Effect of Four Percent Carbon Dioxide During the Second Half of Incubation

on Embryonic Development, Hatching Parameters, and Posthatch Growth

N. Everaert,*1 B. Kamers,† A. Witters,* L. De Smit,* M. Debonne,* E. Decuypere,* and V. Bruggeman*

*Department of Biosystems, Division Livestock-Nutrition-Quality, and †Department of Biosystems, Division Mechatronics,

Biostatistics and Sensors, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium

ABSTRACT In this study, broiler embryos were ex- crease in the CO2-incubated group, demonstrating that
posed during the second half of incubation [embryonic chicken embryos can tolerate high (4%) concentrations of

Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019

day (ED) 10 until ED18] to 4% CO2. The CO2 was set
to reach 2% on ED11 and 4% from ED12 onward. Two
experiments were conducted with the same setup. Em-
bryo weight was measured and partial pressure of CO2
and O2 in the air cell was analyzed at several embryonic
ages. Times of internal pipping, external pipping, and
hatching were recorded. Chicks were raised until d 7
posthatch. Plasma corticosterone, triiodothyronine, and
thyroxine concentrations were determined. Embryonic
growth was not retarded and hatchability did not de-
Key words: high CO2, incubation, tolerance, broiler line
CO2 between ED10 and ED18. In the first experiment,
partial pressure of CO2 in the air cell was significantly
higher in the CO2 group on ED11, ED12, ED13, and ED14,
but disappeared thereafter. This difference was not ob-
served in the second experiment. A change in the hatching
process of the CO2 group was seen. Relative growths of
newly hatched chicks until d 7 posthatch were equal in
the CO2 group and the control group. However, cortico-
sterone and thyroxine concentrations were significantly
higher in the CO2-incubated chicks on d 7 posthatch.
2007 Poultry Science 86:1372–1379

INTRODUCTION
Carbon dioxide is an important gas in embryonic devel-
opment and the incubation of bird eggs. However, partial
pressure of CO2 (pCO2) in the incubator higher than 1%
is often considered to be deleterious (Owen, 1991). Several
older studies contradict this statement and show that the
timing of high CO2 exposure and the level of CO2 play
a crucial role in determining the tolerance of the embryo
for CO2. In several studies, Taylor et al. (1956, 1971) and
Taylor and Kreutziger (1965, 1966, 1969) investigated the
CO2 threshold during several short periods of incubation
of White Leghorn eggs. When eggs were exposed to CO2
during the first 4 d of incubation, no decrease in hatchabil-
ity was found until 1% CO2 (Taylor et al., 1956). When
hatching eggs were exposed to CO2 between d 9 and 12
of incubation, Taylor and Kreutziger (1966) observed a
reduced hatchability in excess of 5% CO2. Hogg (1997)
found an increased hatchability of 2% when Ross Breeder
eggs were exposed to CO2 up to 1.5% on embryonic day
(ED) 10. Results of De Smit et al. (2006) also showed
beneficial effects of a gradual increase of CO2 until ED10
(to 1.5%) on embryonic growth and hatching parameters.
©2007 Poultry Science Association Inc.
Received December 21, 2006.
Accepted March 7, 2007.
1
Corresponding author: nadia.everaert@biw.kuleuven.be
When turkey eggs were exposed to 0.3% CO2 during the
first 10 d of incubation, Gildersleeve and Boeschen (1983)
found lower embryo mortality, lower malpositioning,
and higher hatchability. However, they found decreased
hatchability when these eggs were exposed to CO2 only
during the first 5 d of incubation. All these experiments
revealed an increasing tolerance to CO2 with increasing
embryonic age, although no recent studies exist on the
tolerance threshold for CO2 during the second phase of
incubation. The physiological mechanisms behind this
time-dependent tolerance are not yet understood but are
hypothesized to be related to the increasing buffering
capacity of the embryo with age against acidosis caused
by high levels of dissolved CO2 (Taylor and Kreutziger,
1966). This was shown in an additional study by Everaert
et al. (our unpublished data).
The objectives of this study were to determine the effect
of high CO2 levels during the second half of incubation
in a modern commercial broiler line on embryonic devel-
opment, time to internal pipping (IP), external pipping
(EP), and hatching. In addition, air cell gases, thyroid
hormone, and corticosterone levels were measured be-
cause these are major players in the hatching process
(Visschedijk, 1968; Decuypere et al., 1983; Darras et al.,
1996). Chicks were raised until d 7 to look for possible
persistent effects of chronic CO2 exposure during incuba-
tion on posthatch performance.

