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Poult Sci 2008. 87:1219-1228. doi:10.3382/ps.2007-00435
© 2008 Poultry Science Association
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PRODUCTION, MODELING, AND EDUCATION

Acclimation to Heat During Incubation. 1. Embryonic Morphological Traits, Blood Biochemistry, and Hatching Performance

S. Yalçin*,1, M. Çabuk{dagger}, V. Bruggeman{ddagger}, E. Babacanoglu*, J. Buyse{ddagger}, E. Decuypere{ddagger} and P. B. Siegel§

* Ege University, Faculty of Agriculture, Department of Animal Science, 35100 Izmir, Turkey; {dagger} Celal Bayar University, Akhisar Vocational Training School, 45140 Manisa, Turkey; {ddagger} Laboratory for Physiology, Immunology and Genetics of Domestic Animals, Department of Biosystems, Katholieke Universiteit. Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium; and § Department of Animal and Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306

1 Corresponding author: servet.yalcin{at}ege.edu.tr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eggs obtained from broiler breeders at 32 (young), 42 (middle aged), and 65 wk (old) were used to measure the effects of heat acclimation during incubation on morphological, physiological, and metabolic traits at internal pipping (IP) and at hatch. All eggs were from the same stock, and hatching performance was also evaluated. Eggs from each breeder age were incubated at control (CONT) or 38.5°C for 6 h daily from d 10 to 18 of incubation (HA). On d 10 after heat exposure and on d 14, absolute and proportional weights were significantly lower for HA than CONT embryos. By the time of hatching, HA chicks were heavier than CONT chicks, which suggested accelerated growth. This effect was consistent across ages. Liver and heart weights were lower for HA than CONT chicks. At IP, pH was similar for HA and CONT embryos, whereas pO2 and Na+ were significantly higher and pCO2, HCO3–, and K+ significantly lower for HA than CONT embryos. Blood pH was higher in embryos from older than for younger and mid-aged parents at IP. At hatch there was no effect of heat acclimation for blood HCO3–, Na+, and K+ levels, whereas plasma triglyceride and T3 levels were higher and plasma uric acid, glucose, and lipid peroxidation levels were lower for HA than CONT chicks. Embryonic mortality was similar among parental ages for CONT. In contrast for HA, embryonic mortality from older parents was higher than for younger and middle-aged parents. A delay in external pipping and hatching time with high incubation temperature was consistent across the breeder ages. It was concluded that lower blood pCO2, HCO3–, K+, and higher pO2 at IP stage, plus increased plasma triglyceride concentrations at hatch, indicate adaptive responses of embryos.

Key Words: parental age • heat acclimation • blood biochemistry • embryonic growth • heat stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In poikiloterm embryos deviations from optimum incubation temperatures, which range from 37 to 37.5°C (Decuypere and Michels, 1992), may affect embryo size, organ and skeletal growth, metabolic rate, physiological development, and hatching success (Yalçin and Siegel, 2003; Black and Burggren, 2004; Tazawa et al., 2004). One consequence of lower or higher incubation temperatures is the timing of the onset of key physiological processes. Incubation at lower temperatures slowed embryonic growth and increased the incubation period; however, relative development of embryos was the same as when incubation was at optimum temperatures (Suarez et al., 1996; Black and Burggren, 2004). In contrast, incubation at higher temperatures accelerated embryonic growth and development (Ricklefs, 1987). When eggs were exposed to 39.6°C for 6 h daily from 10 to 18 d of incubation, embryo weights were lower on d 18, whereas at hatch, weights were similar to (Yalçin and Siegel, 2003) or slightly lower than those for controls (Yalçin et al., 2005). Similarly, there were no differences in weights of chicks from eggs incubated at the control temperature or exposed to 39.0°C for either 6 h daily from 11 to 20 d of incubation (Iqbal et al., 1990) or 2 h daily from 14 to 17 d of incubation (Moraes et al., 2004).

A second consequence of lower or higher incubation temperatures involves the thermoregulatory system. During the prenatal period, lower or higher incubation temperatures alter postnatal thermoregulatory systems by inducing epigenetic adaptation to postnatal low or high environmental temperatures (Nichelmann and Tzschentke, 2002). Black and Burggren (2004) reported that whereas a lower temperature had no effect on the relative timing of hatching, it significantly delayed the relative timing of the onset of thermoregulatory ability. Also, in Muscovy ducklings, alterations in neural hypothalamic sensitivity induced by incubation temperatures were observed at d 10 post hatching (Tzschentke and Basta, 2002).

