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Monitoring the development of thermoregulation in poultry embryos and its influence by incubation temperature
c o m p u t e r s a n d e l e c t r o n i c s i n a g r i c u l t u r e 6 4 ( 2 0 0 8 ) 61–71
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Monitoring the development of thermoregulation in poultry embryos and its influence by incubation temperature B. Tzschentke ∗ Humboldt-University of Berlin, Institute of Biology, WG Perinatal Adaptation, Invalidenstr. 43, 10115 Berlin, Germany
a r t i c l e
i n f o
a b s t r a c t
Keywords:
The general purpose of this review is to show the stage of the development of peripheral
Thermoregulation
and central nervous thermoregulatory mechanisms in poultry embryos at the end of
Incubation temperature
incubation, and the impact of long-term changes in incubation temperature. Methods
Poultry embryo
are described which (a) allow continuous measurement of peripheral thermoregulatory
Epigenetic adaptation
mechanisms simultaneously with the body temperature of the embryo, and (b) can be used
Critical period
for identification of changes in the sensitivity of the central controller of body temperature during the development as well as after prenatal temperature experiences. Further, a method for characterisation of ‘critical periods’ in the development of the respective body function is introduced. The results of our investigations were discussed in relation to the following general rules: (a) The development of peripheral and central nervous thermoregulatory mechanisms begins in the course of the prenatal ontogeny. At the end of incubation poultry embryos have all the prerequisites to react to changes in incubation temperature. Regarding the peripheral thermoregulatory mechanisms the most sensitive parameter for characterization of the developmental level of embryonic thermoregulation is the deep body temperature. (b) Functional systems of the organism develop from open loop system without feedback control into closed system controlled by feedback mechanism. Acute changes in the environmental conditions (e.g. incubation temperature) induce as a rule, initially uncoordinated and immediately non-adaptive reactions. Later the uncoordinated (immediately non-adaptive) reactions change into coordinated (adaptive) reactions, probably with closing of the regulatory system (‘critical period’). Environmental manipulation of immature physiological mechanisms could be used for characterization of ‘critical periods’ of the respective system. Monitoring of changes in the reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different perinatal time windows could help to limit ‘critical periods’ in the development of the thermoregulatory system. (c) During this ‘critical periods’, the actual environment modulates the development of the respective physiological control systems for the entire life period. Perinatal epigenetic temperature adaptation could be a tool to adapt poultry embryos or hatchlings to later climatic conditions. For detection of immediate and long-term effects of perinatal epigenetic temperature adaptation (‘imprinting’ of the thermoregulatory system) recordings of changes in neuronal hypothalamic thermosensitivity as well as in neuronal response on temperature stress are useful and have to be verified by identification of the respective effector genes and epigenetic changes in its expression. © 2008 Elsevier B.V. All rights reserved.
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1.
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Introduction
In the hierarchy of the regulatory systems the thermoregulatory system is on a higher level. Depending on the actual environmental situation the thermoregulatory system employs other systems of the body (e.g. heart and circulation, respiration, metabolism) and integrates their activities into appropriate and coordinated reactions for control of body temperature. In poultry the development of thermoregulatory mechanisms and mechanisms of related systems start during incubation (Nichelmann et al., 2001; Nichelmann and Tzschentke, 2002, 2003). It is a prerequisite for the quick maturation of temperature regulation in the early post-hatching phase, which is quite important for the performance of the whole organism. Further, in the early ontogeny during circumscribed time windows, the so-called ‘critical periods’ the ‘imprinting’ of physiological control systems occurs related to the environment of the developing organism (Tzschentke and Plagemann, 2006). In the course of the early development physiological control systems, like the thermoregulatory system, develop from open loop system without feedback control into closed control system regulated by feedback mecha¨ nisms (Dorner, 1974). Obviously, this qualitative change in the operation of regulatory systems is a ‘critical period’ in the development of body functions. Incubation climate and the incubation temperature being the foremost may cause longlasting perinatal epigenetic programming or malprogramming (Tzschentke and Plagemann, 2006). On one hand suboptimum incubation conditions have been related to diseases and disorders during later life (Tzschentke and Plagemann, 2006). On the other hand, knowledge of these mechanisms might be specifically used to induce long-term adaptation of the organism, for instance, to the postnatal climatic conditions (epigenetic temperature adaptation, Nichelmann et al., 1994; Tzschentke and Basta, 2002). In conclusion, a proper management of incubation climate has a high impact for the postnatal development, health and performance in poultry. Prerequisites for the proper management of incubation climate are acquiring knowledge of the physiological needs of poultry embryos and the ‘critical periods’ as well as systematic investigations on long-term effects of chronic environmental changes. This review summarizes investigations, which were carried out in my working group “Perinatal Adaptation”. It is focused on the development of thermoregulation in poultry embryos as well as the impact of chronic changes of incubation temperature. These investigations require methods, which allow continuous measurement of peripheral thermoregulatory mechanisms simultaneously with the body temperature of the embryo. Further, methods for identification of changes in the sensitivity of the central controller of body temperature due to the development as well as prenatal temperature experiences and of ‘critical periods’ in the development of the respective body function are necessary. The methods developed or adapted and used in our group are herewith briefly described.
2.
Materials and methods
2.1.
Incubation of eggs
The experiments were carried out in Muscovy duck (Cairina moschata f. domestica) and in chicken (Gallus gallus f. domestica) embryos during the second half of incubation. Eggs of the Muscovy duck need a total incubation period of 35 days and those of the chicken 21 days. The eggs were incubated at 37.5 ◦ C and at a relative humidity of 70% until the day at which the eggs were transferred into the hatcher, viz. embryonic day (E) 28 in the Muscovy duck and E17 in the chicken. During the incubation period the eggs were subjected to automatic turning. In the hatcher the eggs were incubated at 37.5 ◦ C and at a relative humidity of 90%. Experimental data, which were carried out under normal incubation temperature were used as control for experiments on influence of chronic changes in incubation temperature on the development of thermoregulatory mechanisms. For experiments on chronic influence of changes in incubation temperature on the development of thermoregulation, one group of eggs were incubated at 34.5 ◦ C (cold-incubated group) and another group at 38.5 ◦ C (warm-incubated group) from the day of transfer to the hatcher until hatching. After hatch, the birds were kept during the first 10 days either in the animal house at an ambient temperature of 25 ◦ C with additional infrared lamps (35 ◦ C) or in a temperature gradient channel (10–45 ◦ C). Food and water were given ad libitum.
2.2. Simultaneous measurement of body temperature and O2 -consumption in poultry embryos 2.2.1.
Body temperature
Embryonic body temperature was measured in the allantoic fluid (Taf ) near the embryo using a miniature thermistor probe (Testo GmbH & Co., Lenzkirchen, Germany). On the blunt side of the egg a square hole (2 mm × 2 mm) was drilled into the eggshell without damaging larger blood vessels. Through this hole a thermocouple was inserted into the allantoic fluid between the chorioallantoic membrane and the embryo (Figs. 1 and 2). After internal pipping (from E20 in chicken embryos and E33 in Muscovy duck embryos until hatching) it was also possible to measure the colonic temperature (Tc ). After locating the tail feathers, the cloaca was easily identified and the thermistor probe was inserted to a depth of 1–2 cm. Taf as well as Tc was measured continuously during the entire period of experiments. A comparison between Taf and Tc in single embryos showed only minor differences between Taf and Tc (ranging between 0.0 and 0.2 ◦ C) at constant incubation temperature. Under changing incubation temperature the difference rose to a range of 0.1–0.4 ◦ C (Holland et al., 1998). Details of the method are described by Holland et al. (1998) and Nichelmann and Tzschentke (2003).
2.2.2.
Measurement of O2 -consumption
Oxygen consumption was continuously measured using an oxygen analyser (Magnos 4, Hartmann & Braun, Frankfurt/Main, Germany) based on the paramagnetic principle.
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regression line for each individual embryo Q10 was calculated using the van’t Hoff formula (Precht et al., 1973): Q10 = (HPT2 /HPT1 )10/(T2−T1) (T1 > T2) in steps of 0.1 ◦ C changes in the Taf (Nichelmann et al., 1998).
