Thermal manipulations of broiler embryos—The effect on thermoregulation and development during embryogenesis1

ABSTRACT

This study aimed to elucidate the effects of thermal manipulations (TM) of broiler embryos, during the development of the thyroid and adrenal axis, on embryo development and metabolism. Cobb eggs were divided into 3 treatments: control, 24H–continuous TM at 39.5°C and 65% RH from embryonic day 7 to 16 inclusive, and 12H–intermittent TM for 12 h/d in the same period. Only the 24H treatment negatively affected embryo growth and development, with lower relative weights of embryo, liver, and pipping muscle. During TM, eggshell temperature, heart rate, and oxygen consumption were elevated as embryos were in their ectothermic phase, but from the end of the TM until hatch, these parameters were significantly lower in both treatments than in the control. Moreover, plasma concentrations of the thyroid hormones were significantly lower in the 2 treatments during and after TM, until hatch. Plasma corticosterone concentration of the TM-treated embryos was significantly lower after the TM but significantly higher at hatch. It was concluded that TM during the development of the thyroid and adrenal axis lowered their functional set point, thus lowering metabolic rate during embryogenesis and at hatch.

thermal manipulation, incubation, oxygen consumption, eggshell temperature, embryonic growth

INTRODUCTION

Recent decades were characterized by the development of genetic selection for improved growth rate in meat-type broilers (Havenstein et al., 2003), resulting in 50- to 60-fold increases in BW between hatch and marketing. However, no parallel improvement of the physiological systems that support energy balance (e.g., the cardiovascular and respiratory systems) was established. Increased metabolic heat production that results from the improved growth rate, coupled with the high ambient temperatures that characterize wide areas of the world, lead to serious difficulties in coping with heat, which result in high economic losses from increased morbidity and mortality and decreased quantity and quality of meat production (Sandercock et al., 2001; St-Pierre et al., 2003).

Thermal tolerance in broilers can be obtained by 3 direct responses (Yahav, 2000), one of which is epigenetic temperature adaptation, defined as changes in the regulation of gene activity and expression that are not dependent on gene sequence (National Institutes of Health) and that can occur during early pre- or postnatal ontogeny and may lead to long-lasting physiological memory (Nichelmann and Tzschentke, 2002). In broilers, epigenetic temperature adaptation was found to improve thermotolerance acquisition during prenatal (Piestun et al., 2008a,b) or postnatal ontogeny (Yahav and Hurwitz, 1996): temperature manipulations were used during a critical period when the functional system of temperature regulation develops from a regulation system without negative feedback (prenatal) into a closed control system with a feedback mechanism (postnatal; Loh et al., 2004).

The improvement of thermotolerance acquisition during embryogenesis was based on the hypothesis that thermal manipulation would be most efficient during the critical time window of the hypothalamus-hypophysis-thyroid or adrenal axis, or both, development (Piestun et al., 2008a). Indeed, this has been proved by thermally challenging broilers that had been thermally manipulated during embryogenesis, at marketing age, and evaluating their improvement in heat resistance (Piestun et al., 2008b). However, the nature of the physiological and endocrinological changes that occur in the embryo as a result of the thermal manipulation procedures has not been clarified.

The present study aimed to elucidate the effects of thermal manipulations during the critical phase of the thyroid and adrenal axis development on embryo development with regard to temperature, oxygen consumption, heart rate, plasma thyroid hormones, and corticosterone profile, all of which may shed light on the posthatch acquisition of improved thermotolerance.

MATERIALS AND METHODS

Experimental Design

All procedures in this study were carried out in accordance with the accepted ethical and welfare standards of the Israeli Ethics Committee (IL-005/05). A total of 540 fertile Cobb strain broiler (Gallus domesticus) eggs with an average weight of 62.5 ± 2.5 g were obtained from 1 breeder flock of hens during their period of optimal egg production (38 wk of age). The eggs were randomly divided into 3 incubation treatments: treatment 1, control in which eggs were incubated under 37.8°C and 56% RH throughout the incubation period; treatment 2 (designated as 24H), continuous thermal manipulation (TM) at 39.5°C and 65% RH from embryonic day (E)7 to E16 inclusive (180 to 408 h of incubation; the first day of the incubation was designated as E0); and treatment 3 (designated as 12H), an intermittent TM (39.5°C and 65% RH) for 12 h/d during the same period.

