermoregulatory mechanisms. Specifically, hypothermic
the shell. Reduced embryonic mass-specific VO∑ results because incubation delays the onset of the embryo’s ability to maintain
the embryo continues to grow at the expense of establishing stable VO∑ in the face of acutely declining temperature.
hypoxia within the egg (Romijn and Lokhorst, 1951; The youngest embryos examined from both incubation
Wagensteen et al., 1970; Rahn et al., 1974; Ar et al., 1980; temperatures significantly reduced VO∑ at a Tcrit of 34°C
Tazawa, 1980). If late stage chicken embryos are provided with (Fig.·7A). By stages 41–42 and 43–44, the 35°C embryos
increased O2 (hyperoxic environment), they increase their experienced significant decreases in metabolic activity earlier,
metabolic activity (Tazawa et al., 1992). An examination of the but only at 36°C. The ability of the 38°C embryos to maintain
early stages of development would probably reveal lower VO∑ VO∑ improved with continued development, and by stage
in embryos incubated at 35°C, supporting the slower rate of 43–44 they experienced a significant decline in oxygen
growth and development and the increased length of the consumption only after a full 8°C decline in egg surface
developmental timeline. temperature, thus surpassing the performance reported for
chicken embryos by Tazawa et al. (1988). While some of the
Temperature and thermoregulatory activity statistically significant differences individually may be of
A homeotherm must be able to regulate precisely limited biological significance, collectively these data show
endogenous heat production as well as heat loss in order to that over all incubation conditions, hypothermic incubation
maintain a stable body temperature in the face of fluctuating temperatures produce embryos that are less efficient in
ambient temperatures. Chicken embryos are certainly not responding physiologically to acute Ta decreases.
homeotherms, but at some point in very late development the Important differences exist between measurements of static
embryo makes the transition from poikilothermy to VO∑ made after longer periods of exposure (2·h) to altered
homeothery, using elevated VO∑ to produce heat used to ambient temperature compared with the ‘gradual cooling’
maintain body temperature. Tazawa et al. (1988) slowly cooled experiments. First, after longer exposure, the temperature effect
late-stage embryos from 38°C to 30°C over a period of 8·h, on VO∑ was the same for all stages and both incubation
minimizing the imbalance between heat loss and heat temperatures, with Q10 values ranging from 1.49±0.08 to
production. They noted that embryos as young as stage 43 were 1.57±0.17. Although there is something inherently different
able to maintain a maximal oxygen consumption until the about the 38°C embryos that allows them to better resist gradual
ambient temperature reached 34°C, and proposed that the decreases in ambient temperature, these data suggest this effort
embryo was an apparent poikilotherm, unable to maintain body is short-lived, lasting for only a few hours. The constraints
temperature, only because of the thermal constraints of the egg imposed by the egg environment include a complete lack of
environment, not because they lacked the mechanisms required insulation (Whittow and Tazawa, 1991), high thermal
for regulating metabolic activity. In support of the contentions conductance (Tazawa et al., 1988) and limited diffusion of
of Tazawa et al. (1988), we propose two lines of evidence respiratory gases (Wagensteen et al., 1970; Rahn et al., 1974).
1552 J. L. Black and W. W. Burggren
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We are grateful to Ed Dzialowski and Sheva Khorrami for development in herring (Clupea harengus L.). J. Exp. Biol. 204, 3629-3637.
helpful editorial comments. This work was supported by NSF Wagner-Amos, K. and Seymour, R. S. (2002). Effect of regional changes to
shell conductance on oxygen consumption and growth of chicken embryos.
Grant no. IBN-0128043 to W.W.B. Respir. Physiol. 129, 385-395.
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