Thermal manipulations of turkey embryos: The effect on thermoregulation and development during embryogenesis 1

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1 Thermal manipulations of turkey embryos: The effect on thermoregulation and development during embryogenesis 1 Y. Piestun, 2 I. Zimmerman,, and S. Yahav Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel; and Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel ABSTRACT Previous studies conducted on meat-type chickens in our laboratory showed that thermal manipulations (TMs) of the embryo during the time window of the hypothalamus hypophysis thyroid axis development and maturation significantly reduced the metabolic rates of the embryo and the chicken, improving the posthatch feed conversion rate (FCR). The aim of the present study was to investigate the effect of intermittent TMs during turkey embryogenesis on embryo development. Fertile turkey eggs were divided into three treatments: control; 6H with TM by elevation of temperature and RH by 1.7 C and 9%, respectively, above the control conditions for 6 h/d, from E10 through E22, i.e., 240 through 552 h of incubation; and 12H with TM as above, for 12 h/d, during the same time period. From E0 through E10 and from E23 onward all eggs were incubated under control conditions. The embryo growth rate was not negatively affected by TM. During TM eggshell temperature, the embryonic heart rate and oxygen consumption were elevated by the manipulation while the embryos were in their ectothermic phase. However, by the end of the TM period and until hatch (the endothermic phase) these parameters were significantly lower in both TM treatments than in the control, indicating a lower metabolic rate and heat production. The TM embryos hatched approximately 10 h earlier than the controls, without any negative effects on chick body weight or hatchability. Nevertheless, TM treatments resulted in a higher proportion of chicks with unhealed navels. Body temperature at hatch was significantly lower in the TM chicks than in the controls, suggesting lower heat production and metabolic rate, which might affect the energy requirements for posthatch maintenance. It was concluded that TM during turkey embryogenesis might have altered the thermoregulatory set point, and thus lowered the embryo metabolic rate, which might have a long-lasting posthatch effect. Key words: Turkey, thermal manipulation, incubation, embryo metabolic rate 2015 Poultry Science 94: INTRODUCTION Recent decades have been characterized by the development of genetic selection for improved growth rate in meat-type turkeys (Havenstein et al., 2007). However, the turkey s growth period is still relatively long, with a relatively high feed conversion rate (FCR). Genetic selection programs, as well as nutrition, have improved FCR in broiler chickens by more than 30% since the 1960s (Havenstein et al., 2003), but in turkeys the improvement was by only approximately 20% (Havenstein et al., 2007). Improvement of FCR in turkeys is of great economic importance; females and males are raised for up to 14 and 20 weeks, respectively, and their FCR during the final period of growth is above 4.0 in both C 2015 Poultry Science Association Inc. Received August 20, Accepted October 9, Contribution no. 689/14 from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel. 2 Corresponding author: yogev.p@mail.huji.ac.il males and females. The ability of domesticated poultry to utilize feed is of great interest because it has a large economic effect. Therefore, improving profitability by reducing feed consumption with no effect on performance is of great interest. Incubation air temperatures had been held relatively constant for many decades to eliminate the possible negative effects of temperature changes on embryo development, hatchability, chick quality, and growth performance (Krausova and Peterka, 2007). This contrasts with the incubation process in nature, where incubation temperatures have been found to fluctuate widely (Webb, 1987), and studies demonstrated that cautiously altering incubation temperature during critical phases during embryogenesis might induce favorable changes in the metabolic rate of chickens (Minne and Decuypere,1984). More recent studies have demonstrated a long-lasting effect of intermittent thermal manipulation (TM), e.g., elevation of incubation temperature by 1.7 o C, when applied for 12 h/d during a critical phase of embryogenesis that coincided with 273