Epigenetic heat adaptation involves changes in hormonal and metabolic regulations that enhance heat endurance. Yahav et al. (2004a) concluded that 3 h/d of exposure of eggs to 38.5°C from 16 to 18 d of incubation had a positive effect on thermoregulation by causing a reduction in plasma thyroid hormone concentrations and cloacal temperatures in day-old chicks. Lower cloacal temperatures were also noted at hatch when eggs were acclimated to 39.5°C for 3 h/d from d 8 to 10 or 16 to 18 of incubation (Collin et al., 2007). Moraes et al. (2004) suggested that heat conditioning at 39.0°C for 2 h/d from d 14 to 17 increased blood corticosterone and pO2 and pH levels on d 14 with all levels returning to normal at internal pipping stage (IP). These physiological changes may serve as an epigenetic temperature adaptation because the same mechanisms are employed for coping with postnatal heat stress (Moraes et al., 2004).

In addition to a positive relationship between breeder age and chick weight, breeder age also has a major influence on the ability of broilers to thermoregulate (Weytjens et al., 1999; Yalçin et al., 2005). Parental age influenced daily embryonic heat production and O2 consumption, being higher in embryos from 55- and 59-wk-old parents (Hamidu et al., 2007). The aim of the present study was to measure morphological characteristics during incubation of heat-acclimated embryos from eggs obtained from parents of different ages and to measure physiological and metabolic responses at IP and hatch. Hatching performance was also evaluated. In the present study, eggs were heat acclimated from d 10 to 18 of incubation because the timing of heat acclimation has to be linked to the development of the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-adrenal axis to influence heat production threshold responses (Yahav et al., 2004a). Those axes become functional on d 10 of incubation (Thommes et al., 1977; Thommes 1987).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A total of 1,656 eggs were obtained from a broiler breeder stock (Ross 308) at 3 ages of breeder birds; 32 (young), 42 (middle aged), and 65 wk (old). Breeders from the same farm with 2 flocks per age were fed the same diet and maintained under similar environmental conditions. Samples of 10 eggs (5 egg/breeder age/flock) were randomly selected from each breeder age to measure weights (g) of albumen, yolk, and shell. All eggs were numbered and weighed before incubation.

Incubation and Posthatching Temperature Regime
Eggs from the 3 breeder ages (276 eggs/breeder age/flock) were divided equally into 2 groups and placed into 2 incubators. One incubator (CONT) was maintained at 37.8°C from d 1 to 18 of incubation. In the second incubator eggs were heat acclimated (HA) at 38.5°C for 6 h daily from d 10 to 18 of incubation. From d 18 to hatch, the temperature was 37.5°C for both groups. Relative humidity was maintained at 65% in both incubators. There were 3 replicate trays in each subgroup (46 eggs/tray). From hatching to d 7, chicks from each incubation temperature group/breeder age were raised in the pens with a brooding temperature of 32°C for the first 3 d and 29°C from 4 to 7 d.

Morphological Traits and Cloacal Temperatures
Embryos and chicks were sampled using the same protocol as described above. At the end of the daily 6 h of heat treatment, 10 embryos (5 from each flock) were selected randomly on d 10, 14, and 18 of incubation from each breeder age/incubation temperature subclass. Embryos were killed by cervical dislocation and weighed (mg) without embryonic yolk sac. Liver, heart, and breast (with cage and muscles) were removed and weighed (mg). Length (mm) of beak and third right toe was also measured as an index of growth (Dzialowski et al., 2002). Hatched chicks were removed continuously after feather drying and 10 male chicks/breeder age/incubation temperature (5 chicks from each flock subclass) were sampled. The same traits were measured as soon as the chicks were removed from the incubator. At 18 d and hatch, both lungs were removed and weighed (mg).

Cloacal temperatures (to nearest 0.01°C) of the same chicks were also measured at hatch using a thermocouple thermometer that was inserted approximately 3 cm into the colon. Cloacal temperature measurements were repeated with 12 male chicks on d 7.