1372
 TOLERANCE OF BROILER EMBRYOS TO HIGH CARBON DIOXIDE DURING INCUBATION
MATERIALS AND METHODS
Equipment Setup
Two experiments were conducted with fertile Cobb-
500 eggs from different flocks of 39-wk-olds for each ex-
periment. During the first 9 d, 2 incubators (Pas Reform,
Zeddam, the Netherlands) were set at standard condi-
tions (37.8°C; wet bulb temperature of 29°C; turning of
90°/h). On d 10, the experimental group was placed in
a closed incubator with CO2 input. The CO2 was pro-
grammed to rise gradually until 2% CO2 on ED11, and
to reach 4% by the 12th incubation day. This percentage
was maintained until ED18. In the control group, normal
incubation was continued. Temperature in both incuba-
tors was 37.8°C. The humidity in both incubators was
matched based on wet bulb temperature to prevent differ-
ences in egg weight loss. Temperature, humidity, oxygen,
and CO2 levels in the CO2 incubator were continuously
measured and controlled by a computer with 2 data acqui-
sition boards (PCI-6023E, National Instruments, Zaven-
tem, Belgium). Software was written in the real-time mod-
ule of LabView 8 PDS (spring 2006, National Instru-
ments). Also in the control incubator, continuous
measurements were recorded through this computerized
system by using the same specialized sensors as in the
CO2 incubator [CO2: GMM221 Vaisala, Bonn, Germany;
RH-sensor: Hygrosmart S7000.1, Gefran, Olen, Belgium;
temperature: Pt-100 direct 1/3 DIN, Gefran; O2 (used only
in the CO2 incubator): SST Sensing, MF 010-0-LC, Hon-
eywell, Brussels, Belgium].
Sampling
Before the start of incubation, all eggs were numbered
and weighed. Partial pressure of gases in the air cell was
taken with a gas analyzer (Synthesis 10, Instrumentation
Laboratory, Lexington, MA). This was done by making
a hole in the air cell with a needle (18G). The needle of
the gas analyzer was immediately put into the air cell,
and air was aspirated. This method has previously been
described by Dewil et al. (1996), Buys et al. (1998), and
Tona et al. (2003). The partial pressure of O2 (pO2) to
pCO2 was calculated based on the 2 measured partial
pressures. At the time of sampling, egg and yolk-free
embryo weights were measured. Egg weight was used
to calculate relative egg weight loss to confirm equal hu-
midity in both incubators. Relative embryo weight was
defined as embryo weight relative to initial egg weight.
Hatching Parameters
and Posthatch Growth
On the 18th incubation day, all eggs were candled, and
those with evidence of living embryos were weighed to
calculate relative egg weight loss, and eggs were trans-
ferred to hatching baskets under normal ventilation. From
the 456th hour of incubation until the 504th hour, all
eggs were checked individually every 2 h for IP, EP, or
1373
hatching. The time interval between EP-IP, hatch-IP, and
hatch-EP was calculated. Hatched chicks were weighed.
The hatching percentage was calculated as hatched chicks
to fertile eggs. Chicks were numbered and kept until d
7. Chicks were weighed after hatching and on d 7 to
calculate their individual relative growth.
Experiment 1
A total of 450 Cobb-500 eggs were put in an incubator
under standard conditions. On ED10, 300 eggs were trans-
ferred to the closed incubator, where CO2 was gradually
increased to reach 4% CO2 by the 12th incubation day.
Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019
This 4% CO2 was continued until ED18. The other 150
eggs served as a control group and continued normal
incubation. Samples of air cell gas pressures and egg and
embryo weights were taken on the 11th, 12th, 13th, 14th
and 18th incubation day and at 2 h after the onset of IP.
Experiment 2
A total of 750 Cobb-500 eggs were placed in 2 incuba-
tors under standard conditions. On ED10, all eggs were
equally divided into 2 groups. One half served as a control
under standard incubation conditions and the other half
continued incubation under high CO2, as described for
experiment 1. Fifteen eggs per group were taken daily
(from ED10 until ED18, at IP + 2 h, and at EP) to measure
the partial pressure of gases in the air cell, and egg and
embryo weights. At every sampling day until ED17, blood
was taken from the allantoic vein (oxygen-rich blood) of
the chorioallantoic membrane and collected in heparin-
ized tubes. On ED18, at IP + 2 h, EP, hatching, and d7,
blood was taken from the vena jugularis of the embryo
or chick. Blood samples were centrifuged, and the plasma
was collected and stored at −20°C until assayed for triio-
dothyronine (T3), thyroxine (T4), and corticosterone lev-
els. Concentrations of T3 and T4 were measured in plasma
samples by RIA as described previously (Huybrechts et
al., 1989; Darras et al., 1992). Antisera and T3 and T4
standards were purchased from Byk-Belga (Brussels, Bel-
gium). Intraassay CV were 4.5 and 5.4% for T3 and T4,
respectively. All samples were run in the same assay to
avoid interassay variability. Corticosterone concentra-
tions in plasma samples were measured using a commer-
cially available double-antibody RIA kit (Instrumentation
Laboratory; Decuypere et al., 1983; Meeuwis et al., 1989).
Statistical Analysis
Data were processed using the statistical software pack-
age SAS, version 8.2 (SAS Institute Inc., Cary, NC). A
GLM procedure was used to analyze the effect of embry-
onic age and incubation condition on egg weight loss,
(relative) embryo weight, pCO2 and pO2, and pO2:pCO2 in
the air cell. When the means of the GLM were statistically
different, these means were further compared between
the control and the experimental group by Tukey’s test
per embryonic day. Significance was based on P < 0.05.
1374 EVERAERT ET AL.
Table 1. Mean ± SEM of measured parameters per group from embryonic day (ED) 10 until ED18, at internal (IP) and external (EP) pipping from
1

