Thermal manipulation during embryogenesis has been shown to achieve improvement in thermotolerance up to 10 days of age. However, recent studies have shown that broiler chickens do not exhibit any thermoregulatory advantage during thermal challenge at later ages. These results raise the following questions: could it be that it is not possible to induce long-lasting thermoregulatory memory by thermal manipulation during embryogenesis? Could fine tuning of thermal manipulation result in long term improvement in thermotolerance? Further research is currently underway, aiming to shed light on these questions, and there is evidence that fine tuning might lead to achievement of the targeted goal.
Introduction
Recent decades have seen significant development in genetic selection of meat-type poultry (i.e., broilers and turkeys); this has led to rapid growth, with increased feed efficiency and metabolic rate, and has thus provided the poultry industry with large, rapidly growing birds (Havenstein et al., 2003). Such development necessitates parallel increases in the size and enhancement in the efficiency of functioning of the cardiovascular and respiratory systems. However, inferior development of such major systems has led to a relatively low capability to balance energy expenditure and body water balance under extreme environmental conditions. Thus, acute exposure of chickens to extreme conditions (i.e., hot or cold spells) has resulted in major economic losses (morbidity, mortality and development of metabolic disorders). It is estimated that the global mean surface temperature increased by 0.8 and 1.7°C during the 19th and 20th centuries respectively (U.S. National Climatic Center, 2001). Scientists expect that the average global surface temperature will rise by 0.6-2.5°C during the next 50 years. This situation, in which growth rate (accompanied by increased metabolic heat production) increases from year to year and the future projected increase in global surface temperature, demands an efficient means of economically improving thermotolerance in broiler chickens.
Birds are homeotherms and, therefore, are able to maintain their body temperature within a narrow range. An increase in body temperature above the regulated range, as a result of exposure to environmental conditions and/or excessive metabolic heat production, may lead to a cascade of irreversible thermoregulatory events that could be lethal for the bird. To sustain thermal tolerance and avoid the deleterious consequences of thermal stresses, three direct responses are elicited: the rapid thermal shock response (Parsell and Lindquist, 1994), acclimation (Horowitz, 2002; Yahav et al., 1997b), and thermal manipulations based on epigenetic adaptation during the perinatal period (Nichelmann et al., 1999; Tzschentke et al., 2001).
The last strategy is based on the assumption that environmental factors, especially the ambient temperature (Ta), have a strong influence on the determination of the “set-point” for physiological control systems during “critical developmental phases” (first described as ‘determination rule’; Dörner, 1974). Thermosensitive neurons located in the preoptic anterior hypothalamus (PO/AH) integrate afferent temperature signals from different areas of the body to elicit adequate thermoregulatory responses via the control of physiological, endocrinological, and behavioural responses, and so to keep the core body temperature (Tb) relatively constant (Boulant, 1996). The thermoregulatory response is mediated, to some extent, by the level of metabolism induced/permitted by the thyroid hormones (thyroxine – T4, and triiodothyronine – T3) (McNabb and King, 1993) and the hydration status of the animal which is mediated by arginine vasotocin (AVT) (Saito and Grossmann, 1998).
Changing the sensitivity of the warm- and/or cold-sensitive neurons located in the PO/AH may change the threshold for heat production and/or heat loss of an animal. Heat acclimation in broiler chickens is mediated by reduced metabolic rate, blood volume expansion (Yahav et al., 1997b), and reduced Tb (Yahav et al., 1995). This process requires from 4 to 7 day to be completed in broilers (Yahav, 2000), and is rendered rather impracticable by the early marketing age of broilers, coupled with: (a) the necessity to keep the environmental temperature controlled up to the age of 21 days for brooding; (b) the deleterious effect of heat acclimation on broiler chickens; and (c) the enormous cost of temperature-controlled poultry houses.
Epigenetic adaptation, which has been defined as a lifelong adaptation that occurs during prenatal (embryogenesis) or early post-hatching ontogeny, within critical developmental phases that affect gene expression (Nichelmann and Tzschentke, 2002; Tzschentke and Basta, 2002; Tzschentke et al., 2004), seems to be a suitable means of reaching the goal of improved thermotolerance acquisition in broilers. During early development most functional systems evolve from an open-loop system without feed-back into a closed control system with feed-back (“transformation rule”, Dörner, 1974). Thermal manipulations during the critical phases of this development process may induce alterations in the thermoregulatory control system.
