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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

¹ Corresponding author: nadia.everaert{at}biw.kuleuven.be

	ABSTRACT

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In this study, broiler embryos were exposed during the second half of incubation [embryonic 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. Embryo 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 decrease in the CO₂-incubated group, demonstrating that chicken embryos can tolerate high (4%) concentrations of 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 observed 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, corticosterone and thyroxine concentrations were significantly higher in the CO₂-incubated chicks on d 7 posthatch.

Key Words: high CO₂ • incubation • tolerance • broiler line

	INTRODUCTION

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Carbon dioxide is an important gas in embryonic development 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 hatchability 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. 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 development, time to internal pipping (IP), external pipping (EP), and hatching. In addition, air cell gases, thyroid hormone, and corticosterone levels were measured because 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 incubation on posthatch performance.

	MATERIALS AND METHODS

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Equipment Setup
Two experiments were conducted with fertile Cobb-500 eggs from different flocks of 39-wk-olds for each experiment. During the first 9 d, 2 incubators (Pas Reform, Zeddam, the Netherlands) were set at standard conditions (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 programmed 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 incubators was 37.8°C. The humidity in both incubators was matched based on wet bulb temperature to prevent differences 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 acquisition boards (PCI-6023E, National Instruments, Zaventem, Belgium). Software was written in the real-time module of LabView 8 PDS (spring 2006, National Instruments). 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, Honeywell, 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 humidity 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 transferred 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 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 transferred to the closed incubator, where CO₂ was gradually increased to reach 4% CO₂ by the 12th incubation day. 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 incubators 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 heparinized 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 triiodothyronine (T₃), thyroxine (T₄), and corticosterone levels. 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, Belgium). 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 concentrations in plasma samples were measured using a commercially available double-antibody RIA kit (Instrumentation Laboratory; Decuypere et al., 1983; Meeuwis et al., 1989).

Statistical Analysis
Data were processed using the statistical software package SAS, version 8.2 (SAS Institute Inc., Cary, NC). A GLM procedure was used to analyze the effect of embryonic 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. 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

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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 embryonic 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).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (11K): [in this window] [in a new window]   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.

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 x 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 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).

View larger version (29K): [in this window] [in a new window]   Figure 3. Concentration of plasma corticosterone (ng/mL) from embryos 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.

View larger version (28K): [in this window] [in a new window]   Figure 4. Concentration of plasma thyroxine (T4, 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; asterisk: means between experimental groups differ (P < 0.05) per embryonic day. All values are expressed as mean ± SEM.

View larger version (28K): [in this window] [in a new window]   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.

	DISCUSSION

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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 probably 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 plateau 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 development, the embryo itself produces a considerable amount of CO₂ (Romijn, 1954), which results in a naturally occurring 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-Hasselbalch 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 responsible 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 forward 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 hatching did not differ between the groups. However, the intervals 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, 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 corticosterone 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 dexamethasone (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 supported by the study of Hayward et al. (2006), in which elevated yolk corticosterone decreased the responsiveness of the hypothalamus-pituitary-adrenal axis post-hatch in females.

Differences between the 2 experiments in absolute embryo 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 metabolism 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 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 experiments.

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 concentrations 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.-Vlaanderen, Belgium; G0286.04). V. Bruggeman is a postdoctoral fellow from the F.W.O.-Vlaanderen, Belgium.

Received for publication December 21, 2006. Accepted for publication March 7, 2007.

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