experiment 1 and experiment 22

Experiment 1 Experiment 2

Embryo weight (g) Relative embryo weight (%) Embryo weight (g) Relative embryo weight (%)

Item Control CO2 Control CO2 Control CO2 Control CO2

ED10 3.44 ± 0.057 3.44 ± 0.057 5.02 ± 0.12 5.02 ± 0.12
ED11 4.63 ± 0.098 4.40 ± 0.089 6.77 ± 0.19 7.13 ± 0.18 5.13 ± 0.091 5.16 ± 0.097 7.62 ± 0.13 7.83 ± 0.21
ED12 7.49 ± 0.14 7 ± 0.049 10.02 ± 0.18 10.66 ± 0.32 7.56 ± 0.12 7.33 ± 0.12 11.43 ± 0.33 10.92 ± 0.39
ED13 10.17 ± 0.16a 9.18 ± 0.40b 14.12 ± 0.54 15.42 ± 0.36 11.167 ± 0.20 10.60 ± 0.17 16.54 ± 0.45 15.8 ± 0.31
ED14 13.55 ± 0.17 14.40 ± 0.19 20.82 ± 0.36 21.47 ± 0.44 14.40 ± 0.32 14.63 ± 0.17 22.22 ± 0.65 21.16 ± 0.4
ED15 18.03 ± 0.22 17.49 ± 0.30 27.29 ± 0.45 25.94 ± 0.5
ED16 22.27 ± 0.26 21.03 ± 0.23 33.53 ± 0.41 31.56 ± 0.66
ED17 28.06 ± 0.83 26.51 ± 0.42 39.8 ± 1.49 37.59 ± 0.7
30.17 ± 0.38a 29.19 ± 0.21b 46.56 ± 0.61a 44.51 ± 0.89b ± ± ± ±

Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019

ED18 30.1 0.74 29.66 0.9 43.93 1.09 43.66 1.06
IP 36.35 ± 0.60 35.87 ± 0.56 55.36 ± 0.61 55.51 ± 0.47 37.89 ± 0.47 36.16 ± 0.6 55.27 ± 0.45 54.79 ± 0.47
EP 38.58 ± 0.53 39.17 ± 0.52 56.89 ± 0.52 57.89 ± 0.44

Means between experimental groups differ (P < 0.05) per embryonic day.
a,b

1
CO2 group: on ED10, eggs were transferred to a closed incubator where CO2 was gradually increased to reach 4% CO2 by the 12th incubation
day. This 4% CO2 was continued until ED18. Control group: a continued normal incubation.
2
Wet embryo weight (g) and relative embryo weight (%): (wet embryo weight/initial egg weight) × 100.