2 274 PIESTUN ET AL. the development and maturation of the thyroid and adrenal axis (Piestun et al., 2008a,b, 2013); it improved thermotolerance acquisition by broilers during acute posthatch heat stress (Piestun et al., 2008a,b), with no negative effects on live performance. Furthermore, FCR was improved because of the lower metabolic rate in the embryo and posthatch, which resulted in reduced energy requirements for maintenance (Piestun et al., 2008b, 2011, 2013). The aim of the present study was to implement TM on turkey embryos, which may also have a long-lasting effect on growth performance. 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-237/10). A total of 450 fertile British United Turkeys (B.U.T) (Meleagris gallopavo) eggs with an average weight of 87.5 ± 7.5 g, were obtained from a single breeder flock. The eggs were weighed and divided into three incubation treatments, all with similar weight distributions and average weights: treatment 1 was the control, in which eggs were incubated under a single-stage procedure with temperature gradually falling from 37.8 Con the first day of incubation (E0) to 37.2 C at E27, and constant RH of 56% throughout the incubation period; treatment 2 designated as 6H intermittent thermal manipulation (TM), with temperature 1.7 Cabovethe control temperature and RH of 65% for 6 h/d from E10 through E22, i.e., incubation hours 240 through 552; and treatment 3 designated as 12H intermittent TM,withtemperatureandRHasintreatment2,but for 12 h/d during the same period. The eggs were incubated in two Type 65Hs automatic incubators (Masalles, Barcelona, Spain). From E0 through E10 and from E22 through E26 all eggs were incubated under control conditions, with turning once perhour(bruzualetal.,2000). At E10 the eggs were candled, infertile eggs and those containing early-dead embryos were removed, and eggs from the 6H and 12H treatments were transferred to the TM incubator, from which they were returned intermittently to the control incubators according to the treatment schedules. From E19 until hatch, 10 eggs per treatment were weighed and carefully opened, and the embryos were separated from the yolk sac, wiped, and weighed on a Type E154 analytical scale (Gibertini, Novate, Italy), accurate to ±0.1 mg, and embryo relative weights were expressed as percentages of egg weights. On E26 the eggs were transferred to hatching baskets. Although the experiment was terminated immediately after hatch, body weight (BW) and body temperature (T b )were measured, and blood samples were taken from the jugular vein of 10 randomly selected embryos at external pipping and of 10 chicks approximately 2 h after hatch (Yahav et al., 2004). Measurements and Analysis Generally, to prevent any changes that could have resulted from fluctuations in the environmental temperature, the incubators were kept in temperaturecontrolled chambers during all measurements. The measurements were conducted before and at the end of the thermal manipulation (TM), i.e., from 1 h before elevating or reducing the incubation temperature in the 6H and 12H treatments. Temperature Measurements From E0 onward, the shell temperature (T egg ) of 36 eggs per treatment was measured daily before and at the end of thermal manipulation (TM), with a ThermoScan Type 6022 infrared thermometer (Braun, Kronberg, Germany) (Leksrisompong et al., 2007), and the egg weight was recorded to monitor water loss. Oxygen Consumption To measure the oxygen consumption of the embryos during incubation, 5 eggs from each treatment were placed in a small cylindrical metabolic chamber measuring 15 cm in diameter and height, within a water container that maintained either the control temperature or the thermal manipulation (TM) temperature. From E17 onward, oxygen consumption was measured according to Buffenstein and Yahav (1991) before and at the end of the TM. 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 ml/min (Aalborg Instruments and Controls Inc., Orangeburg, NY). Dried air from the metabolic chamber was monitored for oxygen partial pressure with an Ametek (Pittsburgh, PA) S-3A/I oxygen analyzer. Heart Rate From E12 onward, the heart rate of 12 embryos from each treatment was measured with a Buddy digital embryo heart rate monitor (Avitronics, Torquay, UK). Blood Analysis Radioimmunoassays of thyroxin (T 4 ) and triiodothyronine (T 3 ) were applied to plasma samples with commercial radioimmunoassay kits (Diagnostic Products Corporation DCP, Los Angeles, CA). The intra-assay and interassay variations (CV) ofthet 3 assay were 7.0 and 9.4%, respectively, and those of the T 4 assay were 5.0 and 7.5%, respectively.