Post-hatching thermal manipulations
Broiler chicks complete their brain and body temperature regulation at 10 days post-hatch (Arad and Itsaki-Glucklish, 1991). During this period, body and brain temperatures are regulated at a lower level than in adult chickens. Subsequently, as age increases the difference between body and brain temperatures increases linearly and significantly. The epigenetic response has been successfully modulated by early-age thermal manipulation of post-hatching chicks, by exploiting the incomplete maturation of the thermoregulatory system. Thermal manipulation, involving exposure to 37-38°C at 60-80% relative humidity for 24 hours in 3-day-old broilers, was found to improve acquisition of thermotolerance.
The improvement achieved was manifested in the ability of the thermally manipulated chicks to efficiently reduce heat production during exposure to acute thermal challenge at market age (Yahav and Hurwitz, 1996). This was accompanied by:
a) an alteration in sensible heat loss through convection and radiation (Yahav et al., 2005; Table 1);
b) a significant reduction in the stress level of the thermally manipulated chickens, as indicated by their plasma corticosterone concentration (Figure 1); and
c) pronounced increases in the 27-, 70- and 90-kDa heat-shock proteins (HSPs) in the heart muscle and lung tissue of the thermally non-manipulated chickens, compared with those of the manipulated ones, during thermal challenge (Yahav et al., 1997a). It has been suggested that the induction of HSP was correlated with body temperature, and that the HSP response was not part of the long-term mechanism elicited by the thermal manipulation at an early age.
The reduction in heat production, coupled with an increase in sensible heat loss enabled a relatively slow development of hyperthermia and thus dramatically reduced mortality. Thermal manipulation at 3 days of age also induced compensatory growth, leading to improvement of performance and muscle growth, because of the proliferation of skeletal muscle satellite cells during the manipulation (Halevy et al., 2001).
The effect of thermal manipulation on the PO/AH in the chicks was studied with reference to two genes: the R-Ras3 and brain-derived neurotrophic factor (BDNH).
The R¬Ras3 belongs to the small GTP-binding protein subfamily; it is activated by multiple extracellular stimuli. The Ras subfamily controls a variety of cellular events that culminate in gene transcription. Ras plays an essential role in cell proliferation, differentiation and survival, and it is also significant in modulating synaptic functions. The BDNF binds specifically to the TrK-B receptor and initiates tyrosine phosphorylation, which activates the phosphotyrosine-binding site. This initiates the internal cellular pathway of RAS, and results in transduction of genes involved in neuronal growth and maintenance.
In the chicks' PO/AH, significant increases in expression of the R-Ras3 (Labunskay and Meiri, 2006) and brain-derived neurotrophic factor (BDNH) genes (Katz and Meiri, 2006) were detected during thermal manipulation, which suggests that these genes are involved in thermal manipulation. However, uniform post-hatch temperature manipulation is difficult to achieve, whereas the use of such manipulations during incubation would probably be more efficient and uniform.
Pre-hatching thermal manipulations
The hypotheses underlying these studies are that: (a) during embryogenesis, it is possible to induce long-lasting physiological memory based on epigenetic adaptation; (b) a reasonable definition of long-lasting memory is an alteration in the hypothalamic threshold response to changes in the environment and (c) during sensitive periods in embryogenesis, thermal manipulation involving exposure to high temperatures for specified periods, will elicit improved thermotolerance during the birds’ life span.
In poultry, control elements of the thermoregulatory system can function, but the efficiency of the system is low, possibly as a result of the uniform temperature used in commercial incubation. Endothermic reactions during the late stages of incubation may have a delayed, rather than an immediate influence on the efficiency of thermoregulation (Nichelmann and Tzschentke, 2002). Furthermore, the embryo is surrounded by the chorioallantoic membrane (CAM), which retains the extra-embryonic fluids and protects against deleterious effects on embryo hydration.
In contrast to the uniform temperature of commercial incubation, in nature, incubation conditions are non-uniform, as a result of searching for food, escape from predators, and non¬-uniform nest insulation. This may be one of the reasons why birds in the wild are better able to cope with extreme environmental temperatures.
It was previously reported that exposing embryos to high or low temperatures during incubation improved their capacity to adapt to hot or cold environments, respectively, in the post-hatch phase (Nichelmann et al., 1994; Tzschentke and Basta, 2002; Moraes et al., 2003; Yahav et al., 2004). Three critical parameters have to be considered in the approach to thermal manipulations during chick embryogenesis: a. the critical phase; b. the temperature level; c. duration of exposure.
Determination of the critical phase during embryogenesis, for application of thermal manipulation to improve acquisition of thermotolerance, was based on the hypothesis that the “set point” or “response threshold” of controlling systems can be altered most efficiently during the development/maturation of the hypothalamus-hypophysis-thyroid axis (thermoregulation) and/or the hypothalamus-hypophysis-adrenal axis (stress).