All values are expressed as mean ± SEM. Variances of
hatching time were compared with the Levene test. A
logistic regression model was developed to analyze the
hatching percentage. The incubation treatment (normal
incubation vs. CO2 incubation) served as a categorical
explanatory variable in the model.
RESULTS
Incubation Conditions
The mean egg weight of all eggs was 65.02 ± 0.13 g in
the first experiment, and 68.01 ± 0.15 g in the second
experiment. Between the 10th and 18th incubation day,
the O2 concentration decreased in the CO2 incubator but
did not fall below 19.7% O2. Because humidity was
matched, relative egg weight loss increased with embry-
onic age in both groups in both experiments in a similar
way. In the first experiment, mean relative egg weight
loss at ED18 was 8.07 ± 0.096% (n = 113) and 7.70 ± 0.067%
(n = 246) for the control and CO2 group, respectively, and
was statistically significant (P < 0.0001). In the second
experiment, the relative egg weight loss at ED18 between
the 2 groups did not differ and was 7.57 ± 0.076% (n =
221) and 7.72 ± 0.083% (n = 211) of the control group and
the CO2 group, respectively.
Egg Weight Loss, (Relative) Embryo
Weight, and Partial Pressure
of Gases in the Air Cell
In both experiments, a significant increase in (relative)
embryo weight was seen with increasing development
(Table 1). The air cell pCO2 increased significantly,
whereas the pO2 decreased significantly with embryonic
age in both groups in both experiments (Figure 1, panel
A: experiment 1; Figure 1, panel B: experiment 2).
In the first experiment, there was a significant overall
effect of incubation treatment over incubation time for
(relative) embryo weight, air cell pCO2, and pO2:pCO2 (P
< 0.05). Absolute embryo weight differed between the 2
groups on ED13 and ED18, being significantly higher in
the control group (Table 1). A higher relative embryo
weight of the control group compared with the CO2 group
was seen on ED18. The pCO2 in the air cell was signifi-
cantly higher in the CO2-incubated eggs compared with
the control eggs from ED11 until ED14. A lower pO2 in
the air cell of the CO2 group was observed only on ED12
and ED13. The ratio of pO2:pCO2 was significantly differ-
ent from ED11 until ED14, with a higher ratio in the
control group (Figure 2, panel A). In the second experi-
ment, none of the measured parameters showed a signifi-
cant incubation treatment effect.
Hatching Parameters
and Posthatch Growth
Table 2 shows the results of the hatching parameters,
chick weight at d 7, relative growth, and hatchability of
the chicks of both experiments. In the first experiment,
the hatching percentage was 95 and 96% for the control
and CO2 group, respectively (not significantly different;
Table 2). The times of IP, EP, and hatching were signifi-
cantly advanced in the CO2 group compared with the
control group. Time intervals (EP-IP, hatch-EP, hatch-IP)
did not differ between the groups. The Levene test
showed no difference in the variance of the hatching time,
which is a measure for spread of hatching. Chick weight
at hatching and on d 7, and relative growth from hatching
until d 7 did not differ between groups.
In the second experiment, the hatching percentage was
89% in the control group and 87% in the CO2 group, which
were not significantly different. There was no difference
between the 2 groups in time of onset of IP, EP, and
hatching, or in duration of time from IP until EP (Table
2). However, the interval between hatch-IP and hatch-EP
was significantly shorter in the CO2 group (P-value of
0.0007 and 0.0071, respectively). Variances of hatching
 TOLERANCE OF BROILER EMBRYOS TO HIGH CARBON DIOXIDE DURING INCUBATION
Figure 1. Changes in the partial pressure of CO2 (pCO2) and O2 (pO2)
in the air cell from embryonic day (ED) 10 (before the start of CO2)
until ED18 and at internal pipping (IP) from CO2 and the control group.
CO2 in the incubator increased gradually from ED10 to reach 4% at
ED12. This level was continued until ED18. Panel A: experiment 1;
panel B: experiment 2. Filled symbols: CO2 group; open symbols: control
group; solid line: pCO2; dashed line: pO2; asterisk: means between
experimental groups differ (P < 0.05) per embryonic day. All values are
expressed as mean ± SEM.
time did not differ between the 2 groups. No difference
was seen in chick weight at hatching and at d 7 and in
relative growth between the 2 groups.
Hormone Levels
Figure 3 shows the corticosterone concentrations of
both groups of the second experiment per embryonic day
from ED14, at IP, EP and hatching and on d 7 of the
chick’s life. There was no general age effect (P = 0.1247),
but there was a significant effect of incubation treatment
(0.0021; significant interaction age × treatment; P =
0.0041). In the control group, the corticosterone increased
from ED14 onward to reach a first maximum level at
ED16 (17.11 ± 1.31 ng/mL). Thereafter, the level decreased
but reached a second peak of 15 ng/mL at ED18, and
declined again to levels of approximately 7 to 8 ng/mL
at IP. In the CO2 group, a similar but more smoothed
age-related pattern was observed without a peak at ED16,
resulting in a significant higher level in the control group
1375
Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019
Figure 2. Changes in the partial pressure of CO2 to O2 (pO2:pCO2)
in the air cell from embryonic day (ED) 10 (before the start of CO2)
until ED18 and at internal pipping (IP) from CO2 and the control group.
CO2 in the incubator increased gradually from ED10 to reach 4% at
ED12. This level was continued until ED18. Panel A: experiment 1;
panel B: experiment 2. Filled symbols: CO2 group; open symbols: control
group; asterisk: means between experimental groups differ (P < 0.05)
per embryonic day. All values are expressed as mean ± SEM.
on that day. At EP, hatching, and d 7, corticosterone levels
tended to be higher in the CO2 chicks, and were significant
at d 7. For T4, there were significant age (P < 0.0001),
group, (P = 0.0462), and interaction effects (P = 0.0273;
Figure 4). In both groups, T4 levels decreased from IP
until d 7. However, this decrease was more gradual in
the CO2 group, resulting in a significant higher T4 at d 7
compared with the control group. There was no effect of
age or treatment, and no interaction for levels of T3 from
IP until d 7 (Figure 5).
DISCUSSION
The most striking observation of this study was the
remarkable embryonic tolerance for CO2 in the incuba-
1376 EVERAERT ET AL.
Table 2. Hatching parameters of the first and second experiment for both groups1: time (h) of internal pipping
(IP), external pipping (EP), and hatching; chick weight at hatching (g); duration (h) of IP (EP-IP), duration of
EP (hatch-EP), duration of hatching (hatch-IP); chick weight on d 7 (g); relative growth after 1 wk2 (%); and
hatchability (%)3