The eggs were incubated in 2 Type 65Hs automatic incubators (Masalles, Barcelona, Spain). From E0 to E7 and from E17 to E18 (all days inclusive), all eggs were incubated in the control incubator under regular conditions of 37.8°C, 56% RH, and turning once per hour (Bruzual et al., 2000). At E7, the eggs were candled, infertile eggs and those containing early dead embryos were removed, and eggs from the 2 TM treatments were transferred to the TM incubator. The 24H eggs were kept in this incubator until E16 (inclusive), whereas those of the 12H treatment were transferred between the TM and the control incubators every 12 h.

From the start of the second half of the incubation period (E11), each day until hatch, 10 eggs per treatment were weighed, carefully opened, and a blood sample was drawn from the allantoic veins. The embryos were separated from the yolk sack, wiped, and weighed on a Type E154 analytical scale with an accuracy of ±0.1 mg (Gibertini, Novate, Italy). Embryo relative weight was expressed as a percentage of egg weight. The liver and the pipping muscle (musculus complexus) were removed and weighed, each day from E16 onward, and expressed as a relative weight of embryo dry weight. On E18, the eggs were transferred into hatching baskets. Day E19 includes embryos at their internal pipping. Although the experiment was terminated immediately after hatch, BW and body temperature were measured and blood samples were taken from the brachial vein of 10 randomly selected chicks approximately 2 h after hatch (Yahav et al., 2004).

Measurements and Analysis—Temperature Measurements

In general, to prevent any changes that could have resulted from fluctuations in environmental temperature, the incubators were kept in temperature- and RH-controlled rooms during the measurements. From E0 onward, 15 eggs per treatment were taken, eggshell temperature (T_egg) was measured every 12 h with a ThermoScan type 6022 infrared thermometer (Braun, Kronberg, Germany; Leksrisompong et al., 2007), and egg weight was recorded daily to monitor water loss.

Oxygen Consumption

To measure oxygen consumption of the embryos during incubation, every 12 h from E7 onward, eggs (n = 5) from each treatment were placed in a small cylindrical metabolic chamber that measured 7 cm in diameter and height, which was placed in a water container that maintained either the control temperature (37.8°C) or the TM temperature (39.5°C). Oxygen consumption was measured according to Buffenstein and Yahav (1991). Briefly, dried air was pumped into the metabolic chamber at a flow rate of 50 mL/min, using a flow meter with a range of 0 to 60.56 mL/min (Aalborg Instruments and Controls Inc., Orangeburg, NY). Dried air from the metabolic chamber was monitored for oxygen partial pressure with an S-3A/I oxygen analyzer (Ametek, Pittsburgh, PA).

Heart Rate

From E7 onward, the heart rates of 15 embryos from each treatment were measured with a Buddy digital embryo heart rate monitor (Avitronics, Torquay, UK) every 12 h (i.e., from 1 h before the start of the temperature alternations in the 12H treatment).

Blood Analysis

Radioimmunoassays of thyroxin (T₄) and triiodothyronine (T₃) were applied to plasma samples with commercial RIA kits (Diagnostic Products Corporation, Los Angeles, CA; Yahav et al., 2004). The intraassay and interassay variations (CV) of the T₃ assay were 7.0 and 9.4%, respectively, and those of the T₄ assay were 5.0 and 7.5%, respectively. Plasma corticosterone concentrations were measured with the RIA kit with the ImmuChem Double Antibody (ICN Biomedical Inc., Diagnostics Division, Orangeburg, NY; Yahav et al., 2004).

Statistical Analysis

The data were subjected to 1-way ANOVA and to the all-pairs Tukey-Kramer honestly significant difference test, by means of the JMP software (SAS Institute, Cary, NC). Means were considered significantly different at P ≤ 0.05.

RESULTS

Egg Weight

Egg weights decreased continuously by approximately 0.2 to 0.3 g/d from the beginning of incubation, and by E19 they had fallen to 92.07, 92.67, and 92.66% of the initial weights in the control, 12H, and 24H treatments, respectively. There were no significant differences between treatments in egg weight loss during incubation (data not shown).