2.3. Simultaneous measurement of body temperature and respiration parameters as well as body temperature and blood flow 2.3.1.
Recording of respiratory parameters
Breathing by lung occurs after internal pipping. The breathing activity induces pressure fluctuations in the air cell of the egg. These pressure fluctuations can be registered using a Statham element (Hugo Sachs Electronic KG, March, Germany) in a tube which is inserted in the air cell (Fig. 2). The recordings furnish information about the respiratory rate, the relative tidal volume and relative respiratory minute volume (Nichelmann and Tzschentke, 2003) in relation to changes in the body temperature. Fig. 1 – Schematic diagram of the metabolic chamber for single egg measurement (from Janke et al., 2004).
For these measurements single eggs were placed into the metabolic chambers (Fig. 1). The metabolic chambers (volume 290 ml) were watertight and consist of acrylic glass. The respective ambient (incubation) temperature was regulated by a temperature controlled water bath. After a minimum of 2 h of acclimation of the embryos to the respective ambient temperature, oxygen consumption simultaneously with ambient temperature and Taf were recorded for 1 h. Oxygen consumption (vol%) was measured as difference between oxygen concentration of the air inflow and outflow of the climatic chambers taking into account the actual gas flow (l/h). The influence of evaporation-water loss was removed by drying the gas streams before analysing. Detailed information on the methodology are published in Janke et al. (2004), Nichelmann et al. (1998, 2001) and Nichelmann and Tzschentke (2002, 2003).
2.2.3.
Calculation of heat production
Assuming a respiratory quotient of 0.72 (Decuypere, 1984), which corresponds to a caloric heat equivalent of 19.7 J ml−1 oxygen consumption, heat production (HP) was calculated on the basis of oxygen consumption, egg mass and caloric heat equivalent.
2.2.4.
Investigation of thermoregulatory heat production
To investigate the development of endothermic reactions acute temperature stimulations were applied by decrease (34.5–31.5 ◦ C) of ambient (incubation) temperature from the normal level (37.5 ◦ C) for 3 h using different water baths. Changes in HP were analysed in relation to simultaneously recorded body temperature (Taf ). As no net-increase in HP with decreasing body temperature was observed in embryos it was possible to estimate thermoregulatory counter reactions during cooling using the Q10 method. A quadratic regression was used to describe the relationship between HP and Taf . From the
2.3.2. Recording of the blood flow in the chorioallantoic membrane Blood flow in the chorioallantoic membrane was measured by MBF3 laser-Doppler instrument (Moor Instrument Company Ltd., Devon, GB). The laser-Doppler probe was placed directly on the egg membrane (Fig. 2). Before positioning the probe the egg was candled, to find an area with a large number of small blood vessels. Then a 5 mm × 5 mm piece of the egg shell was removed. The following parameters could be measured: mean red blood cell flow rate (flux), the red blood cell concentration and the mean red blood cell speed. These parameters were measured simultaneously with the embryonic temperature. Detailed information is furnished in Nichelmann and Tzschentke (2003).
2.4.
Recordings of neuronal thermosensitivity
2.4.1. Extracellular recordings of neuronal activity in relation to temperature stimulation in embryonic and post-hatching period Extracellular recordings of single cell activity were carried out in brain slices (400 m) from neurons of the preoptic area of the anterior hypothalamus (PO/AH). The slices were placed into a recording chamber (Schmid et al., 1993) and continuously perfused by artificial cerebrospinal fluid. From beginning of the experiment, the bath temperature in the recording chamber was maintained at 39 ◦ C in the embryos or 40 ◦ C in the birds post-hatching and continuously controlled by a small thermocouple. This temperature approximately corresponds to the deep body temperature in poultry at a later stage of embryonic development if incubated at the normal 37.5 ◦ C (Janke et al., 2002) or during the first days post-hatching (Tzschentke and Nichelmann, 1999). Neuronal activity and slice temperature were recorded by conventional electrophysiological equipment and stored on a personal computer using a 1401 interface (Cambridge Electronic Design) (CED) and the CED software Spike 2. To identify the thermosensitivity of single neurons, the bath temperature was sinusoidally changed to a maximum of ±3 ◦ C and a velocity of about 0.02 ◦ C s−1 . Temperature sensitivity was calculated by a
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Fig. 2 – Methods for recording of body temperature and different physiological parameters simultaneously. The focus in this figure is on localisation of thermistor probes for measurement of the temperature of the allantoic fluid (Taf ) and colonic temperature (Tc ), laser-Doppler-probe for estimation of the blood flow in the chorioallantoic membrane, ECG electrodes and tube with pressure differential sensor for recording of breathing activity in the avian egg.
computer program, adapted under Spike 2. The temperature response curve of each neuron was evaluated by relating firing rate to slice temperature and fitted to a piecewise and/or rectilinear regression function (Vieht, 1989). The thermosensitivity of a neuron was defined by a temperature coefficient of greater than or equal to 0.6 imp s−1 ◦ C−1 for warm-sensitive (WS) neurons and less than or equal to −0.6 imp s−1 ◦ C−1 for cold-sensitive (CS) neurons. All other neurons are named as temperature insensitive (TI) according to this definition (Nakashima et al., 1987). Further details of the methodology are described in Tzschentke and Basta (2002) and Tzschentke et al. (2004).
2.4.2. Changes in sensitivity of central thermoregulatory mechanisms in embryonic and post-hatching period For characterisation of the neuronal hypothalamic thermosensitivity the proportion of WS, CS and TI neurons in the PO/AH was determined in relation to all neurons investigated (Tzschentke and Basta, 2000, 2002). For instance, an increase in the proportion of CS neurons and a decrease in WS neurons in relation to all neurons investigated was used as a sign of elevation in total neuronal cold sensitivity of the PO/AH.
2.4.3.
Investigation of c-fos expression in poultry embryos
Compared to single cell recordings, the c-fos method can exhibit activity of numerous neurons at one time and thus demonstrate neuronal networks. It is expressed within a short time after changes in the environment of the organism. Because of this, the c-fos gene is considered as an immediate early gene. In the nucleus of the cell, c-fos can be detected by immunohistochemical method. In our investigations, on the last day of incubation acute heat stress (42.5 ◦ C) for 90 min was applied before starting the experi-
ment. Then the extracted embryos were anaesthetized and transcardial perfusion was performed. Brains were dissected and 20-m brain sections were made using a cryostat. In the PO/AH region of the slices c-fos expression was immunohistochemically detected. Analysis was made by light microscopy and digital photography (magnification of 50-fold). C-fos positive neurons were counted in a standardised area of the PO/AH using a rectangle mask. Due to the lack of stereotaxic data of the brain of the chicken embryo, the width of the rectangle was set proportional to the brain of the adult chicken at 990 m for all embryos. Stereotaxic data of the adult chicken brain were taken from Kuenzel and Masson (1988). For details see Janke and Tzschentke (2006).
2.5.
Identification of ‘critical periods’
Environmental manipulation of immature physiological mechanisms may be a physiological tool for characterization of ‘critical periods’ (Tzschentke and Plagemann, 2006). A typical reaction pattern of physiological mechanisms on acute and chronic environmental stimulation was found during the perinatal period (Tzschentke and Basta, 2002; Tzschentke et al., 2004). More details on this tool are given in this review under Section 3.
3.
Results and discussion
The methods applicated in this study are useful for basic investigations of special aspects of the development of the thermoregulatory system in single bird embryos. Altogether, with our different studies we could demonstrate fundamental developmental processes in chicken embryos using the
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thermoregulatory system as an example, which could be summarized in the following rules.
3.1. The development of peripheral as well as central nervous thermoregulatory mechanisms start in the course of the prenatal ontogeny To demonstrate, whether poultry embryos are already able to activate peripheral thermoregulatory mechanisms with changes in incubation temperature and if this activation is effective enough to regulate the embryonic body temperature, are only possible using simultaneous measurements of the embryonic body temperature and the respective parameter (e.g. HP, blood flow). With our methods the early activation of thermoregulatory mechanisms as well as differences in the developmental status between heat production and heat loss mechanisms in poultry embryos could be shown (Nichelmann and Tzschentke, 2003). Finally, during the late prenatal development poultry embryos have all prerequisites (peripheral and central nervous mechanisms) to react to changes in incubation temperature.