Experiment 1 Experiment 2
Item Control CO2 Control CO2

IP (h) 467.2 ± 0.72 a

462.9 ± 0.56b
463.5 ± 0.56 464.6 ± 0.61
EP (h) 477.3 ± 0.68a 472.8 ± 0.52b 470.6 ± 0.58 471.9 ± 0.6
Hatch (h) 488.3 ± 0.54a 483.6 ± 0.42b 485.8 ± 0.5 486.3 ± 0.6
Chick weight (g) 49.16 ± 0.35 49.47 ± 0.21 51.53 ± 0.26 52.04 ± 0.25
EP-IP (h) 10.7 ± 0.57 11 ± 0.40 8.3 ± 0.43 8.8 ± 0.41
Hatch-EP (h) 11 ± 0.48 10.7 ± 0.4 13.54 ± 0.39a 11.5 ± 0.40b
Hatch-IP (h) 21.2 ± 0.46 21.1 ± 0.4 21.9 ± 0.35a 20.4 ± 0.42b
Chick weight on d 7 (g) 126.56 ± 3.81 125.96 ± 1.50 155.4 ± 1.71 155.06 ± 1.87

Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019

Relative growth (%) 163.18 ± 8.58 155.72 ± 3.07 200.89 ± 3.74 197.86 ± 4.12
Hatchability (%) 95 96 89 87
Means between experimental groups differ (P < 0.05) per embryonic day.
a,b
1
CO2 group: on ED10, eggs were transferred to a closed incubator where CO2 gradually increased to reach
4% CO2 by the 12th incubation day. This 4% CO2 was continued until ED18. Control group: a continued normal
incubation.
2
(weight on d 7 − weight at hatching) × 100/weight at hatching.
3
(hatched chicks × 100/fertile eggs).