Blood Biochemical Traits
At IP stage and hatch, blood samples were collected from the jugular vein of 10 embryos and chicks, respectively, into heparinized syringes to measure pH, partial gas pressures (pO2 and pCO2), HCO3, and Na+ and K+ ions. Gases and ions were immediately determined using an automated blood gas (MEDICA Easy Blood gas analyzer), and ion analyzer (MEDICA, EasyLtyte), respectively. On day of hatch, blood was also taken to measure triglycerides, glucose, uric acid, creatine kinase, 3,5,3'-triiodothyronine (T3), thyroxine (T4), corticosterone, and lipid peroxidation (malondialdehyde, MDA) levels. On d 7, blood analyses were repeated on a sample of 10 male chicks from each group. Plasma samples obtained by centrifugation were frozen and stored at –20°C pending analysis. Commercial colorimetric diagnostic kits were used to measure glucose (IL Test kit, No. 182508–00), uric acid (IL Test kit, No. 181685–00), and creatine kinase (IL Test kit, No. 181605–90), using the Monarch 2000 Chemistry system Model 760 (Instrumentation Laboratories, B-1930, Zaventem, Belgium). Plasma lipid peroxidation was estimated by spectrophotometric determination of thiobarbituric acid reacting substances as described in detail by Lin et al. (2004). Plasma T3 and T4 concentrations were measured by radioimmunoassay as described by Darras et al. (1992). Intra-assay coefficients of variation were 4.5 and 5.4% for T3 and T4, respectively. Antisera and T3 and T4 standards were purchased from Byk-Belga (Belgium). All samples were run in the same assay to avoid interassay variability.

Hatching Performance
Eggs were individually checked every 2 h between 464 and 500 h of incubation, and the number of embryos pipped externally and chicks hatched were recorded. Hatchability was defined as the percentage of number of chicks per eggs set. Eggs that failed to hatch were broken out, and fertility was determined. If the unhatched eggs were fertile, they were examined macroscopically and assigned to one of the following categories: early-deads (before 7 d), mid-deads (8 to 18 d), late deads (after 19 d), and pips (pipped shell but not emerged). Because there were no mid-dead and only a few early dead, all embryonic mortalities were combined before statistical analyses.

Statistical Analyses
Data from the 2 flocks/breeder age were pooled because differences between flocks/breeder age were not significant. Data for morphological traits were subjected to ANOVA with 3 breeder ages, 2 incubation temperatures, 4 embryonic ages, and the interactions among them as main effects using the GLM procedure (SAS, 1999). Effects of embryonic age were also measured over time by polynomial regressions. For blood traits and cloacal temperatures the same model was used, except embryonic age was 2 (IP and hatch). For blood gases, ions, and cloacal temperatures chick age was 2 (at hatch and d 7 posthatch). Because parental age and incubation temperature interacted with embryonic age, incubation temperature and parent age were also analyzed within each embryonic age. The same model was used excluding embryonic age for hatching performance. When more than 2 means were involved, comparisons post ANOVA were performed by Tukey. Significance was based on P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eggs obtained from parental flocks at 42 and 65 wk of breeder age were significantly heavier than those obtained from parental flocks of 32 wk of age (Table 1Go). Proportional and absolute yolk weights increased significantly with age of breeder parent (65 > 42 > 32 wk). Weights of albumen were significantly lower in eggs from 32-wk-old than 42- and 65-wk-old parents, which did not differ. There was no age of breeder effect for the proportional albumen weight. Shell weight, but not % shell, increased with age of breeder. Yolk to albumen ratios were 57.1, 60.9, and 72.6%, respectively, being significantly higher in eggs from 64-wk-old than 32- or 42-wk-old breeders, which did not differ.


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  		Table 1. Egg, yolk, albumen, and shell weights by parental age
 
Morphological Traits
Analyses of variance and distributions for embryo, organ, and breast weights, and beak and third toe lengths are presented by incubation temperature, parental age, and embryonic age in Table 2Go. Embryonic age interacted with incubation temperature for all traits measured, except proportional breast weight. Parental age x embryonic age interactions were also significant for embryo, organs, and breast weights, and beak length. Embryo, liver, and breast weights and beak and toe lengths increased quadratically from 10 to 21 d. Increments in heart weight were linear.


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  		Table 2. Analyses of variance (P-values) for embryo, organ, and breast weights and beak and third toe lengths by incubation temperature,1 parental age,2 and embryonic age at 10, 14, and 18 d of incubation and at hatch
 
After heat exposure on d 10 and 14, absolute and proportional weights of HA embryos were significantly lower for HA than CONT embryos. On d 18, embryo weights were similar between incubation groups. By the time of hatching, HA chicks were heavier than CONT chicks (Table 3Go). Yolk weights were also significantly heavier for HA than for CONT chicks, whereas yolk free body weights were similar (Table 4Go).