Until mid-incubation the thyroid gland possesses only limited ability to synthesize hormones. This period is characterized by the synthesis of monoiodotyrosine on E8 (i.e. 8th day of embryogenesis), of diiodotyrosine on E9, and by synthesis of T4 and thyroid stimulating hormone (TSH) on E10. The linkage of the hypothalamic-pituitary-thyroid axis is formed between E10.5 and E11.5. Levels of T3 start increasing on E12 and increase significantly prior to hatching, in preparation for their role in the final maturation of many tissues and in the physiological integration of hatching. Therefore, application of thermal manipulations during the sensitive period of development of the axis (Reynes et al., 2003) might affect the heat production threshold “set point”. Epple et al. (1997) suggested the embryo to be susceptible to stress. Therefore, increasing the incubation Ta during and/or after the hypothalamic-hypophyseal-adrenal axis has been activated (Wise and Frye, 1975) might affect the stress response of the post-hatch chick.
In recent experiments (Yahav et al., 2004; Collin et al., 2006) the thermal manipulation of chick embryos, at 39.5°C for 3 h, on days 8 to 10 or 16 to 18 improved hatchability in the E16-E18 chicks, but did not affect the body weight of the hatched chicks. Also, it significantly reduced their metabolic rate, as indicated by body temperature (Tb) (Table 2). A parallel experiment conducted by Tona et al. (unpublished data) revealed a significant decline in the measured pre-hatching oxygen consumption of the thermally manipulated embryos, which proved that the reduction in metabolic rate was a result of the thermal manipulations.
Challenging the chicks (41°C for 6 h on day 3 of age) revealed significantly improved thermotolerance in birds thermally manipulated during embryogenesis. The improved thermotolerance was indicated by a significantly lower metabolic rate, and coincided with non-significant and significant declines in the stress levels in the E8-E10 and E16-E18 embryos, respectively (Table 3).
In other experiments, a prolonged thermal manipulation (38.5°C) was applied to layer-¬strain chicken embryos from E18 until the end of incubation. On the last day of incubation the thermally manipulated embryos showed a significantly higher level of heat production than the controls (Loh et al., 2004). Similar effects were found in Muscovy duck embryos that experienced thermal manipulation from E29 until hatch. Prolonged exposure of Muscovy duck embryos to warm (38.5°C) or cold (34.5°C) conditions induced changes in the thermosensitivity of PO/AH neurons (Loh et al., 2004), which persisted until 10 days post-hatch (Tzschentke and Basta, 2002). Furthermore, during the first 10 days post-hatch Muscovy ducklings and turkeys that had been exposed to thermal manipulations during embryogenesis exhibited changes in heat production and in their preferred ambient temperatures (Nichelmann et al., 1994). Janke and Tzschentke (2006) found that layer embryos that had been exposed to high or low incubation temperatures (38.5 or 34.5°C, respectively) on E18, and were heat stressed on E20 differed in their expression of c-Fos in the hypothalamus on the last day of incubation.
Most reported studies have demonstrated an improvement in thermotolerance during the first 10 days post-hatch. However, chicks that had been exposed to thermal manipulation during embryogenesis and then raised to marketing age, did not exhibit a long-lasting improvement in thermotolerance. Although thermally treated chicks had significantly lower Tb immediately post-hatch than untreated chicks, the difference persisted only until 4 or 5 weeks of age, after which it diminished. Furthermore, heat challenge at 42 days of age of thermally manipulated chickens during embryogenesis did not reveal any thermal advantages in these chickens (Collin et al., 2006; Tona et al., unpublished data). Piestone et al. (unpublished data) found that adopting E7 to E16 as the “critical phase” for thermal manipulation of chick embryos significantly enhanced thermotolerance but also caused teratogenic effects which were found to depend on the duration of exposure.
These conflicting results raised the questions of whether long-lasting thermal memory can be imparted by thermal manipulation during embryogenesis, and whether it is only a question of correct choice of the critical period. In mammals, it seems that for different control systems, as well as for specific functions of a system, e.g., as exhibited in the development of the mammalian visual system (Harwerth et al., 1986), several different, partially overlapping “critical phases” were found. Furthermore, species-specific differences have to be taken into consideration.
There is accumulating evidence that the epigenetic adaptation approach, and its association with changes in the environment in mammals and avian species, with emphasis on tuning the level and duration of stress to coincide with the “critical phase”, will elicit an efficient epigenetic adaptation. This complex issue needs to be intensively studied to shed light on epigenetic adaptation in domestic poultry.
References are available on request.
From Proceedings of the “19th Australian Poultry Science Symposium”, New South Wales, Australia.
S. YAHAV
Institute of Animal Science
ARO the Volcani Center,
Israel