Temperature, Heart Rate, and Oxygen Consumption of the Embryo

Eggshell temperatures were similar to the incubator temperature until E7 (Figure 1). From E7 to E10, T_egg of the control remained similar to the incubator temperature, whereas that of 24H was similar to the elevated incubation temperature. From E10 onward, T_egg of the control and 24H rose progressively above the incubator temperature, whereas that in 12H fluctuated between the control and the elevated temperature, being similar every 12 h to the T_egg of the control and the 24H treatment, respectively. During TM, the T_egg levels of 12H and 24H were significantly higher than that of the control by approximately 1.5°C. From E18 onward, T_egg of the 12H- and 24H-treated embryos was lower to significantly lower than that of the controls. In addition, body temperature at hatch of both temperature treatment chicks was significantly lower than that of the control chicks by approximately 0.5°C (data not shown).

Figure 1

Eggshell temperature of broiler embryos (n = 15) incubated under control conditions (37.8°C, 56% RH) or thermally manipulated conditions (39.5°C, 65% RH) from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

Heart rate was measurable only from E14.5 onward. As illustrated in Figure 2, the heart rate of the control embryos was stable at an average of 283 to 290 beats per minute until E18, when a significant increase to 330 beats per minute was monitored before and during internal and external pipping. During the TM period, the heart rate of the 24H-treated embryos was significantly higher than that of the controls, but after the termination of TM and from E18 onward, it was significantly lower than that of the controls. The 12H-treated embryos exhibited a similar pattern to that of the 24H ones, but it was less pronounced.

Figure 2

Heart rate of broiler embryos (n = 15) incubated under control (37.8°C, 56% RH) or thermally manipulated (39.5°C, 65% RH) conditions from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

Oxygen consumption was detectable from E10 onward (Figure 3). In the control embryos, oxygen consumption gradually increased as the embryos grew and developed, until E17; it then remained on a plateau until E19, after which it increased dramatically until hatch. The 12H-treated embryos exhibited a similar pattern of oxygen consumption to that of the 24H ones, with fluctuations that followed the TM pattern. They also exhibited a consumption plateau from E17 to E19, but at a significantly lower level than that of the control. The 24H-treated embryos exhibited higher oxygen consumption than the controls until E15, after which it was similar to that of the controls until the end of the TM period. This was followed by significantly lower levels during the plateau period until pipping, during which a significant increase in oxygen consumption was observed.

Figure 3

Oxygen consumption of broiler embryos (n = 5) incubated under control (37.8°C, 56% RH) or thermally manipulated (39.5°C, 65% RH) conditions from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

Hormonal Analysis

Plasma T₄ concentration increased in all 3 treatments as the embryos developed (Figure 4). In the control embryos, it increased dramatically from E17 to E18, after which there was a more moderate increase until pipping, followed by a significant decrease after hatch. During most of the period of monitoring (E13 onward), plasma T₄ concentration was significantly lower in the TM-treated embryos than in the controls, with no differences between the TM treatments. Moreover, after hatch, the plasma T₄ concentration was significantly lower in the 24H than in the 12H treatment, and both were significantly lower than that in the controls.

Figure 4

Plasma thyroxin (T₄) concentration of broiler embryos (n = 10) incubated under control (37.8°C, 56% RH) or thermally manipulated (39.5°C, 65% RH) conditions from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

Plasma T₃ concentration increased with age (up to E18) in all 3 treatments (Figure 5). Before hatch, from E18 until pipping, it increased dramatically, and after hatch it declined in all 3 treatments. Throughout the period of measurement, the plasma T₃ concentration of the TM-treated embryos was lower to significantly lower than that of the controls. At posthatch, it was significantly lower in the treated chicks than in the controls (data not shown).

Figure 5

Plasma triiodothyronine (T₃) concentration of broiler embryos (n = 10) incubated under control (37.8°C, 56% RH) or thermally manipulated (39.5°C, 65% RH) conditions from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Small figure illustrates E13 to E19 in a different scale. Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

The plasma corticosterone concentration in the controls did not change significantly until E19, when a dramatic increase was observed, then followed by a significant decline at hatch. After the TM period, the plasma corticosterone concentration of the TM-treated embryos was significantly lower than that of the controls and from E19 onward, it increased and was significantly higher in the TM groups than in the control at hatch (Figure 6).