3.1.1.
Heat production
Long-term recordings of O2 -consumption indicated that in all precocial and altricial bird species investigated the development of HP under normal incubation temperature (37.5 ◦ C) follows an exponential function (Prinzinger and Dietz, 1995). Initially a continuous increase in HP is observed. After approximately 80% of incubation time, stagnation in HP occurs (plateau phase). At the end of the plateau phase the embryo pierces the chorioallantoic and inner shell membrane (internal pipping) and starts respiration through lungs (Tazawa and Whittow, 2000). After internal pipping until hatch there is a large increase in HP. Similar developmental pattern of HP was found in our experiments (Nichelmann et al., 1998; Janke et al., 2002). Even though there is a similar qualitative developmental pattern, quantitative differences (Fig. 3) in the development of HP could be found not only between poultry species (Janke et al., 2002) but also between poultry breeds (e.g. different high yielded chicken breeds, Janke et al., 2004). If HP is recorded simultaneously with embryonic body temperature (Taf ) a strong linear relationship between both parameters could be observed (Fig. 3). Under constant incubation temperatures this relationship could be described by a highly significant linear correlation (Janke et al., 2002). The relationship between HP and Taf changed absolutely, if acute changes in the incubation temperature occurred. With changing incubation temperature, e.g. during cooling, the relationship between HP and Taf can be described by quadratic regressions (Nichelmann et al., 1998). During acute decrease in incubation temperature Taf also shows a similar decrease with the difference in incubation temperature but Taf is always higher than ambient temperature. On the other hand, HP only decreases moderately (Fig. 4). A drop in body temperature, due to low incubation temperature mostly causes a decrease of net HP but the decrease is lower as assumed by the van’t Hoff rule (Nichelmann et al., 2001). To come to this conclusion is only possible after simultaneous recordings of HP and embryonic body temperature. Using the Q10 method (Nichelmann et al., 1998), an endothermic counter
Fig. 3 – Course of heat production (A) and body temperature (B) of two broiler chicken lines (Ross 308, Ross 508) and a layer chicken line (Lohmann White Leghorn) from day 9 and 11 of incubation until hatch. Means represent values of 6 embryos. The error bars represent standard deviations (from Janke et al., 2004).
reaction was found. The Q10 method enabled us to calculate the effect of thermoregulatory heat production with decreasing body temperature. It is a possibility to investigate the early development of endothermy in poultry embryos, which have no net-increase in HP after decrease in body temperature. In our investigations such endothermic counter reaction were already obtained before internal pipping in Muscovy duck and chicken embryos (Nichelmann et al., 1998). With increasing embryonic age the cold load induced decrease in HP, diminishes and near hatching time in some embryos a short-term
Fig. 4 – Course of body temperature (temperature of allantoic fluid) and heat production before and during 3 h cooling in a single Muscovy duck embryo on day 34 of incubation (Nichelmann and Tzschentke, 1999).
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Fig. 5 – Influence of increase in incubation temperature (ambient temperature, Ta ) on the course of body temperature (colonic temperature, Tc ) and blood flow in the chorioallantoic membrane in a single chicken embryo after internal pipping (modified from Holland et al., 1998).
increase in HP was observed. However, poultry embryos are not able to keep body temperature constant under cold load. In comparison with the heat loss mechanisms, in embryos efficiency of thermoregulatory heat production is very low. An increase in high-energy costly thermoregulatory HP to keep body temperature constant is not necessary for the survival of the bird embryos (Nichelmann and Tzschentke, 2001, 2002; Tzschentke, 2003) because precocial bird embryos show a high thermal tolerance, which protects them to some extent from disturbances by cooling (Whittow and Tazawa, 1991; Tazawa and Whittow, 2000). The phase of ‘full-blown’ homoeothermy starts during the first days of post-hatching (Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Tazawa et al., 2004).
3.1.2. Heat loss mechanisms 3.1.2.1. Changes in the blood flow of the chorioallantoic membrane. In poultry embryos non-specific changes in the blood flow of the chorioallantoic membrane due to changes in incubation temperature could be found already during the last third of incubation time. But at end of the plateau phase, blood flow increases with increasing incubation temperature or decreases with decreasing ambient temperature. In chicken embryos after internal pipping, for instance, the body core temperature remained constant for more than 40 min after the beginning of increase in ambient temperature up to 40.5 ◦ C (Fig. 5) by activating this heat loss mechanism (Nichelmann and Tzschentke, 2003). This result could be only found by measurement of the deep body temperature in the colon of the embryo. In these experiments, simultaneous measurements of Taf were not sensitive enough for the monitoring of the short-term regulation of the body temperature by changes in the blood flow of the chorioallantoic membrane (Holland et al., 1998).
3.1.2.2. Changes in respiration. First rhythmic contractions of the respiratory muscles without ventilation of the lung can be monitored already before internal pipping. One goal of this movement is to consolidate the morphology and function of the respiratory tract (Tazawa, 1987; Murzenok et al., 1997). The lung ventilation occurs after internal pipping. The developmental level of the respiration as a heat loss mechanism
Fig. 6 – Influence of colonic temperature (body temperature) on respiratory rate, tidal volume and relative respiratory minute volume in a single Muscovy duck embryo on day 34 of incubation (Nichelmann and Tzschentke, 1999). Respiratory rate is given in n min−1 , tidal volume and relative respiratory minute volume in arbitrary units.
in thermoregulation can be only investigated by simultaneous measurements of the respiratory rate and the deep body temperature. Between internal and external pipping, panting reactions were found in Muscovy duck embryos when body core temperature increased. In Muscovy duck embryos panting reactions show similar characteristics to adult birds (two phases of panting, Arad and Marder, 1983). At body core temperatures between 38.5 and 40.5 ◦ C respiratory rate increased and the tidal volume decreased. Above 40.5 ◦ C, the second phase of panting starts characterised by a decrease in respiratory rate and an increase in tidal volume (Fig. 6). At the later stage of incubation heat loss mechanisms (blood flow, respiration) in poultry embryo seem to be more effective in relation to control of body temperature than the heat production mechanisms. At the end of incubation both mechanisms show to a degree similar characteristics to postnatal birds.
3.1.3. Development of central nervous thermoregulatory mechanisms In poultry embryos thermoregulation is only possible if central nervous thermoregulatory mechanisms are developed early.
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mosensitivity of the PO/AH from the ‘juvenile’ to the ‘adult’ type (Tzschentke and Basta, 2000), which is characterised by a high warm sensitivity and a low cold sensitivity (Nakashima et al., 1987). Similar developmental pattern was also found in chicken during early postnatal development (Sallagundala et al., 2006). Besides the single cell recordings, we used the neuronal c-fos expression for demonstration of the activity of neuronal networks. Chicken embryos that were heat stressed (42.5 ◦ C for 90 min) on the last day of incubation show a clear expression of neuronal c-fos in the PO/AH (Janke and Tzschentke, 2006).
3.2. Acute changes in the environmental conditions induce as a rule, first uncoordinated and immediately non-adaptive reactions
Fig. 7 – Example of a hypothalamic cold sensitive neurone in a Muscovy duck embryo on day 22 of incubation (TC = thermal coefficient) imp s−1 ◦ C−1 , from Tzschentke et al. (2004).
We could find thermosensitive neurons in brain slices of Muscovy duck embryos using the method of extracellular single neuron recording. In this species thermosensitive PO/AH neurons were found on E22 (Fig. 7) and E23 that show characteristics similar to post-hatching (Tzschentke and Basta, 2000), growing (own experiments, unpublished data) and adult birds (Nakashima et al., 1987) as well as mammals (Schmid and Pierau, 1993). On E28 and E33 the proportion of CS, WS and TI neurons in relation to all neurons investigated was very constant and not significantly different from that in hatchlings (Tzschentke and Basta, 2000; Tzschentke et al., 2004). In contrast to the growing and adult ducks as well as mammals, the neuronal hypothalamic thermosensitivity in embryos and ducklings during the first days of post-hatching is characterised by a high neuronal cold sensitivity (Fig. 8). A qualitative change occurs between days 5 and 10 in the neuronal ther-
Fig. 8 – Influence of age on proportion of warm-, cold- and temperature-insensitive neurons in relation to all neurons investigated in their respective age groups in the preoptic area of the anterior hypothalamus of Muscovy ducks (modified from Tzschentke and Basta, 2000). Asterisks represent significance at the level of *p < 0.05 (D: embryonic day, d: day post-hatching).