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  		Table 3. Mean embryo weights (g and % of egg) and organ and breast weights (g and % of embryo weight) and beak and third toe lengths by embryonic age, incubation temperature,1 and parental age
 

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  		Table 4. Mean residual yolk sac weight (g), chick weights (g) without residual yolk sac and weights of organ and breast (% of chick weight without residual yolk sac) by incubation temperature1 and parental age
 
Although absolute weights of liver were similar, proportional liver weights of HA embryos were heavier after 6 h of heat exposure on d 10 than CONT embryos. Liver weights were lower for HA than CONT embryos on d 14. The HA embryos had heavier absolute and proportional liver weights than CONT on d 18, and their liver weights were lower at hatch. Absolute heart weights were lower for HA than CONT embryos from 10 to 21 d of incubation. Although proportional heart weights were similar between incubation temperature groups, HA chicks had lower proportional heart weights than CONT at hatch. Lower proportional liver and heart weights obtained from HA incubation did not change when weights were expressed relative to chick weight without residual yolk sac (Table 4Go). Proportional and absolute lungs were heavier on d 18 but not at hatch for HA than CONT embryos. Absolute weights of breast were similar between groups on d 10 and 14 (Table 3Go). Proportional breast weights were heavier for HA than CONT embryos on d 14 and 18. At hatch, absolute and proportional weights were similar between incubation groups.

At d 10 and 18, HA embryos had significantly shorter beaks than CONT. This difference disappeared at hatch. Although on d 10 and at the end of heat acclimation HA embryos had significantly longer toes than CONT, at hatch HA chicks had significantly shorter toes than CONT.

Parental age affected embryo weight, with embryos being heavier from older than from younger flocks. During the embryonic period and on day of hatch, absolute weights of liver, heart, and breast were also heavier in chicks from older parents (Table 3Go). There were no differences among parental ages for proportional organ and breast weights. Organ weights relative to chick weight without yolk sac did not change the lack of effect of parental age. At 14 to 18 d of incubation, embryos from 32-wk-old breeders had shorter beaks than those from older breeders. At hatch, however, the longest beaks were from the 42-wk-old breeders. Toe length followed the same pattern as that for beaks except that there was no effect of parental age at hatch.

Blood Biochemistry and Cloacal Temperatures
Embryonic age (IP stage and hatch) significantly affected blood gases and ions with the exception of pH (Table 5Go). Blood pO2 and Na+ were significantly higher while pCO2, HCO3–, and K+ were significantly lower at hatch than at IP. At IP, pH was similar between HA and CONT embryos, whereas pO2 and Na+ were significantly higher and pCO2, HCO3–, and K+ significantly lower for HA than CONT (Table 6Go). On day of hatch, pH was significantly higher for HA than CONT chicks. Blood pO2 and pCO2 of chicks differed significantly, being lower in HA than CONT. No effect of heat acclimation on newly hatched chicks was observed for blood HCO3–, Na+, and K+ levels. At IP, blood pH was higher in embryos from older parents than for those from younger and mid-aged parents. Blood pCO2 and K+ were highest in embryos from flocks 42 wk of age and lowest in those from parents 65 wk of age. On day of hatch, blood pH and HCO3–were significantly lower for chicks from 65-wk-old than for those from younger parents. There was no parental age effect on blood pCO2 nor for blood pO2 and Na+ on d IP or hatch (Table 6Go).


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  		Table 5. Analyses of variance (P-values) for blood pH, pO2, pCO2, HCO3–, Na+, and K+ by incubation temperature,1 parental age,2 and embryonic age (at time of internal pipping and at hatch)
 

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  		Table 6. Mean blood pH, pO2, pCO2, HCO3–, Na, and K of chicks by incubation temperature1 and parental age at time of internal pipping (IP) and hatch
 