Figure 6

Plasma corticosterone concentration of broiler embryos (n = 10) incubated under control (37.8°C, 56% RH) or thermally manipulated (39.5°C, 65% RH) conditions from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

Embryo Development

Figure 7 illustrates the effect of TM on the embryo relative weight, which in general increased with time. Continuous TM (24H) negatively affected the embryo relative weight, which was clearly demonstrated during the second half of embryogenesis. In contrast, intermitted TM (12H) elicited a positive response, with the relative weight of the embryos being significantly higher than that of the controls on E11, E12, E14, and E16. The 24H treatment significantly depressed liver relative weights on E14, E15, and E16; they were 2.0, 2.05, and 2.17%, respectively, in the control and 1.9, 1.93, and 2.04%, respectively, in the 24H treatment. This pattern was found before hatch also, on E18 and E19, when the liver relative weights were 2.19 and 2%, respectively, in the controls and 2.06 and 1.91%, respectively, in the 24H treatment.

Figure 7

Embryo weight as a percentage of egg weight (n = 10) incubated under control (37.8°C, 56% RH) or thermally manipulated conditions (39.5°C, 65% RH) from embryonic day (E)7 to E16 inclusive (180 until 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment). Within each day of incubation, different letters indicate significant differences (P ≤ 0.05) among treatments.

In addition, the 24H treatment depressed the pipping muscle growth on E18 and E19, and its weight and relative weight were significantly lower compared with the control and 12H groups (Table 1). In the 12H treatment, no negative effect was found on the relative weights of either the liver or the pipping muscle. Hatched chicks of the 24H group were characterized by a whiteish, faded color, short down, and a significantly large number of chicks with a nonabsorbed yolk sac and rough navel.

Pipping muscle weight and pipping muscle relative weight (percentage of embryo weight) in chick embryos incubated under control conditions (37.8°C, 56% RH) or thermally manipulated conditions (39.5°C, 65% RH) from embryonic day (E)7 to E16 inclusive (180 to 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment)

Table 1

Pipping muscle weight and pipping muscle relative weight (percentage of embryo weight) in chick embryos incubated under control conditions (37.8°C, 56% RH) or thermally manipulated conditions (39.5°C, 65% RH) from embryonic day (E)7 to E16 inclusive (180 to 408 h of incubation), continuously (24H treatment) or intermittently (12H treatment)

Embryonic day	Pipping muscle weight (g)				Pipping muscle relative weight (%)
	Control	12H	24H		Control	12H	24H
E16	0.12 ± 0.01a	0.13 ± 0.01a	0.12 ± 0.01a		0.62 ± 0.02a	0.61 ± 0.02a	0.67 ± 0.03a
E17	0.17 ± 0.01a	0.19 ± 0.01a	0.19 ± 0.02a		0.71 ± 0.02a	0.76 ± 0.03a	0.83 ± 0.04a
E18	0.31 ± 0.01a	0.34 ± 0.01a	0.28 ± 0.01b		1.09 ± 0.02a	1.14 ± 0.03a	0.91 ± 0.04b
E19	0.54 ± 0.03a	0.54 ± 0.03a	0.4 ± 0.05b		1.24 ± 0.06a	1.2 ± 0.12a	0.94 ± 0.08b
External pipping	0.70 ± 0.03a	0.69 ± 0.07a	0.68 ± 0.05a		1.39 ± 0.07a	1.34 ± 0.08a	1.41 ± 0.08a
Hatch	0.54 ± 0.03b	0.53 ± 0.02a	0.55 ± 0.03a		1.17 ± 0.06a	1.14 ± 0.04a	1.24 ± 0.07a

^a,bIn each row, different superscript letters designate significant difference (P ≤ 0.05) among treatments. n = 10.