To investigate the developmental level of the thermoregulatory system (open loop system without feedback mechanisms or closed system with developed feedback mechanisms) acute changes in ambient (incubation) temperature have to be applied. The reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different time windows of the perinatal period show a typical pattern. First uncoordinated and immediately non-adaptive reactions occur. Later the uncoordinated (immediately non-adaptive) reactions change into coordinated (adaptive) reactions, probably with closing of the regulatory system. First, during the development of physiological control systems it seems to be unimportant for the organism that a distinct adaptable reaction of physiological mechanisms on various environmental influences occurs, but rather that any reaction occurs seems to be important for the adaptability during the later life (Nichelmann et al., 1999, 2001; Tzschentke et al., 2004). These first reactions seem to be important for the ‘training’ of the respective function to develop feedback mechanisms. For instance, in chicken embryos the blood flow increased or decreased while warming or cooling on E15 until E19 (proximate non-adaptive). After this period, the reaction became proximate adaptive; on E20 and E21, the blood flow in the chorioallantoic membrane increased during warming and decreased during cooling, as expected (Fig. 9, Nichelmann and Tzschentke, 2003). Similar changes in the blood flow during cooling or warming were also found in Muscovy duck embryos at the end of incubation (Tzschentke, 2002). Also in other systems first proximate non-adaptive reactions on acute or chronic environmental stimulation was found in the course of the perinatal period. In Muscovy duck embryos, for instance, for different groups of reactions on acoustic stimulation could be classified; heart rate increase, heart rate decrease, increase or decrease in heart rate variability with¨ out a change in the mean heart rate (Hochel et al., 2002). Also after chronic changes in the incubation temperature at the end of embryonic development first proximate nonadaptive changes occurred in the heat production (Loh et al., 2004), neuronal hypothalamic thermosensitivity (Tzschentke and Basta, 2002) and neuronal c-fos expression after acute temperature application (Janke and Tzschentke, 2006). Obviously, the development of regulatory systems from an open loop system without feedback into a closed control sys-
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Fig. 9 – Influence of warming (38.5 ◦ C) and cooling (35.5 ◦ C) on blood flow in the chorioallantoic membrane of 15- to 21-day-old chicken embryos. Each column represents the reaction in one individual embryo, expressed in Flux, which is given in arbitrary units. The blood flow was measured using the laser-Doppler method (from Nichelmann and Tzschentke, 1999, 2003).
tem with feedback is a ‘critical period’ in the ontogeny of physiological control systems, which is of high importance for the later development in poultry (see Section 3.3). The changes in the reaction pattern on environmental influences could be typical for this phase. In conclusion, environmental manipulation of immature physiological mechanisms may be a physiological tool for characterization of ‘critical periods’ of the respective system (Tzschentke and Plagemann, 2006).
3.3. During critical developmental phases, a long-term adaptation to an actual environment occurs via epigenetic adaptation processes Our hypothesis states that, in the course of the perinatal period, ‘imprinting’ of physiological control systems occur which, probably is realized by both neural ‘imprinting’ at the microstructural level (e.g., in terms of synaptic plasticity) as well as by a lasting environment-induced modification of the genome (Tzschentke and Plagemann, 2006). During ‘critical periods’ of development of physiological control systems (mentioned in the last paragraph), the actual level at which physiological parameters occur may pre-determine the ‘set-point’ (‘set ranges’) of the respective physiological control system during the entire life period, possibly through acquired changes in the expression of related effector genes (Fig. 10). On one hand, this mechanism seems to be a possible basis for perinatal malprogramming which, e.g., causes metabolic disorders and cardiovascular diseases as well as behavioural disorders during later life in mammals including man (Plagemann, 2004) as well as in birds (Schwabl, 1996, 1997; Ruitenbeek et al., 2000). On the other hand, knowledge and better understanding of these mechanisms might be specifically used to induce long-term adaptation of an organism, for instance, to postnatal climatic conditions (epigenetic temperature adaptation; Nichelmann et al., 1994, 1999; Tzschentke and Basta, 2002; Tzschentke et al., 2004). In chicken and other precocial birds epigenetic temperature adaptation can be induced by changes in incubation temperature at the end of embryonic development (Decuypere, 1984; Minne and Decuypere, 1984; Nichelmann et al., 1994; Tzschentke and Nichelmann, 1997; Tzschentke and Basta,
2002; Loh et al., 2004) as well as by thermal conditioning during the first days after hatching (Yahav and Plavnik, 1999; Yahav, 2000). Altogether, prenatal temperature experiences induce postnatal warm or cold adaptation (Tzschentke et al., 2004) (Fig. 11). On the first day of post-hatching Muscovy ducklings incubated at lower temperatures than normal, for instance, have a 56% higher heat production and a higher deep body temperature under cold load as compared to controls (1 h at 10 ◦ C). Cold-incubated birds are able to control their actual deep body temperature at this set-point, in contrary to those incubated at 37.5 ◦ C, which display a lower heat production (Nichelmann et al., 1994; Tzschentke et al., 2004). Further, Muscovy ducklings incubated at a low temperature preferred a significantly lower temperature than birds incubated at the normal incubation temperature during the first 10 days of post-hatching. This supports the hypothesis that avian prenatal cold experience leads to a downward shift of the thermoregulatory set-point (Tzschentke and Nichelmann, 1999).
Fig. 10 – Basic concept of induction of epigenetic perinatal malprogramming or epigenetic adaptation processes, such as epigenetic temperature adaptation by environmental factors during ‘critical periods’ of early development.
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Fig. 11 – Epigenetic temperature adaptation in Muscovy duck embryos. Embryos were incubated from day 28 of incubation until hatching in either warmer or colder temperatures than the usual 37.5 ◦ C. A: Changes in the neuronal hypothalamic thermosensitivity at day 10 post-hatching induced by changes in incubation temperature (significant differences, *p < 0.05, 2 -test). For characterization of the neuronal hypothalamic thermosensitivity the proportion of warm sensitive, cold sensitive and temperature insensitive neurons in the PO/AH was determined in relation to all neurons (n = 80 neurons) investigated in the respective incubation groups. B: HP and C: colonic temperature of hatchlings from cold (34.5 ◦ C) and normal (37.5 ◦ C) incubated eggs after 1 h exposure to 10 ◦ C (significant differences, *p < 0.05, t-test).
On the other hand, the preferred ambient temperature in 1- to 10-day-old turkeys is higher after a prenatal heat load (38.5 ◦ C) than in birds incubated at the normal temperature (37.5 ◦ C). This indicates an elevation of the thermoregulatory set-point after prenatal heat load. In our experiments changes in the neuronal thermosensitivity of the hypothalamic control centre of the thermoregulatory system reflect the changes in peripheral thermoregulatory mechanisms after prenatal temperature experiences. Prenatal cold experience increases on day 10 of post-hatching the neuronal hypothalamic warm sensitivity and prenatal heat experiences increase neuronal hypothalamic cold sensitivity. In differentially incubated birds the change in the levels of HP and in neuronal hypothalamic thermosensitivity (Loh et al., 2004) and in neuronal hypothalamic c-fos expression (Janke and Tzschentke, 2006) occur already before hatching. But these changes are proximate non-adaptive during this developmental period. It is interesting to note, that the embryonic body temperature is more influenced by chronic changes in incubation temperature than the HP. If the HP is measured at temperatures at which the embryos were adapted, the HP in cold-incubated (34.5 ◦ C) embryos, for instance, was not lower than in the control, which was incubated at 37.5 ◦ C. On other hand the body temperature was 3 ◦ C lower in the cold-incubated than in the control group (Loh et al., 2004). But
this lower body temperature could be a prerequisite for a lower thermoregulatory set-point during the post-hatching period. In experiments with warm-incubated chicken and Muscovy duck embryos (38.5 ◦ C during the last third of incubation) Taf was approximately 1 ◦ C higher than in the control. This could be a prerequisite for a higher thermoregulatory set-point during the entire life (Loh et al., 2004).