3 THERMAL MANIPULATIONS EFFECTS ON TURKEY EMBRYOS 275 Figure 1. Egg shell temperature of turkey embryos (n = 36) incubated under control conditions or thermal manipulation (TM) from E10 through E22, for 6 (6H) or 12h/d (12H). Within each day of incubation, different superscripts indicate significant differences (P 0.05) among treatments. Statistical Analysis The data were subjected to one-way ANOVA and to the all-pairs Tukey Kramer HSD test using JMP software (SAS Institute, Cary, NC). Means were considered significantly different at P Egg Weight RESULTS Egg weights decreased continuously by approximately 0.4 to 0.6 g/d from the initial egg weight at the beginning of incubation, and by E26 they reached 89.01, 89.07, and 89.26% of the initial weights in the control, 6H, and 12H treatments, respectively. There were no significant differences between treatments in egg weight loss during incubation (data not shown). Embryo Temperature, Heart Rate, and Oxygen Consumption Egg shell temperatures were similar to the incubator temperature until E10 (Figure 1). The T egg of the control remained similar to that of the incubator from E10 through E14 and rose progressively above the incubator temperature from E15 onward. T egg in the thermal manipulation (TM) treatments fluctuated between the control and the elevated temperature. During TM, T egg levels in treatments 6H and 12H were significantly higher than in the control, but from E23 onward T egg of 6H- and 12H-treated embryos was significantly lower than that of the controls; at external pipping it was significantly lower than that of the control by approximately 0.7 o C, and the body temperature of the hatched TM chicks was significantly lower than that of the control chicks (Table 1). Heart rate was measurable only from E12 onward because of technical limitations. As illustrated in Figure 2, the heart rate of the control embryos was stable at an average of 240 to 250 beats per minute (bpm) until E24, when a significant increase to 260 bpm was monitored prior to and during internal pipping. During TM the heart rate of TM-treated embryos was significantly higher than that of the controls, but after the treatment was terminated (from E23 onward), it was significantly lower than that of the controls. Oxygen consumption was detectable from E17 onward (Figure 3). In the control embryos, oxygen consumption gradually increased as the embryos grew and developed, until E23. It then remained on a plateau until E25, after which it increased dramatically until hatch. The TM-treated embryos exhibited significantly higher oxygen consumption during the growth period until the plateau phase, which started 2 d before that in the control. The duration of the plateau was longer

4 276 PIESTUN ET AL. Table 1. Body temperature (n = 120), body weight (n = 120), and plasma thyroid hormone concentrations (n = 10) of embryos and chicks incubated under control conditions or thermal manipulation (TM) from E10 through E22, for 6 (6H) or 12 h/d (12H). Body temperature ( o C) Plasma thyroid hormone concentration (ng/ml) Body weight (g) External pipping Hatch T 4 T 3 Hatch (Egg shell temperature) External Hatch External Hatch pipping pipping Control ± 0.06 a ± 0.06 a 11.7 ± 1.22 a 7.38 ± 0.83 a 3.71 ± 0.39 a 3.37 ± ± H ± 0.08 b ± 0.05 b 7.18 ± 0.82 b 5.22 ± 0.59 b 3.36 ± 0.21 b 3.28 ± ± H ± 0.06 b 39.1 ± 0.05 b 7.56 ± 0.75 b 5.15 ± 0.61 b 2.37 ± 0.26 b 2.95 ± ± 0.32 In each column, different superscripts indicate significant differences (P 0.05) among treatments. Figure 2. Heart rate of turkey embryos (n = 12) incubated under control conditions or thermal manipulation (TM) from E10 through E22, for 6 (6H) or 12 h/d (12H). Within each day of incubation, different superscripts indicate significant differences (P 0.05) among treatments. for the TM-treated embryos (from E21 through E25) and significantly lower than that in the control. This was followed by significantly lower levels during internal and external pipping, during which significant increases in oxygen consumption were observed in all treatments. Hormonal Analysis Thermal manipulation (TM)-treated embryos exhibited significantly lower plasma thyroid hormone concentrations than controls during pipping and at hatch (Table 1). Plasma T 4 concentrations were significantly lower in the TM-treated embryos than in the controls, with no differences between the TM treatments. Plasma T 3 concentration of the TM-treated embryos was lower to significantly lower than that of the controls. Embryo Development Figure 4 illustrates the effect of thermal manipulation (TM) on the embryo relative weight, which in general increased with time. The TM treatment had no negative effect on the embryo relative weight and during embryonic days 23 through 26 was significantly higher for the 12H embryos. No effect was found on the relative weights of the shell, yolk sac, breast muscle, liver, heart, or pipping muscle (data not shown). Onset of hatch was similar for all treatments. Nevertheless, hatching of the TM chicks was faster than that of controls, with 50% of hatch being 10 and 12 h earlier in the 6H and 12H treatments, respectively. No significant difference in hatchability was found between the treatments; it was 85, 92, and 90% for the control, 6H, and 12H chicks, respectively. Chick body weight was not affected by TM, but TM had a negative effect on chick quality rough-navel rates in the TM chicks