Analyses of variance for plasma characteristics and cloacal temperatures by incubation temperature, parental age and chick age (at hatch and d 7 posthatch) are presented in Table 7Go. Plasma glucose, corticosterone, and MDA levels increased from hatch to 7 d, whereas creatine kinase level decreased. Triglycerides and T3 levels were similar at hatch and on d 7 (Table 8Go). Plasma triglycerides and T3 were significantly higher in HA than CONT chicks at hatch, and significantly lower in HA than CONT on d 7. This reversal resulted in a significant incubation temperature x chick age interaction (Table 7Go). A significant parental age by age of chick interaction for plasma T3 levels occurred because at hatch, the highest T3 levels were for chicks from parents 42 wk of age, whereas on d 7, T3 from 32 wk of age parents were higher than in those from parents 65 wk of age (Table 8Go). No significant effect of heat acclimation on chicks was observed for plasma T4. Plasma T4 was highest in chicks from parents 65 wk old on day of hatch and at 7 d. A significant incubation temperature by chick age interaction for T3/T4 (Table 7Go) resulted from HA chicks having higher T3/T4 at hatch, and lower T3/T4 on d 7 (Table 8Go). Although, on day of hatch T3/T4 was lower in chicks from 32-wk-old parents than for those from middle-aged parents, those chicks had similar T3/T4 on d 7, which led a significant interaction.


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  		Table 7. Analyses of variance (P-values) for plasma triglycerides, uric acid, glucose, creatine kinase, T3, T4, T3/T4, corticosterone, and lipid peroxidation levels and cloacal temperatures of chicks by incubation temperature, parental age,1 and age at hatch and d 7 posthatch
 

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  		Table 8. Mean plasma triglycerides, uric acid, glucose, creatine kinase, T3, T4, corticosterone, lipid peroxidation levels, and cloacal temperatures of chicks by incubation temperature1 and parental age at hatch and d 7 posthatch
 
The significant incubation temperature by chick age interaction for plasma uric acid levels (Table 7Go) resulted from CONT chicks having similar uric acid levels at hatch and d 7, whereas uric acid levels of HA chicks were lower at hatch than on d 7 (Table 8Go). At 7 d, parental age effects were significant for uric acid, being higher for chicks from 62-wk-old parents. Although glucose levels decreased in HA chicks at hatch and 7 d, a significant incubation temperature x parental age interaction was apparent because chicks from 42-wk-old parents had similar glucose levels at CONT and HA incubation (208 and 198 mg/dL, respectively), at hatch and d 7 (data not shown).

Incubation temperature and parental age had no effect on plasma creatine kinase and corticosterone concentrations. However, a significant incubation temperature by parental age interaction for corticosterone was because heat acclimation decreased corticosterone levels in chicks from 42-wk-old parents (35.72 vs. 24.17 ng/dL for CONT and HA) and increased levels in chicks from 64-wk-old parents (25.52 vs. 36.96 ng/dL; data not shown).

Plasma lipid peroxidation was lower for HA than CONT at IP and hatch. Parental age had no effect on peroxidation level (Table 8Go).

At hatch, cloacal temperatures were slightly but not significantly (P = 0.07) higher for HA than CONT chicks. On d 7, HA chicks had significantly higher cloacal temperatures than CONT. There was no effect of parental age on cloacal temperatures.

Hatching Performance
There was a significant parental age by incubation temperature interaction for embryonic mortalities. Embryonic mortalities were similar among parental ages when eggs were incubated at control temperatures (averaged 0.82%). In contrast, embryonic mortality from older parents was 2.47% when eggs were incubated at higher temperatures and 0.16 and 0.0% in eggs from younger and middle-aged parents, respectively. Neither incubation temperature nor parental age had an effect on mortalities in the pipping stage, which averaged 2.31%. Counting of external pips started at 464 h of incubation. At this time, about 40% of embryos in CONT eggs had already started external pipping. External pipping was completed at 484 and 490 h for CONT and HA embryos, respectively. The CONT chicks started to hatch almost 6 h earlier than HA chicks. On average 50% of chicks from CONT completed hatching at 478 h of incubation in contrast to 484 h for the HA group. At 488 and 494 h, hatching was completed for CONT and HA chicks, respectively. Incubation treatment had no effect on hatchability (85.2 vs. 83.3% for CONT and HA) and hatchability of fertile eggs (95.8 vs. 93.3% for CONT and HA). Hatchability decreased as parental age increased from 32 to 65 wk being 92.1, 87.3, and 73.4% for younger, middle-aged, and older parents, respectively. Hatchability of fertile eggs was 97.3, 95.6, and 90.7% for 32-, 42-, and 64-wk-old parents. Fertility was 94.6, 91.2 and 80.9% for the eggs from younger, middle-aged, and older parents (P = 0.016), respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The effect of age of breeder that we observed for chick weight was consistent with those of Shanawany (1984) and O’Sullivan et al. (1991) who reported an increase in relative embryo weight with parental age. Proportional weights of yolk increased significantly with parental age, whereas there was no change in proportional albumen and shell weights separately. Decreases in albumen and shell weight with parental age (67.5, 65.9, and 63.8% for 32-, 42-, and 65-wk-old parents) balanced with increases in yolk weight. Yolk:albumen ratios suggested a large increase in yolk weight relative to albumen weight with age of parent, an observation consistent with that of O’Sullivan et al. (1991) and Hamidu et al. (2007).