4.
Conclusions
In poultry embryos autonomic and central nervous thermoregulatory mechanisms are developed. Latest at the end of incubation poultry embryos have all prerequisites to react to changes in incubation temperature. During this developmental period some physiological mechanisms show to certain degree similar characteristics to postnatal birds. Regarding the autonomic thermoregulatory mechanisms we could come to this conclusion only by simultaneous recordings of the respective mechanism and the embryonic body temperature. The most sensitive parameter for characterization of the developmental level of embryonic thermoregulation after acute changes in incubation temperature is the deep body temperature (colonic temperature). But its measurement is limited to the post-internal pipping period. During earlier developmen-
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tal stages the internal egg temperature (Taf ) can be used as a very good measure of the embryonic body temperature under constant incubation temperatures. However, for characterization of the thermoregulatory ability under acute temperature changes Taf measurements are limited. During critical developmental periods epigenetic adaptation processes can induce a long-term adaptation to the actual environment. Perinatal epigenetic temperature adaptation could be a tool to adapt poultry embryos or hatchlings to later climatic conditions. For the specific use of perinatal epigenetic temperature adaptation in practice more investigations on the basic mechanisms of ‘imprinting’ of physiological control systems and on the problem of ‘critical periods’ are necessary. Environmental manipulation of immature physiological mechanisms could be used for characterization of ‘critical periods’ of the respective system. Monitoring of changes in the reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different perinatal time windows could help to limit ‘critical periods’ in the development of the thermoregulatory system. For detection of immediate and long-term effects of perinatal epigenetic temperature adaptation (‘imprinting’ of the thermoregulatory system) changes in plasticity of the central controller of thermoregulation in the hypothalamus are important. Recordings of changes in neuronal hypothalamic thermosensitivity as well as in neuronal response on temperature stress are useful tools and have to be verified by identification of the respective effector genes and epigenetic changes in its expression.
Acknowledgements Research projects carried out in our working group “Perinatal Adapation” were supported by grants of the Deutsche Forschungsgemeinschaft (Ni 336/3-1; TZ 6/2-1, 6/2-2, 6/6-4, 6/10, JA 1440/1-1).
references
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Tzschentke, B., Basta, D., 2000. Development of hypothalamic neuronal thermosensitivity in birds during the perinatal period. J. Therm. Biol. 25, 119–123. Tzschentke, B., Basta, D., 2002. Early development of neuronal hypothalamic thermosensitivity in birds: influence of epigenetic temperature adaptation. Comp. Biochem. Physiol. 131A, 825–832. Tzschentke, B., Nichelmann, M., 1997. Influence of prenatal and postnatal acclimation on nervous and peripheral thermoregulation. Ann. NY Acad. Sci. 813, 87–94. Tzschentke, B., Nichelmann, M., 1999. Development of avian thermoregulatory system during the early postnatal period: development of the thermoregulatory set-point. Ornis Fen. 76, 189–198. Tzschentke, B., Plagemann, A., 2006. Imprinting and critical periods in early development. World’s Poult. Sci. J. 62, 626– 637. Tzschentke, B., Basta, D., Janke, O., Maier, I., 2004. Characteristics of early development of body functions and epigenetic adaptation to the environment in poultry: focused on development of central nervous mechanisms. Avian Poult. Biol. Rev. 15 (Suppl. 3/4), 107–118. Vieht, E., 1989. Fitting piecewise linear regression functions to biological responses. J. Appl. Physiol. 67, 390–396. Whittow, G.C., Tazawa, H., 1991. The early development of thermoregulation in birds. Physiol. Zool. 64, 1371–1390. Yahav, S., 2000. Domestic fowl—strategies to confront environmental conditions. Avian Poult. Biol. Rev. 11, 81–95. Yahav, S., Plavnik, I., 1999. Effects of early-age thermal conditioning and food restriction on performance and thermotolerance of male broiler fowl. Br. Poult. Sci. 40, 120–126.
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Monitoring the development of thermoregulation in poultry embryos and its influence by incubation temperature B. Tzschentke ∗ Humboldt-University of Berlin, Institute of Biology, WG Perinatal Adaptation, Invalidenstr. 43, 10115 Berlin, Germany
a r t i c l e
i n f o
a b s t r a c t
Keywords:
The general purpose of this review is to show the stage of the development of peripheral
Thermoregulation
and central nervous thermoregulatory mechanisms in poultry embryos at the end of
Incubation temperature
incubation, and the impact of long-term changes in incubation temperature. Methods
Poultry embryo
are described which (a) allow continuous measurement of peripheral thermoregulatory
Epigenetic adaptation
mechanisms simultaneously with the body temperature of the embryo, and (b) can be used
Critical period
for identification of changes in the sensitivity of the central controller of body temperature during the development as well as after prenatal temperature experiences. Further, a method for characterisation of ‘critical periods’ in the development of the respective body function is introduced. The results of our investigations were discussed in relation to the following general rules: (a) The development of peripheral and central nervous thermoregulatory mechanisms begins in the course of the prenatal ontogeny. At the end of incubation poultry embryos have all the prerequisites to react to changes in incubation temperature. Regarding the peripheral thermoregulatory mechanisms the most sensitive parameter for characterization of the developmental level of embryonic thermoregulation is the deep body temperature. (b) Functional systems of the organism develop from open loop system without feedback control into closed system controlled by feedback mechanism. Acute changes in the environmental conditions (e.g. incubation temperature) induce as a rule, initially uncoordinated and immediately non-adaptive reactions. Later the uncoordinated (immediately non-adaptive) reactions change into coordinated (adaptive) reactions, probably with closing of the regulatory system (‘critical period’). Environmental manipulation of immature physiological mechanisms could be used for characterization of ‘critical periods’ of the respective system. Monitoring of changes in the reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different perinatal time windows could help to limit ‘critical periods’ in the development of the thermoregulatory system. (c) During this ‘critical periods’, the actual environment modulates the development of the respective physiological control systems for the entire life period. Perinatal epigenetic temperature adaptation could be a tool to adapt poultry embryos or hatchlings to later climatic conditions. For detection of immediate and long-term effects of perinatal epigenetic temperature adaptation (‘imprinting’ of the thermoregulatory system) recordings of changes in neuronal hypothalamic thermosensitivity as well as in neuronal response on temperature stress are useful and have to be verified by identification of the respective effector genes and epigenetic changes in its expression. © 2008 Elsevier B.V. All rights reserved.
∗
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1.
c o m p u t e r s a n d e l e c t r o n i c s i n a g r i c u l t u r e 6 4 ( 2 0 0 8 ) 61–71
Introduction
In the hierarchy of the regulatory systems the thermoregulatory system is on a higher level. Depending on the actual environmental situation the thermoregulatory system employs other systems of the body (e.g. heart and circulation, respiration, metabolism) and integrates their activities into appropriate and coordinated reactions for control of body temperature. In poultry the development of thermoregulatory mechanisms and mechanisms of related systems start during incubation (Nichelmann et al., 2001; Nichelmann and Tzschentke, 2002, 2003). It is a prerequisite for the quick maturation of temperature regulation in the early post-hatching phase, which is quite important for the performance of the whole organism. Further, in the early ontogeny during circumscribed time windows, the so-called ‘critical periods’ the ‘imprinting’ of physiological control systems occurs related to the environment of the developing organism (Tzschentke and Plagemann, 2006). In the course of the early development physiological control systems, like the thermoregulatory system, develop from open loop system without feedback control into closed control system regulated by feedback mecha¨ nisms (Dorner, 1974). Obviously, this qualitative change in the operation of regulatory systems is a ‘critical period’ in the development of body functions. Incubation climate and the incubation temperature being the foremost may cause longlasting perinatal epigenetic programming or malprogramming (Tzschentke and Plagemann, 2006). On one hand suboptimum incubation conditions have been related to diseases and disorders during later life (Tzschentke and Plagemann, 2006). On the other hand, knowledge of these mechanisms might be specifically used to induce long-term adaptation of the organism, for instance, to the postnatal climatic conditions (epigenetic temperature adaptation, Nichelmann et al., 1994; Tzschentke and Basta, 2002). In conclusion, a proper management of incubation climate has a high impact for the postnatal development, health and performance in poultry. Prerequisites for the proper management of incubation climate are acquiring knowledge of the physiological needs of poultry embryos and the ‘critical periods’ as well as systematic investigations on long-term effects of chronic environmental changes. This review summarizes investigations, which were carried out in my working group “Perinatal Adaptation”. It is focused on the development of thermoregulation in poultry embryos as well as the impact of chronic changes of incubation temperature. These investigations require methods, which allow continuous measurement of peripheral thermoregulatory mechanisms simultaneously with the body temperature of the embryo. Further, methods for identification of changes in the sensitivity of the central controller of body temperature due to the development as well as prenatal temperature experiences and of ‘critical periods’ in the development of the respective body function are necessary. The methods developed or adapted and used in our group are herewith briefly described.