DISCUSSION

General

This study demonstrated for the first time some of the physiological and endocrinological changes that occur in broiler embryos as a result of elevating incubation temperature during the development of the thyroid (metabolism) and adrenal (stress) axis. The application of TM from E7 to E16 inclusive was selected after Piestun et al. (2008a) demonstrated its long-lasting improvement of thermotolerance acquisition in broilers.

The use of elevated incubation temperature was coupled with the increase of RH to 65%, to avoid an excessive water loss from the eggs. In fact, no significant differences were observed among egg weights from preincubation up to E19 (internal pipping) in any of the 3 treatments. Nevertheless, the RH elevation caused a slight decline in the partial pressure of oxygen in the incubator, but by only 7.1 mmHg, which was not considered to represent a hypoxic environment for the embryos (Chan and Burggren, 2005).

T_egg

Eggshell temperature is well known to be an accurate measure for the embryo body temperature (Leksrisompong et al., 2007). The temperature of the embryo is considered to be affected by air temperature and the exchange of heat between the egg and its microenvironment (Van Brecht et al., 2005), which is dependent on the heat production by the embryo and the heat loss, which in commercial incubators is mainly by convection (Van Brecht et al., 2003). The fowl embryo produces heat at a rate that increases with embryogenesis so that, at a certain point, the T_egg is raised above the incubation temperature because heat production exceeds heat loss (Whittow and Tazawa, 2000). In the present study, in all 3 treatments, the T_egg was approximately equal to the incubation temperature until E10, after which it increased daily as the embryo heat production increased. The T_egg in the intermittent TM treatment (12H) rose to that of the 24H treatment and fell to that of the control in parallel with the TM fluctuations. In addition, 12 h after the TM ended (E17), the T_egg in both TM treatments were higher than that of the control. However, from E18 onward, T_egg in both TM treatments fell below that of the controls despite exposure to the same incubation temperature, which suggests an ectothermic response by the embryo (Van Brecht et al., 2005) until E18, followed by an endothermic response by the end of embryogenesis (Tazawa et al., 1988; Whittow and Tazawa, 1991; Janke et al., 2002). The lower T_egg clearly indicates a lower metabolic rate, with a consequent decrease in heat production. Reduced heat production by the hatched TM chicks was previously demonstrated by Yahav et al. (2004) and Piestun et al. (2008a,b). In the present study, lower metabolism was also confirmed by monitoring oxygen consumption, heart rate, and plasma concentrations of thyroid hormones.

Oxygen Consumption

In the present study, in agreement with findings of other authors (Prinzinger and Dietz, 1995; Dietz and Prinzinger, 1997; Whittow and Tazawa, 2000; Janke et al., 2002), oxygen consumption during embryogenesis increased in parallel with embryo growth and development until E17. It remains constant thereafter while the additional oxygen demand of the growing embryo is supplied by the increased oxygen-carrying capacity and oxygen affinity of the hemoglobin of the embryo (Tazawa, 1980), and stored glycogen, mainly from the hepatic source (Freeman, 1969), serves as an alternative energy source (Pearce, 1971; O’Dea et al., 2004). A sharp increase in oxygen consumption was observed during the period in which the chick embryo switched to lung respiration and started to consume atmospheric oxygen in preparation for hatch.

The TM caused elevated oxygen consumption in both treatments, which suggests that the metabolic rate increased in coincidence with the elevation in incubation temperature, as expected from an ectothermic organism and in accordance with Janke et al. (2002), who demonstrated that embryos in mid-incubation responded to increased incubation temperature by elevating their heat production. In the present study, upon termination of TM, the TM-treated embryos exhibited the same pattern of a plateau phase followed by a sharp increase in oxygen consumption before hatch, but with significantly lower levels than that in the control, indicating a reduction in their metabolic rate.

Heart Rate

In contrast to the increased T_egg during the second half of incubation, which coincided with the increases in oxygen consumption and plasma thyroid hormone concentrations, the heart rate of the control embryos did not increase, and any changes were barely observed in the present study or in others (Whittow and Tazawa, 2000). However, as internal pipping progressed and the chick embryo switched to lung respiration, the heart rate increased dramatically, in coincidence with the increases in oxygen consumption and plasma thyroid hormone levels, indicating a sharp increase in metabolic rate (Nichelmann et al., 1998; Whittow and Tazawa, 2000; Janke et al., 2002; Tona et al., 2004; Lu et al., 2007) that resulted in a further increase in T_egg. In the present study, both TM treatments resulted in elevation of heart rate, which suggests that incubation temperature strongly affected heart rate, which is consistent with the ectothermic status of the embryo. By the end of the TM treatment, the heart rates of both TM-treated embryos declined below that of the control, again suggesting a lower metabolic rate of the TM embryos.