tor—up to 4% between ED10 and ED18 by the embryos
of a modern commercial broiler line. This was supported
by the normal embryonic growth and hatching of these
CO2-incubated embryos. Moreover, there was no signifi-
cantly higher embryonic mortality, thereby confirming
the conclusions of Taylor and Kreutziger (1966, 1969) for a
modern broiler line; there was a high resistance of chicken
Figure 3. Concentration of plasma corticosterone (ng/mL) from em-
bryos on embryonic day (ED) 14 until ED18, at internal pipping (IP),
external pipping (EP), and hatching, and from chicks on d 7 from the
CO2-incubated group and control group (experiment 2). CO2 in the
incubator increased gradually from ED10 to reach 4% at ED12. This
level was continued until ED18. Solid bars: CO2 group; hatched bars:
control group; asterisk: means between experimental groups differ (P
< 0.05) per embryonic day. All values are expressed as mean ± SEM.
embryos of a layer type line to hypercapnia. In these
studies, we demonstrated that a progressive increase in
tolerance to higher levels of CO2 occurs in the chicken
embryo toward the end of d 16 of incubation.
Based on the research of Taylor and Kreutziger (1966),
we can assume that the small decrease in O2 concentration
to reach 19.7%, as observed in our experiment, most prob-
ably did not have an effect on embryonic development.
When changing the oxygen percentage between 15 and
50% O2 in the incubator between d 9 and 12, normal
hatchabilities were found. Moreover, synergistic effects
were obtained when either high or low O2 levels were
combined with high CO2 (Taylor and Kreutziger, 1966).
Moreover, relative egg weight losses between d 11 and
18 were not different (experiment 1), or were very small
(0.3% on ED18; experiment 2), between the control and
CO2 groups. Thus, the observed differences between the
control and CO2 groups for a number of parameters can
most likely be ascribed to CO2 differences alone and not
differences in O2 or humidity in the incubator.
During embryonic development, the pO2 in the air cell
decreases and the pCO2 increases because of the increased
O2 consumption and CO2 production, together with a
limitation in the diffusion of these gases through the egg-
shell. The course of the pO2 and pCO2 in the air cell has
been described by Romijn and Roos (1938) and Tazawa
et al. (1980). Our results during normal development are
in agreement with these findings, hence resulting in a
decreased ratio of pO2:pCO2 as development progresses.
The exchange of both gases increases until the maximum
exchange rate through the porous eggshell is achieved.
Thereafter, the ratio remains unchanged (called the pla-
teau phase; Bamelis, 2003), and was reached at ED16. A
significant increase of pCO2 in the air cell (experiment 1)
was seen during the first days of exposure to high CO2,
but it disappeared thereafter. At this stage of develop-
ment, the embryo itself produces a considerable amount
 TOLERANCE OF BROILER EMBRYOS TO HIGH CARBON DIOXIDE DURING INCUBATION
Figure 4. Concentration of plasma thyroxine (T4, ng/mL) from em-
bryos at internal pipping (IP), external pipping (EP), and hatching, and
from chicks on d 7 from the CO2-incubated group and control group
(experiment 2). CO2 in the incubator increased gradually from ED10 to
reach 4% at ED12. This level was continued until ED18. Solid bars: CO2
group; hatched bars: control group; asterisk: means between experimen-
tal groups differ (P < 0.05) per embryonic day. All values are expressed
as mean ± SEM.
of CO2 (Romijn, 1954), which results in a naturally oc-
curring acidosis status of the embryo. This acidosis is
the result of diffusion limitations of gases through the
membranes and shell, as well as the Henderson-Hassel-
balch equilibrium reaction, resulting in bicarbonate and
proton formation. Taylor and Kreutziger (1966) suggested
that the additional acidification that depends on increased
CO2 tension would accelerate calcium mobilization,
which in turn would increase the bicarbonate buffering
capacity. This blood buffering system could be responsi-
ble for the disappearance of the difference in pCO2 in
the air cell at later stages of development. In additional
research, this increase in buffering capacity of the embryo
to high CO2 is shown (our unpublished data).
In the first experiment, there was a parallel shift for-
ward of the hatching time points (IP, EP, hatching), which
led to earlier hatching events of the CO2 group. A possible
relation with hormonal parameters could not be shown
because no blood samples were taken in this experiment.
In the second experiment, the times of IP, EP, and hatch-
ing did not differ between the groups. However, the inter-
vals between hatch-IP and hatch-EP were significantly
shorter in the CO2 group, which can be related to the
higher T3 concentration in the CO2 group at IP, as seen
in the studies of Tona et al. (in press) and De Smit et al.
(2006). Corticosteroids are known to stimulate thyroid
metabolism in the chicken embryo, which is related to
the hatching process (Decuypere et al., 1991). However,
1377
Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019
Figure 5. Concentration of plasma triiodothyronine (T3, ng/mL) from