2.
Materials and methods
2.1.
Incubation of eggs
The experiments were carried out in Muscovy duck (Cairina moschata f. domestica) and in chicken (Gallus gallus f. domestica) embryos during the second half of incubation. Eggs of the Muscovy duck need a total incubation period of 35 days and those of the chicken 21 days. The eggs were incubated at 37.5 ◦ C and at a relative humidity of 70% until the day at which the eggs were transferred into the hatcher, viz. embryonic day (E) 28 in the Muscovy duck and E17 in the chicken. During the incubation period the eggs were subjected to automatic turning. In the hatcher the eggs were incubated at 37.5 ◦ C and at a relative humidity of 90%. Experimental data, which were carried out under normal incubation temperature were used as control for experiments on influence of chronic changes in incubation temperature on the development of thermoregulatory mechanisms. For experiments on chronic influence of changes in incubation temperature on the development of thermoregulation, one group of eggs were incubated at 34.5 ◦ C (cold-incubated group) and another group at 38.5 ◦ C (warm-incubated group) from the day of transfer to the hatcher until hatching. After hatch, the birds were kept during the first 10 days either in the animal house at an ambient temperature of 25 ◦ C with additional infrared lamps (35 ◦ C) or in a temperature gradient channel (10–45 ◦ C). Food and water were given ad libitum.
2.2. Simultaneous measurement of body temperature and O2 -consumption in poultry embryos 2.2.1.
Body temperature
Embryonic body temperature was measured in the allantoic fluid (Taf ) near the embryo using a miniature thermistor probe (Testo GmbH & Co., Lenzkirchen, Germany). On the blunt side of the egg a square hole (2 mm × 2 mm) was drilled into the eggshell without damaging larger blood vessels. Through this hole a thermocouple was inserted into the allantoic fluid between the chorioallantoic membrane and the embryo (Figs. 1 and 2). After internal pipping (from E20 in chicken embryos and E33 in Muscovy duck embryos until hatching) it was also possible to measure the colonic temperature (Tc ). After locating the tail feathers, the cloaca was easily identified and the thermistor probe was inserted to a depth of 1–2 cm. Taf as well as Tc was measured continuously during the entire period of experiments. A comparison between Taf and Tc in single embryos showed only minor differences between Taf and Tc (ranging between 0.0 and 0.2 ◦ C) at constant incubation temperature. Under changing incubation temperature the difference rose to a range of 0.1–0.4 ◦ C (Holland et al., 1998). Details of the method are described by Holland et al. (1998) and Nichelmann and Tzschentke (2003).
2.2.2.
Measurement of O2 -consumption
Oxygen consumption was continuously measured using an oxygen analyser (Magnos 4, Hartmann & Braun, Frankfurt/Main, Germany) based on the paramagnetic principle.
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regression line for each individual embryo Q10 was calculated using the van’t Hoff formula (Precht et al., 1973): Q10 = (HPT2 /HPT1 )10/(T2−T1) (T1 > T2) in steps of 0.1 ◦ C changes in the Taf (Nichelmann et al., 1998).
2.3. Simultaneous measurement of body temperature and respiration parameters as well as body temperature and blood flow 2.3.1.
Recording of respiratory parameters
Breathing by lung occurs after internal pipping. The breathing activity induces pressure fluctuations in the air cell of the egg. These pressure fluctuations can be registered using a Statham element (Hugo Sachs Electronic KG, March, Germany) in a tube which is inserted in the air cell (Fig. 2). The recordings furnish information about the respiratory rate, the relative tidal volume and relative respiratory minute volume (Nichelmann and Tzschentke, 2003) in relation to changes in the body temperature. Fig. 1 – Schematic diagram of the metabolic chamber for single egg measurement (from Janke et al., 2004).
For these measurements single eggs were placed into the metabolic chambers (Fig. 1). The metabolic chambers (volume 290 ml) were watertight and consist of acrylic glass. The respective ambient (incubation) temperature was regulated by a temperature controlled water bath. After a minimum of 2 h of acclimation of the embryos to the respective ambient temperature, oxygen consumption simultaneously with ambient temperature and Taf were recorded for 1 h. Oxygen consumption (vol%) was measured as difference between oxygen concentration of the air inflow and outflow of the climatic chambers taking into account the actual gas flow (l/h). The influence of evaporation-water loss was removed by drying the gas streams before analysing. Detailed information on the methodology are published in Janke et al. (2004), Nichelmann et al. (1998, 2001) and Nichelmann and Tzschentke (2002, 2003).
2.2.3.
Calculation of heat production
Assuming a respiratory quotient of 0.72 (Decuypere, 1984), which corresponds to a caloric heat equivalent of 19.7 J ml−1 oxygen consumption, heat production (HP) was calculated on the basis of oxygen consumption, egg mass and caloric heat equivalent.
2.2.4.
Investigation of thermoregulatory heat production
To investigate the development of endothermic reactions acute temperature stimulations were applied by decrease (34.5–31.5 ◦ C) of ambient (incubation) temperature from the normal level (37.5 ◦ C) for 3 h using different water baths. Changes in HP were analysed in relation to simultaneously recorded body temperature (Taf ). As no net-increase in HP with decreasing body temperature was observed in embryos it was possible to estimate thermoregulatory counter reactions during cooling using the Q10 method. A quadratic regression was used to describe the relationship between HP and Taf . From the
2.3.2. Recording of the blood flow in the chorioallantoic membrane Blood flow in the chorioallantoic membrane was measured by MBF3 laser-Doppler instrument (Moor Instrument Company Ltd., Devon, GB). The laser-Doppler probe was placed directly on the egg membrane (Fig. 2). Before positioning the probe the egg was candled, to find an area with a large number of small blood vessels. Then a 5 mm × 5 mm piece of the egg shell was removed. The following parameters could be measured: mean red blood cell flow rate (flux), the red blood cell concentration and the mean red blood cell speed. These parameters were measured simultaneously with the embryonic temperature. Detailed information is furnished in Nichelmann and Tzschentke (2003).
2.4.