Heat acclimation during incubation influenced organ weights in the developing embryo and the chick at hatch. Lower liver and heart weights for HA than CONT were observed during early acclimation without changes in proportional growth of heart and liver. During the last 4 d of acclimation (from 14 to 18 d of incubation), however, embryo growth accelerated (CONT embryos gained 18.17 g, whereas HA embryos gained 19.71 g), indicating compensatory growth. From 14 to 18 d of incubation, liver, lung, and breast weights increased faster than embryo weight resulting in a significant increased percentage of these organs, hence a differential proportional growth. Heavier weights at hatch for HA than CONT (13.0 vs. 10.9 g) chicks meant that the former gained more weight from the end of acclimation to the time of hatch. In previous studies, lower embryo weights at 18 d of incubation were found when embryos were heat acclimated from 10 to 18 d, although hatch weights were similar to (Yalçin and Siegel, 2003) or lighter than controls (Yalçin et al., 2005). These differences were probably due to the HA temperature which was 39.6°C in the previous experiments. Hulet et al. (2007) also reported heavier chick weights at hatch when eggs were incubated at a constant temperature of 39.0°C from d 14 to 21. Although chicks were heavier, liver and heart weights did not follow the same pattern as body weight. This result did not change when liver and heart weights were expressed as chick weight without yolk. Lower liver weights for HA than CONT chicks may reflect physiological differences in yolk mobilization, which is consistent with higher residual yolk sac weights in HA than CONT chicks. Less yolk utilization in HA than CONT chicks was in agreement with results reported by Wineland et al. (2000a, b). The smaller hearts may be explained by a reduction in cardiac cell development with higher incubation temperatures (Wineland et al., 2000a,b; Leksrisompong et al., 2007), reduced hematocrits and higher skin temperatures, or both, facilitating heat dissipation (Lindquist, 1986; Yahav et al., 1997).

At the IP stage, the higher blood pO2 combined with lower pCO2 in HA was not consistent with that reported by Moraes et al. (2004) who found no differences in blood gases on d 16 and 17 when eggs were acclimated at 39.0°C from 13 to 17 d of incubation. At IP stage, reductions in blood pCO2 and HCO3– were not accompanied by increases in blood pH. Blood concentrations of Na+ were higher and K+ lower at IP for HA than CONT embryos. These results are consistent with previous reports (Ait-Boulahsen et al., 1989; Deyhim and Teeter, 1995) showing that heat stress decreased plasma concentration of K+ and increased Na+, which may be a consequence of lower HCO3– in HA embryos and an exchange of H+ to K+ from the intracellular compartment. Lower blood HCO3– of chicks from older breeders may be due to numbers of pores in larger eggs. High porosity decreased retention of carbon dioxide within the egg, which was accompanied by a smaller increase in blood pCO2 and HCO3– (Tullett and Burton, 1985).