5 THERMAL MANIPULATIONS EFFECTS ON TURKEY EMBRYOS 277 Figure 3. Oxygen consumption of turkey embryos (n = 5) incubated under control conditions or thermal manipulation (TM) for 6 (6H) or 12 h/d (12H) from E10 through E22. Within each day of incubation, different superscripts indicate significant differences (P 0.05) among treatments. Figure 4. Embryo weight as percentage of egg weight (n = 10) during incubation under control conditions or thermal manipulation (TM) from E10 through E22, for 6 (6H) or 12 h/d (12H). Within each day of incubation, different superscripts indicate significant differences (P 0.05) among treatments. were 6 and 11% higher than controls in 6H and 12H, respectively. General DISCUSSION It is well known that exposing eggs to high temperature may result in teratogenic effects, i.e., abnormal morphological development and malfunction of physiological systems such as heart and liver (Thinh et al., 1977; Primmett et al., 1988; Buckiova et al., 1998), early onset of hatch (Leksrisompong et al., 2007), and reduced chick quality (Piestun et al., 2009). To avoid deleterious effects, conducting thermal manipulation (TM) during incubation requires consideration of 3 major aspects: critical phase, level, and duration. The time window in which to apply TM during embryogenesis should be chosen to match the specific physiological system to be affected. In the present study, the TM was designed

6 278 PIESTUN ET AL. to alter the energy-balance axis in light of the findings of Piestun et al. (2009). Changes in incubation temperature must avoid negative effects on embryo development or chick quality, and the duration of TM should be long enough to stimulate an effect on thermoregulation. A preliminary study was conducted to adapt the TM employed for broiler embryos (Piestun et al., 2009, 2013) to suit turkey embryos; it included characterization of metabolic parameters oxygen consumption, eggshell temperature, heart rate, and plasma thyroid hormone concentrations throughout the turkey s embryogenesis. The results indicated that development of the energy-balance axis and heat production in the turkey embryo were approximately 33% slower than those in the broiler embryo. The TM time window for broilers was from E7 through E16 (Piestun et al., 2008); therefore, the TM for turkeys was set as E10 through E22. Temperature elevation during this period was similar to that employed previously for broilers, but the duration was set at 6 or 12 h/d, in light of the higher sensitivity of turkey embryos to elevated temperature and their higher egg weight than that of broilers, which results in higher heat accumulation in the egg. Also, TM was coupled with an increase in RH to 65% to avoid excessive water loss from the eggs. In fact, no significant differences in egg weight loss were observed among the 3 treatments, during incubation up to E26 (internal pipping). Nevertheless, the RH elevation caused a slight decline in the partial pressure of oxygen in the incubator, but by only 7.1 mm Hg, which was not considered to represent a hypoxic environment for the embryos (Chan and Burggren, 2005). Embryo Metabolic Rate The thermal manipulations (TMs) caused elevated oxygen consumption in both TM treatments, which suggests that the metabolic rate increased in parallel with the elevation of incubation temperature, as expected in an ectothermic organism. This is 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, the plateau phase of the TM embryos started earlier and lasted longer than in the control, suggesting that these embryos developed rapidly and reached the period within incubation during which the capacity of the chorioalantoic membrane to supply oxygen reaches its limit (O Dea et al., 2004). The occurrence of the enlarged plateau in parallel with lower levels of oxygen consumption during the endothermic phase of TM embryos indicates a reduction in metabolic rate, similar to previous findings in broilers (Piestun et al., 2009). In the present study, the reduction in the metabolic rate was also confirmed by monitoring eggshell temperature, heart rate, and plasma thyroid hormone concentrations. Eggshell temperature is widely known to be an accurate indication of the embryo T b (Leksrisompong et al., 2007), which in turn reflects embryo heat production (Van Brecht et al., 2003). The chicken embryo produces heat at a rate that increases with the progress of embryogenesis, so that at a certain point T egg is elevated above the incubation temperature because heat production exceeds heat loss (Tazawa and Whittow, 2000). In the present study, in all 3 treatments, T egg was approximately equal to the incubation temperature until E14, after which it increased daily as embryo heat production increased. The T egg in the TM treatments rose above that in the controls and fluctuated according to the