Recordings of neuronal thermosensitivity
2.4.1. Extracellular recordings of neuronal activity in relation to temperature stimulation in embryonic and post-hatching period Extracellular recordings of single cell activity were carried out in brain slices (400 m) from neurons of the preoptic area of the anterior hypothalamus (PO/AH). The slices were placed into a recording chamber (Schmid et al., 1993) and continuously perfused by artificial cerebrospinal fluid. From beginning of the experiment, the bath temperature in the recording chamber was maintained at 39 ◦ C in the embryos or 40 ◦ C in the birds post-hatching and continuously controlled by a small thermocouple. This temperature approximately corresponds to the deep body temperature in poultry at a later stage of embryonic development if incubated at the normal 37.5 ◦ C (Janke et al., 2002) or during the first days post-hatching (Tzschentke and Nichelmann, 1999). Neuronal activity and slice temperature were recorded by conventional electrophysiological equipment and stored on a personal computer using a 1401 interface (Cambridge Electronic Design) (CED) and the CED software Spike 2. To identify the thermosensitivity of single neurons, the bath temperature was sinusoidally changed to a maximum of ±3 ◦ C and a velocity of about 0.02 ◦ C s−1 . Temperature sensitivity was calculated by a
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Fig. 2 – Methods for recording of body temperature and different physiological parameters simultaneously. The focus in this figure is on localisation of thermistor probes for measurement of the temperature of the allantoic fluid (Taf ) and colonic temperature (Tc ), laser-Doppler-probe for estimation of the blood flow in the chorioallantoic membrane, ECG electrodes and tube with pressure differential sensor for recording of breathing activity in the avian egg.
computer program, adapted under Spike 2. The temperature response curve of each neuron was evaluated by relating firing rate to slice temperature and fitted to a piecewise and/or rectilinear regression function (Vieht, 1989). The thermosensitivity of a neuron was defined by a temperature coefficient of greater than or equal to 0.6 imp s−1 ◦ C−1 for warm-sensitive (WS) neurons and less than or equal to −0.6 imp s−1 ◦ C−1 for cold-sensitive (CS) neurons. All other neurons are named as temperature insensitive (TI) according to this definition (Nakashima et al., 1987). Further details of the methodology are described in Tzschentke and Basta (2002) and Tzschentke et al. (2004).
2.4.2. Changes in sensitivity of central thermoregulatory mechanisms in embryonic and post-hatching period For characterisation of the neuronal hypothalamic thermosensitivity the proportion of WS, CS and TI neurons in the PO/AH was determined in relation to all neurons investigated (Tzschentke and Basta, 2000, 2002). For instance, an increase in the proportion of CS neurons and a decrease in WS neurons in relation to all neurons investigated was used as a sign of elevation in total neuronal cold sensitivity of the PO/AH.
2.4.3.
Investigation of c-fos expression in poultry embryos
Compared to single cell recordings, the c-fos method can exhibit activity of numerous neurons at one time and thus demonstrate neuronal networks. It is expressed within a short time after changes in the environment of the organism. Because of this, the c-fos gene is considered as an immediate early gene. In the nucleus of the cell, c-fos can be detected by immunohistochemical method. In our investigations, on the last day of incubation acute heat stress (42.5 ◦ C) for 90 min was applied before starting the experi-
ment. Then the extracted embryos were anaesthetized and transcardial perfusion was performed. Brains were dissected and 20-m brain sections were made using a cryostat. In the PO/AH region of the slices c-fos expression was immunohistochemically detected. Analysis was made by light microscopy and digital photography (magnification of 50-fold). C-fos positive neurons were counted in a standardised area of the PO/AH using a rectangle mask. Due to the lack of stereotaxic data of the brain of the chicken embryo, the width of the rectangle was set proportional to the brain of the adult chicken at 990 m for all embryos. Stereotaxic data of the adult chicken brain were taken from Kuenzel and Masson (1988). For details see Janke and Tzschentke (2006).
2.5.
Identification of ‘critical periods’
Environmental manipulation of immature physiological mechanisms may be a physiological tool for characterization of ‘critical periods’ (Tzschentke and Plagemann, 2006). A typical reaction pattern of physiological mechanisms on acute and chronic environmental stimulation was found during the perinatal period (Tzschentke and Basta, 2002; Tzschentke et al., 2004). More details on this tool are given in this review under Section 3.
3.
Results and discussion
The methods applicated in this study are useful for basic investigations of special aspects of the development of the thermoregulatory system in single bird embryos. Altogether, with our different studies we could demonstrate fundamental developmental processes in chicken embryos using the
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thermoregulatory system as an example, which could be summarized in the following rules.
3.1. The development of peripheral as well as central nervous thermoregulatory mechanisms start in the course of the prenatal ontogeny To demonstrate, whether poultry embryos are already able to activate peripheral thermoregulatory mechanisms with changes in incubation temperature and if this activation is effective enough to regulate the embryonic body temperature, are only possible using simultaneous measurements of the embryonic body temperature and the respective parameter (e.g. HP, blood flow). With our methods the early activation of thermoregulatory mechanisms as well as differences in the developmental status between heat production and heat loss mechanisms in poultry embryos could be shown (Nichelmann and Tzschentke, 2003). Finally, during the late prenatal development poultry embryos have all prerequisites (peripheral and central nervous mechanisms) to react to changes in incubation temperature.
3.1.1.
Heat production
Long-term recordings of O2 -consumption indicated that in all precocial and altricial bird species investigated the development of HP under normal incubation temperature (37.5 ◦ C) follows an exponential function (Prinzinger and Dietz, 1995). Initially a continuous increase in HP is observed. After approximately 80% of incubation time, stagnation in HP occurs (plateau phase). At the end of the plateau phase the embryo pierces the chorioallantoic and inner shell membrane (internal pipping) and starts respiration through lungs (Tazawa and Whittow, 2000). After internal pipping until hatch there is a large increase in HP. Similar developmental pattern of HP was found in our experiments (Nichelmann et al., 1998; Janke et al., 2002). Even though there is a similar qualitative developmental pattern, quantitative differences (Fig. 3) in the development of HP could be found not only between poultry species (Janke et al., 2002) but also between poultry breeds (e.g. different high yielded chicken breeds, Janke et al., 2004). If HP is recorded simultaneously with embryonic body temperature (Taf ) a strong linear relationship between both parameters could be observed (Fig. 3). Under constant incubation temperatures this relationship could be described by a highly significant linear correlation (Janke et al., 2002). The relationship between HP and Taf changed absolutely, if acute changes in the incubation temperature occurred. With changing incubation temperature, e.g. during cooling, the relationship between HP and Taf can be described by quadratic regressions (Nichelmann et al., 1998). During acute decrease in incubation temperature Taf also shows a similar decrease with the difference in incubation temperature but Taf is always higher than ambient temperature. On the other hand, HP only decreases moderately (Fig. 4). A drop in body temperature, due to low incubation temperature mostly causes a decrease of net HP but the decrease is lower as assumed by the van’t Hoff rule (Nichelmann et al., 2001). To come to this conclusion is only possible after simultaneous recordings of HP and embryonic body temperature. Using the Q10 method (Nichelmann et al., 1998), an endothermic counter
Fig. 3 – Course of heat production (A) and body temperature (B) of two broiler chicken lines (Ross 308, Ross 508) and a layer chicken line (Lohmann White Leghorn) from day 9 and 11 of incubation until hatch. Means represent values of 6 embryos. The error bars represent standard deviations (from Janke et al., 2004).
reaction was found. The Q10 method enabled us to calculate the effect of thermoregulatory heat production with decreasing body temperature. It is a possibility to investigate the early development of endothermy in poultry embryos, which have no net-increase in HP after decrease in body temperature. In our investigations such endothermic counter reaction were already obtained before internal pipping in Muscovy duck and chicken embryos (Nichelmann et al., 1998). With increasing embryonic age the cold load induced decrease in HP, diminishes and near hatching time in some embryos a short-term
Fig. 4 – Course of body temperature (temperature of allantoic fluid) and heat production before and during 3 h cooling in a single Muscovy duck embryo on day 34 of incubation (Nichelmann and Tzschentke, 1999).
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Fig. 5 – Influence of increase in incubation temperature (ambient temperature, Ta ) on the course of body temperature (colonic temperature, Tc ) and blood flow in the chorioallantoic membrane in a single chicken embryo after internal pipping (modified from Holland et al., 1998).
increase in HP was observed. However, poultry embryos are not able to keep body temperature constant under cold load. In comparison with the heat loss mechanisms, in embryos efficiency of thermoregulatory heat production is very low. An increase in high-energy costly thermoregulatory HP to keep body temperature constant is not necessary for the survival of the bird embryos (Nichelmann and Tzschentke, 2001, 2002; Tzschentke, 2003) because precocial bird embryos show a high thermal tolerance, which protects them to some extent from disturbances by cooling (Whittow and Tazawa, 1991; Tazawa and Whittow, 2000). The phase of ‘full-blown’ homoeothermy starts during the first days of post-hatching (Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Tazawa et al., 2004).