Thyroid Hormones

The pattern of developmental changes in plasma thyroid hormone concentrations, in which T₃ increased moderately until E18, corroborates the findings of Kameda et al. (1986), Prati et al. (1992), and Lu et al. (2007). Triiodothyronine fills a major role during the critical time of hatching and is essential for the supplementation of additional energy demands during hatch. Indeed, a sharp increase of this hormone was observed in this study, during the internal and external pipping (Mallon and Betz, 1982; Decuypere et al., 1992). In the present study, after hatch, plasma T₃ declined (Figure 5), suggesting a lower rate of metabolism as the chick had completed the harsh process (Tona et al., 2004).

In parallel to the plasma T₃ concentration, plasma T₄ concentration increased significantly during mid-incubation, as demonstrated by Bellabarba et al. (1988) and Lu et al. (2007), followed by a sharp increase at E17 and reaching a peak during external pipping.

Although the same pattern of changes in plasma hormone concentrations was seen in both the TM and the control, TM caused significantly lower levels of both T₃ and T₄ plasma concentrations than in the control. Changes in the thyroid hormone levels as a result of changes in ambient temperature were found elsewhere to be related to a balance between central and peripheral control mechanisms (Kühn et al., 1984). This reduction in circulating thyroid hormones could have been attributed to the following: a) change in the hypothalamic-pituitary threshold level to secrete less thyroid-stimulating hormone; b) reduced thyroid gland activity, which would reduce the secretion of T₄; or c) decreased peripheral deiodination, which would reduce T₃ production. In light of the lower plasma T₄ levels of the TM embryos, it seems that the activity of the thyroid gland was reduced to some extent, indicating a lowering of the thyroid function set point; however, this issue remains to be elucidated.

At pipping, T₃ increases more sharply than T₄, whereas reverse T₃ decreases (Kühn et al., 1984), However, production of the latter increases with increasing ambient temperature. If these findings are taken together, it can be speculated that the lower concentrations of T₃ in TM treatments resulted also from alteration in the biochemical pathway of T₄, which suggests increased deiodination of T₄ into reverse T₃ rather than T₃. Moreover, a reduction in plasma T₃ concentration could be a result of lower plasma concentration of corticosterone, which modulates the conversion of T₄ to T₃ (Decuypere et al., 1983; Neeuwis et al., 1989). Indeed, in the present study, we showed that the plasma corticosterone levels of the TM embryos were significantly lower than those of the controls.

In the present study, TM caused increases in egg temperature, heart rate, and oxygen consumption but decreases in thyroid hormone concentrations. In ectotherms, metabolic rate and heat production appear to be regulated independently of the plasma levels of thyroid hormones (Lutterschmidt and Hutchison, 2003). For instance, in the African clawed frog, Xenopus laevis, thyroid hormones influenced the mean preferred ambient temperature while having no effect on the metabolic rate (Dupré et al., 1986). Because the chicken embryo exhibits ectothermic characteristics (Decuypere and Michels, 1992) and its metabolic rate is directly related to its temperature until late incubation (Freeman, 1971; Whittow and Tazawa, 1991), these parameters are governed by ambient temperature. The response of the thyroid hormones to TM can be explained by the fact that the thyroid hormonal secretion axis is being formed during this period, and its feedback mechanism has not yet matured. Only after E18, during the endothermic phase, did the axis respond according to physiological expectations.

Previous studies showed that lower levels of T₃ in the TM groups persist posthatch and during chick growth (our unpublished data) up to marketing age (Piestun et al., 2008b) and were involved in the acquisition of improved thermotolerance. Thus, it can be speculated that TM had altered the hypothalamic-pituitary-thyroid axis set point and thereby caused a reduction in heat production.