embryos at internal pipping (IP), external pipping (EP), and hatching,
and from chicks on d 7 from the CO2-incubated group and control
group (experiment 2). CO2 in the incubator increased gradually from
ED10 to reach 4% at ED12. This level was continued until ED18. Solid
bars: CO2 group; hatched bars: control group. All values are expressed
as mean ± SEM.
in this study, the concentrations of corticosterone during
the hatching process did not differ between the 2 groups,
which is difficult to explain, taking the results of T3 into
account. On the other hand, chicks from the CO2 group
on d 7 had significantly higher concentrations of cortico-
sterone and T4, indicating some long-lasting effects of
this CO2 treatment during embryonic life, as has been
observed for other embryonic treatments (Tona et al., in
press). When certain changes are induced in the direct
environment of the embryo by administration of dexa-
methasone (Tona et al., in press) or higher CO2 (in our
case, during the sensitive period of the establishment of
the hypothalamus-pituitary-adrenal axis), the regulation
of corticostone and thyroid hormones during the post-
hatch period seems to be affected. This is further sup-
ported by the study of Hayward et al. (2006), in which
elevated yolk corticosterone decreased the respon-
siveness of the hypothalamus-pituitary-adrenal axis post-
hatch in females.
Differences between the 2 experiments in absolute em-
bryo weight, pCO2 in the air cell, hatching percentage,
and hatching time could be due to a number of factors,
such as eggs being from different flocks, different storage
conditions on the farm, and differences in management
of the breeders (Decuypere et al., 2001; Tona et al., 2005).
A lower embryo weight in the control group in the first
experiment from ED11 until ED14 suggests a lower me-
tabolism in the embryo, thus a lower production of CO2,
as supported by the study of Tona et al. (2004). This can
be seen in the lower pCO2 in the air cell in the control
1378 EVERAERT ET AL.
group between the 2 experiments from ED11 until ED14.
Another reason for the difference in partial pressure of
O2 and CO2 between the 2 experiments could be due to
the different eggshell permeabilities between the 2 flocks
(experiments 1 and 2). Chick weight at hatching was
higher in the second experiment, as was egg weight at
the start of incubation. Because there is a strong positive
correlation between egg weight and chick weight (Suarez
et al., 1997), the difference in egg weight explains the
chick weight difference. Because both groups in the first
experiment hatched early, we considered ED20 as d 0 of
the chick’s life. In the second experiment, ED21 was d 0
for the hatched chick. This means that d 7 was about 1
d later in time in the second experiment than in the first
experiment. This methodological bias in fixing time
points for weighing chicks explains the difference in chick
weight at d 7 and the relative growth between the 2 exper-
iments.
We can conclude that broiler embryos can tolerate 4%
CO2 between d 10 and 18 of incubation without any effect
on pre- and postnatal growth, embryonic mortality, and
hatchability. Higher CO2 from ED10 until ED18 changed
the timing and time intervals of the hatching process,
although this was not consistent between experiments.
The significantly higher corticosterone and T4 concentra-
tions at d 7 posthatch point to a possible carryover effect.
The search for underlying mechanisms explaining the
tolerance for high CO2 warrants further investigation.
ACKNOWLEDGMENTS
Nadia Everaert and L. De Smit are supported by the
Fund for Scientific Research Flanders (F.W.O.-Vlaande-
ren, Belgium; G0286.04). V. Bruggeman is a postdoctoral
fellow from the F.W.O.-Vlaanderen, Belgium.
REFERENCES
Bamelis, F. 2003. Non invasive assessment of eggshell conduc-
tance and different developmental stages during incubation
of eggs. PhD Thesis (no. 587). Katholieke Univ. Leuven,
Belgium.
Buys, N., E. Dewil, E. Gonzales, and E. Decuypere. 1998. Differ-
ent CO2 levels during incubation interact with G time and
ascites susceptibility in two broiler lines selected for different
growth rate. Avian Pathol. 27:605–612.
Darras, V. M., T. J. Visser, L. R. Berghman, and E. R. Kühn.
1992. Ontogeny of type I and type III deiodinase activities
in embryonic and post-hatch chicks: Relationship with
changes in plasma triiodothyronine and growth hormone
levels. Comp. Biochem. Physiol. A 103:131–136.
Darras, V. M., S. P. Kotanen, K. L. Geris, L. R. Berghman, and
E. R. Kühn. 1996. Plasma thyroid hormone levels and iodoth-
yronine deiodinase activity following an acute glucocorticoid
challenge in embryonic compared with posthatch chickens.
Gen. Comp. Endocrinol. 104:203–212.
Decuypere, E., E. Dewil, and E. R. Kühn. 1991. The hatching
process and the role of hormones. Pages 239–256 in Avian
Incubation. S. G. Tullett, ed. Butterworth-Heinemann, Lon-
don, UK.
Decuypere, E., S. G. Scanes, and E. R. Kühn. 1983. Effects of
glucocorticoids on circulating concentrations of thyroxine
(T4) and triiodothyronine (T3) and on peripheral monodeiodi-
nation in pre- and post-hatching chickens. Horm. Metab. Res.
15:233–236.
Decuypere, E., K. Tona, V. Bruggeman, and F. Bamelis. 2001.
The day-old chick: A crucial hinge between breeders and