treatment duration. Interestingly, after the TM ended, at E22, T egg in both TM treatments was lower than that in the control, despite exposure to the same incubation temperature, which suggests an ectothermic response by the embryo (Van Brecht et al., 2005) through E22, followed by an endothermic response as the end of embryogenesis approached (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, as indicated by the lower cloacal temperature at hatch. Reduced heat production by the hatched TM chicks was previously demonstrated by Yahav et al. (2004) and Piestun et al. (2008a,b, 2009). In contrast to the increased eggshell temperature during the second half of incubation, which coincided with the increase in oxygen consumption, the heart rate of the control embryos tended to decrease gradually with increasing embryonic age, indicating higher cardiac output (Mulder et al., 1998). In the present study, both TM treatments elicited an elevation of heart rate, suggesting that incubation temperature strongly affected heart rate, which is consistent with the embryo s ectothermic status. By the end of the TM treatment, the heart rates of both TM-treated embryos declined below that of the control, highlighting the lower metabolic rate of the TM embryos. This may be compensated by increased cardiac output; previous studies showed an increased stroke volume for heat-acclimated hearts because of changes in the intrinsic properties of the left ventricle (Horowitz et al., 1986). Plasma T 4 and T 3 concentrations increased sharply during internal and external pipping (Mallon and Betz, 1982; Decuypere and Michels, 1992) and then decreased after hatch, suggesting that the metabolic rate decreased as the chick completed this harsh process (Tona et al., 2004). In the present study, TM caused significantly lower plasma concentrations of both T 4 and T 3 than those in the control at pipping and hatch. Kühn et al. (1984) found that changes in the thyroid hormone levels that resulted from changes in ambient temperature were related to a balance between central and peripheral control mechanisms. This reduction in circulating thyroid hormones could have been attributed to a) a change in the hypothalamic pituitary threshold level to secrete less thyroid-stimulating

7 THERMAL MANIPULATIONS EFFECTS ON TURKEY EMBRYOS 279 hormone; b) reduced thyroid gland activity, which would reduce secretion of T 4 ; or c) decreased peripheral deiodination, which would reduce T 3 production. In light of the lower plasma T 4 levels of the TM embryos, it seems that the activity of the thyroid gland was reduced to some extent. Moreover, at pipping, T 3 increased more sharply than T 4, whereas reverse T 3 decreased (Kühn et al., 1984). However, production of the latter changed according to ambient temperature, and it can be speculated that the lower concentrations of T 3 in the TM embryos resulted also from alteration in the biochemical pathway of T 4, which suggests increased deiodination of T 4 into reverse T 3 rather than T 3.In conclusion, it may be suggested that TM resulted in a lower thyroid function set point (Piestun et al., 2009); however, this issue remains to be elucidated. Embryo Development Embryo development can be expressed in terms of yolk-free embryo weight (Hill, 2001). It had previously been reported that the elevation of incubation temperature at a very early stage accelerated growth and development (Romanoff, 1960; Ricklefs, 1987) and had a negative effect on body weight (BW) at hatch,and other studies also reported accelerated development and lower BW at hatch as a result of elevated incubation temperature (Webb, 1987; Gladys et al., 2000; Leksrisompong et al., 2007). The results of the present study showed that thermal manipulation (TM) had no negative effect on embryo growth but had a positive effect on the embryo relative weight at various days from E23 till hatch. Elevating the incubation temperature can also impair chick quality, possibly manifested in short down, generally abnormal and unhealthy appearance possibly due to poor absorption of the yolk sac pigments, and unhealed navels (Leksrisompong et al., 2007). In the present study no negative effects of TM on chick BW and general appearance were observed, indicating that intermittent TM allows embryos to dissipate excessive heat and to avoid teratogenic consequences (Piestun et al., 2009); nevertheless, the proportion of chicks with unhealed navels at hatch increased with TM duration (6H and 12H), suggesting the necessity of fine-tuning TM duration. Previous studies on broilers reported a longlasting effect of TM in reducing energy demands for maintenance and thereby improving feed conversion rate (FCR) (Piestun et al., 2009, 2011, 2013). The present study demonstrated an effect of TM on the metabolic rate of turkey embryos, which might affect energy requirements for posthatch maintenance. It can be concluded that intermittent TM reduced the metabolic rate in turkey embryos during development and at hatch. This might have a long-lasting effect, but it remains to be elucidated. 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