Corticosterone

In consistency with other studies, in the present study, the corticosterone plasma level increased dramatically during internal pipping (Tona et al., 2004) and declined at hatch (Moraes et al., 2004). In the present study, after TM, corticosterone levels of the TM-treated embryos were significantly lower than those of the controls, indicating a downward shift in the adrenal function during the last days of incubation. Interestingly, plasma concentrations of this hormone at hatch were significantly higher in the TM-treated chicks than in the controls, suggesting greater stress during hatch. Although no hatchability differences were observed between the intermittent TM group (12H) and the control, those in the 24H group had hatching difficulties that resulted in only 60% hatchability. This could be attributed to a less developed pipping muscle (musculus complexus) exhibited on days E18 and E19 (Table 1) because this muscle has a critical role in the hatching process (Christensen et al., 2001).

Although no significant differences in plasma corticosterone were observed between the chicks after hatch (data not shown), these levels were significantly lower in the TM treatments than in the controls after heat challenge (Piestun et al., 2008a,b). This can be attributed to lower stress that resulted from the better thermotolerance of the TM chickens.

Embryo Development

Embryo development can be expressed in terms of yolk-free embryo weight (Hill, 2001). It has been previously reported that elevation of incubation temperature at a very early stage accelerated growth and development (Romanoff, 1960; Ricklefs, 1987) and had a negative effect on BW at hatch. In the present study, as the embryo grew dramatically during the second half of embryogenesis it was clearly seen that continuous thermal manipulation (24H treatment) affected its growth negatively. This is consistent with other reports of accelerated development and lower BW at hatch as a result of elevated incubation temperature (Webb, 1987; Gladys et al., 2000; Leksrisompong et al., 2007). Previous studies demonstrated that heat manipulation during mid-incubation influenced organ weights in the developing embryo and at hatch (Yalcin et al., 2008). The results of the present study demonstrate that continuous TM (24H) depressed development of the liver and reduced its glycogen content (data not shown). Low liver glycogen content was associated with depressed embryo survival (Christensen et al., 1993, 1999) and with reduced chick weight at hatch and posthatch (Christensen et al., 2000). Indeed, the BW of the 24H chicks was significantly lower than that of the controls at hatch (Piestun et al., 2008a,b) and during growth up to marketing (Piestun et al., 2008b). The most remarkable characteristic of the hatching chicks from the 24H treatment was their faded whiteish color and short down. This is in agreement with the findings of Leksrisompong et al. (2007), who reported white-colored chicks with a generally abnormal and unhealthy appearance that might have been due to poor absorption of the yolk sack pigments, as a result of their reduced metabolic rate. In contrast to the continuous TM (24H), the intermittent TM (12H) did not affect embryo growth but had a positive effect on the embryo relative weight at various days during TM. This coincides with the findings of Yalcin et al. (2008), who reported that intermittent TM for 6 h per day between E10 and E18 did not have a deleterious effect on embryo growth and chick weight. Taken together, these findings indicate that continuous TM from E7 to E16 seems to be too severe to allow regular embryonic development, whereas intermittent TM allows embryos to dissipate excessive heat and to avoid teratogenic consequences.

Previous studies (Piestun et al. 2008a,b), reported a long-lasting effect of TM in terms of improved thermotolerance acquisition of the chicken posthatch (Piestun et al., 2008b) and during its lifespan (Piestun et al., 2008a). In continuation along this research route, the present study demonstrated that TM during the development of the thyroid and adrenal axis lowered their functional set point, thereby lowering the prehatch metabolic rate, which indicates the possibility of epigenetic thermal adaptation.

1

Contribution from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel. No. 539-09.

ACKNOWLEDGMENTS

This research was supported by research grant no. IS-3836-06R from the United States-Israel Binational Agricultural Research and Development Fund (BARD, Bet Dagan, Israel) and by grant no. 356-380 from the Egg and Poultry Board of Israel (Tel-Aviv). We thank D. Shinder, M. Ruzal, B. Gill, and P. Shudnovskey (Institute of Animal Science, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel) and S. Reicher and S. Fehl (Department of Animal Sciences, Faculty of Agriculture, the Hebrew University of Jerusalem, Rehovot, Israel) for technical assistance.