broilers. World’s Poult. Sci. J. 57:127–138.
De Smit, L., V. Bruggeman, J. K. Tona, M. Debonne, O. Onagbe-
san, L. Arckens, J. De Baerdemaeker, and E. Decuypere. 2006.
Embryonic developmental plasticity of the chick: Increased
CO2 during early stages of incubation changes the develop-
mental trajectories during prenatal and postnatal growth.
Comp. Biochem. Physiol. A 145:166–175.
Dewil, E., N. Buys, G. A. A. Albers, and E. Decuypere. 1996.
Different characteristics in chick embryos of two broiler lines
differing in susceptibility to ascites. Br. Poult. Sci. 37:1003–
1013.
Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019
Gildersleeve, R. P., and D. P. Boeschen. 1983. The effects of
incubator carbon dioxide level on turkey hatchability. Poult.
Sci. 62:779–784.
Hayward, L. S., J. B. Richardson, M. N. Grogan, and J. C. Wing-
field. 2006. Sex differences in the organizational effects of
corticosterone in the egg yolk of quail. Gen. Comp. Endocri-
nol. 146:144–148.
Hogg, A. 1997. Single stage incubation trials. Poult. Avian Biol.
Rev. 8:168. (Abstr.)
Huybrechts, L. M., R. Michielsen, V. M. Darras, F. C. Buonomo,
E. R. Kühn, and E. Decuypere. 1989. Effect of the sex-linked
dwarf gene on thyrotrophic and somatotrophic axes in the
chick embryo. Reprod. Nutr. Dev. 29:219–226.
Meeuwis, R., R. Michielsen, E. Decuypere, and E. R. Kühn. 1989.
Thyrotrophic activity of the ovine corticotropin-releasing fac-
tor in the chick embryo. Gen. Comp. Endocrinol. 76:357–363.
Owen, J. 1991. Principles and problems of incubator design.
Pages 205–224 in Avian Incubation. S. G. Tullet, ed. Butter-
worth-Heinemann, London, UK.
Suarez, M. E., H. R. Wilson, F. B. Mather, C. J. Wilcox, and B.
N. McPherson. 1997. Effect of strain and age of the broiler
breeder female on incubation time and chick weight. Poult.
Sci. 76:1029–1036.
Romijn, C. 1954. Untersuchungen über künstliche Bebrütung
von Hühnereiern. Arch. Geflügelk. 18:173–183.
Romijn, C., and J. Roos. 1938. The air space of the hen’s egg
and its changes during the period of incubation. J. Physiol.
94:365–379.
Taylor, L. W., and G. O. Kreutziger. 1965. The gaseous environ-
ment of the chick embryo in relation to its development and
hatchability. 2. Effect of carbon dioxide and oxygen levels
during the period of the fifth through the eighth days of
incubation. Poult. Sci. 44:98–106.
Taylor, L. W., and G. O. Kreutziger. 1966. The gaseous environ-
ment of the chick embryo in relation to its development and
hatchability 3. Effect of carbon dioxide and oxygen levels
during the period of the ninth through the twelfth days of
incubation. Poult. Sci. 45:867–884.
Taylor, L. W., and G. O. Kreutziger. 1969. The gaseous environ-
ment of the chick embryo in relation to its development and
hatchability. 4. Effect of carbon dioxide and oxygen levels
during the period of the thirteenth through the sixteenth
days of incubation. Poult. Sci. 48:871–877.
Taylor, L. W., G. O. Kreutziger, and G. L. Abercrombie. 1971.
The gaseous environment of the chick embryo in relation to
its development and hatchability. 5. Effect of carbon dioxide
and oxygen levels during the terminal days of incubation.
Poult. Sci. 50:66–78.
Taylor, L. W., R. A. Sjodin, and C. A. Gunns. 1956. The gaseous
environment of the chick embryo in relation to its develop-
ment and hatchability 1. Effect of carbon dioxide and oxygen
levels during the first four days of incubation upon hatchabil-
ity. Poult. Sci. 35:1206–1215.
Tazawa, H., A. Ar, H. Rahn, and J. Piiper. 1980. Repetitive and
simultaneous sampling from the air cell and blood vessels
in the chick embryo. Respir. Physiol. 39:265–272.
 TOLERANCE OF BROILER EMBRYOS TO HIGH CARBON DIOXIDE DURING INCUBATION
Tona, K., V. Bruggeman, O. Onagbesan, F. Bamelis, M. Gbeassor,
K. Mertens, and E. Decuypere. 2005. Day-old chick quality:
Relationship to hatching egg quality, adequate incubation
practice and prediction of broiler performance. Avian Poult.
Biol. Rev. 16:109–119.
Tona, K., R. D. Malheiros, F. Bamelis, C. Careghi, V. M. B.
Moraes, O. M. Onagbesan, E. Decuypere, and V. Bruggeman.
2003. Effects of storage time on incubating egg gas pressure,
thyroid hormones, and corticosterone levels in embryos and
on their hatching parameters. Poult. Sci. 82:840–845.
Tona, K., O. Onagbesan, V. Bruggeman, L. De Smit, D. Fi-
gueiredo, and E. Decuypere. Non-ventilation during early
incubation in combination with dexamethasone administra-
1379
tion during late incubation 1. Effects on physiological hor-
mone levels, incubation duration and hatching events.
Domest. Anim. Endocrinol. doi:10.1016/j.domaniend.
2006.04.002.
Tona, K., O. M. Onagbesan, Y. Jego, B. Kamers, E. Decuypere,
and V. Bruggeman. 2004. Comparison of embryo physiologi-
cal parameters during incubation, chick quality, and growth
performance of three lines of broiler breeders differing in
genetic composition and growth rate. Poult. Sci. 83:507–513.
Visschedijk, A. H. J. 1968. The air space and embryonic respira-
tion 3. The balance between oxygen and carbon dioxide in
the air space of the incubating chicken egg and its role in
stimulating pipping. Br. Poult. Sci. 9:197–210.
Downloaded from https://academic.oup.com/ps/article-abstract/86/7/1372/1623364 by guest on 26 November 2019