Literature on plasma T3 levels of heat-acclimated embryos and chicks is inconsistent. Whereas HA at 39.0°C from 13 to 17 d of incubation for 2 h/d did not affect concentration of T3 of day-old chicks (Moraes et al., 2004), lower levels of T3 chicks were reported when eggs were incubated at 38.5°C for 3 h/d from 16 to 18 d of incubation (Yahav et al., 2004a). In our study, higher levels of T3- were observed in HA chicks at hatch. The duration of HA in our experiment was longer than that used in the other studies (i.e., embryos were exposed to a total of 54 h of higher temperatures, whereas it was 9 to 10 h in the other studies). On the other hand, it was suggested that heat-induced effects appear to start after the onset of heat acclimation (Horowitz et al., 1986), which is consistent with lower embryo weights observed on d 14 of incubation, immediately after the 6-h heat treatment. Although we did not measure plasma T3 on d 14, the higher levels in HA than CONT chicks at hatch suggests that less T3 was necessary for oxidative metabolism which led to less T3 taken up into the cells and thus higher T3 remaining in the plasma. The conversion of T4 to T3 during embryonic development may be more rapid in HA than CONT chicks, such that differences in levels could not be noted (Tona et al., 2004). The increases in plasma T3 levels may also be due to inhibition of hepatic D3 expression. Although we did not measure hepatic D3 level, degradation of T3 by D3 is an important factor regulating of plasma T3 level (Decuypere and Kühn, 1985; Darras et al., 2000). We may conclude that HA embryos were different physiologically from CONT embryos. This thesis is supported by lower MDA levels observed in HA than control chicks. Increased plasma triglycerides and glucose are associated with high T3 (Nikkila and Kekki, 1972).

Results for length of incubation period when eggs were incubated at fluctuating higher temperatures are inconsistent. A reduction in length of incubation could be due to increased incubation temperatures up to 39.0°C for 6 h/d between 11 and 20 d of incubation (Iqbal et al., 1990) or constant 39.5°C from 14 to 20 d (Leksrisompong et al., 2007). However, no effect on hatching time was observed when eggs were incubated at 39.5 or 41°C from 8 to 10 or 16 to 18 d of incubation (Yahav et al., 2004b), nor a delay in hatching process for eggs incubated at 39.0°C for 2 h/d between 13 and 17 d of incubation (Moraes et al., 2004). Differences among studies may be due to genetic stock, length of the acclimation period, timing of acclimation, and incubation temperature per se, which may alter chronological time to complete development. In the present study, exposure of eggs to 38.5°C 6h/d from 10 to 18 d of incubation delayed external pipping as well as the total incubation period. Because counting of external pipping started at 464 h of incubation and at that time 40% of CONT embryos had pipped externally, we do not know the exact time of external pipping of CONT embryos. Length of incubation, however, was delayed almost 6 h, and total time spent for hatching was similar to CONT. Even though the HA chicks were heavier at hatch, we may conclude that the delay in hatching indicates that fluctuating temperatures between 10 and 18 d of incubation reduced metabolism. When the embryo draws relatively more energy from the anaerobic system under high temperatures, heat production and thus embryo development decreases (Lourens et al., 2006). Larger residual yolks and heavier body weights of HA than CONT chicks were consistent with late hatching (Joseph and Moran, 2005; Lourens et al., 2007).

Lower embryonic weights during the first half of treatment and higher pO2 and lower pCO2 at IP are indicators of lower metabolism, which could also delay the hatching process. The delay in external pipping and hatching time with high incubation temperature was consistent across the breeder ages. Our results also showed that embryos could tolerate the higher temperature without influencing percent hatchability.

Previously, similar or lower cloacal temperatures were observed in HA chicks than in controls (Yahav et al., 2004b; Collin et al., 2007). On d 7 cloacal temperatures were significantly higher for HA than CONT chicks as was blood pH (7.30 vs. 7.25). These results may suggest increased thermoregulatory set points (Tzschentke and Nichelmann, 1999; Janke et al., 2002), adaptive body functions, or both, that are not yet well understood.

In conclusion, exposure of embryos to 38.5°C from 10 to 18 d of incubation for 6 h/d decreased weight gain of embryos from 10 to 14 d, which thereafter was accelerated resulting in the acclimated chicks being heavier at hatch. This effect was consistent across breeder flocks of several ages. Higher cloacal temperatures and blood pH at 7 d posthatch may be indicators of contributions to the thermoregulatory set point of HA chicks. Moreover, lower blood pCO2, HCO3–, K+, and higher pO2 at IP stage, plus increased plasma triglyceride concentrations at hatch, and reversal changes in triglycerides and T3 levels from hatch to 7 d of incubation could also indicate adaptive responses of embryos.


   ACKNOWLEDGMENTS
 
This research was supported by TUBITAK (Project No.: 155 O 044) and Ege University Scientific Research Projects (project no.: 2005 ZRF 039). Veerle Bruggeman is a postdoctoral fellow of the F.W.O.-Vlaanderen (Belgium).

Received for publication October 23, 2007. Accepted for publication March 1, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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