3.1.2. Heat loss mechanisms 3.1.2.1. Changes in the blood flow of the chorioallantoic membrane. In poultry embryos non-specific changes in the blood flow of the chorioallantoic membrane due to changes in incubation temperature could be found already during the last third of incubation time. But at end of the plateau phase, blood flow increases with increasing incubation temperature or decreases with decreasing ambient temperature. In chicken embryos after internal pipping, for instance, the body core temperature remained constant for more than 40 min after the beginning of increase in ambient temperature up to 40.5 ◦ C (Fig. 5) by activating this heat loss mechanism (Nichelmann and Tzschentke, 2003). This result could be only found by measurement of the deep body temperature in the colon of the embryo. In these experiments, simultaneous measurements of Taf were not sensitive enough for the monitoring of the short-term regulation of the body temperature by changes in the blood flow of the chorioallantoic membrane (Holland et al., 1998).
3.1.2.2. Changes in respiration. First rhythmic contractions of the respiratory muscles without ventilation of the lung can be monitored already before internal pipping. One goal of this movement is to consolidate the morphology and function of the respiratory tract (Tazawa, 1987; Murzenok et al., 1997). The lung ventilation occurs after internal pipping. The developmental level of the respiration as a heat loss mechanism
Fig. 6 – Influence of colonic temperature (body temperature) on respiratory rate, tidal volume and relative respiratory minute volume in a single Muscovy duck embryo on day 34 of incubation (Nichelmann and Tzschentke, 1999). Respiratory rate is given in n min−1 , tidal volume and relative respiratory minute volume in arbitrary units.
in thermoregulation can be only investigated by simultaneous measurements of the respiratory rate and the deep body temperature. Between internal and external pipping, panting reactions were found in Muscovy duck embryos when body core temperature increased. In Muscovy duck embryos panting reactions show similar characteristics to adult birds (two phases of panting, Arad and Marder, 1983). At body core temperatures between 38.5 and 40.5 ◦ C respiratory rate increased and the tidal volume decreased. Above 40.5 ◦ C, the second phase of panting starts characterised by a decrease in respiratory rate and an increase in tidal volume (Fig. 6). At the later stage of incubation heat loss mechanisms (blood flow, respiration) in poultry embryo seem to be more effective in relation to control of body temperature than the heat production mechanisms. At the end of incubation both mechanisms show to a degree similar characteristics to postnatal birds.
3.1.3. Development of central nervous thermoregulatory mechanisms In poultry embryos thermoregulation is only possible if central nervous thermoregulatory mechanisms are developed early.
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mosensitivity of the PO/AH from the ‘juvenile’ to the ‘adult’ type (Tzschentke and Basta, 2000), which is characterised by a high warm sensitivity and a low cold sensitivity (Nakashima et al., 1987). Similar developmental pattern was also found in chicken during early postnatal development (Sallagundala et al., 2006). Besides the single cell recordings, we used the neuronal c-fos expression for demonstration of the activity of neuronal networks. Chicken embryos that were heat stressed (42.5 ◦ C for 90 min) on the last day of incubation show a clear expression of neuronal c-fos in the PO/AH (Janke and Tzschentke, 2006).
3.2. Acute changes in the environmental conditions induce as a rule, first uncoordinated and immediately non-adaptive reactions
Fig. 7 – Example of a hypothalamic cold sensitive neurone in a Muscovy duck embryo on day 22 of incubation (TC = thermal coefficient) imp s−1 ◦ C−1 , from Tzschentke et al. (2004).
We could find thermosensitive neurons in brain slices of Muscovy duck embryos using the method of extracellular single neuron recording. In this species thermosensitive PO/AH neurons were found on E22 (Fig. 7) and E23 that show characteristics similar to post-hatching (Tzschentke and Basta, 2000), growing (own experiments, unpublished data) and adult birds (Nakashima et al., 1987) as well as mammals (Schmid and Pierau, 1993). On E28 and E33 the proportion of CS, WS and TI neurons in relation to all neurons investigated was very constant and not significantly different from that in hatchlings (Tzschentke and Basta, 2000; Tzschentke et al., 2004). In contrast to the growing and adult ducks as well as mammals, the neuronal hypothalamic thermosensitivity in embryos and ducklings during the first days of post-hatching is characterised by a high neuronal cold sensitivity (Fig. 8). A qualitative change occurs between days 5 and 10 in the neuronal ther-
Fig. 8 – Influence of age on proportion of warm-, cold- and temperature-insensitive neurons in relation to all neurons investigated in their respective age groups in the preoptic area of the anterior hypothalamus of Muscovy ducks (modified from Tzschentke and Basta, 2000). Asterisks represent significance at the level of *p < 0.05 (D: embryonic day, d: day post-hatching).
To investigate the developmental level of the thermoregulatory system (open loop system without feedback mechanisms or closed system with developed feedback mechanisms) acute changes in ambient (incubation) temperature have to be applied. The reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different time windows of the perinatal period show a typical pattern. First uncoordinated and immediately non-adaptive reactions occur. Later the uncoordinated (immediately non-adaptive) reactions change into coordinated (adaptive) reactions, probably with closing of the regulatory system. First, during the development of physiological control systems it seems to be unimportant for the organism that a distinct adaptable reaction of physiological mechanisms on various environmental influences occurs, but rather that any reaction occurs seems to be important for the adaptability during the later life (Nichelmann et al., 1999, 2001; Tzschentke et al., 2004). These first reactions seem to be important for the ‘training’ of the respective function to develop feedback mechanisms. For instance, in chicken embryos the blood flow increased or decreased while warming or cooling on E15 until E19 (proximate non-adaptive). After this period, the reaction became proximate adaptive; on E20 and E21, the blood flow in the chorioallantoic membrane increased during warming and decreased during cooling, as expected (Fig. 9, Nichelmann and Tzschentke, 2003). Similar changes in the blood flow during cooling or warming were also found in Muscovy duck embryos at the end of incubation (Tzschentke, 2002). Also in other systems first proximate non-adaptive reactions on acute or chronic environmental stimulation was found in the course of the perinatal period. In Muscovy duck embryos, for instance, for different groups of reactions on acoustic stimulation could be classified; heart rate increase, heart rate decrease, increase or decrease in heart rate variability with¨ out a change in the mean heart rate (Hochel et al., 2002). Also after chronic changes in the incubation temperature at the end of embryonic development first proximate nonadaptive changes occurred in the heat production (Loh et al., 2004), neuronal hypothalamic thermosensitivity (Tzschentke and Basta, 2002) and neuronal c-fos expression after acute temperature application (Janke and Tzschentke, 2006). Obviously, the development of regulatory systems from an open loop system without feedback into a closed control sys-
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Fig. 9 – Influence of warming (38.5 ◦ C) and cooling (35.5 ◦ C) on blood flow in the chorioallantoic membrane of 15- to 21-day-old chicken embryos. Each column represents the reaction in one individual embryo, expressed in Flux, which is given in arbitrary units. The blood flow was measured using the laser-Doppler method (from Nichelmann and Tzschentke, 1999, 2003).
tem with feedback is a ‘critical period’ in the ontogeny of physiological control systems, which is of high importance for the later development in poultry (see Section 3.3). The changes in the reaction pattern on environmental influences could be typical for this phase. In conclusion, environmental manipulation of immature physiological mechanisms may be a physiological tool for characterization of ‘critical periods’ of the respective system (Tzschentke and Plagemann, 2006).
3.3. During critical developmental phases, a long-term adaptation to an actual environment occurs via epigenetic adaptation processes Our hypothesis states that, in the course of the perinatal period, ‘imprinting’ of physiological control systems occur which, probably is realized by both neural ‘imprinting’ at the microstructural level (e.g., in terms of synaptic plasticity) as well as by a lasting environment-induced modification of the genome (Tzschentke and Plagemann, 2006). During ‘critical periods’ of development of physiological control systems (mentioned in the last paragraph), the actual level at which physiological parameters occur may pre-determine the ‘set-point’ (‘set ranges’) of the respective physiological control system during the entire life period, possibly through acquired changes in the expression of related effector genes (Fig. 10). On one hand, this mechanism seems to be a possible basis for perinatal malprogramming which, e.g., causes metabolic disorders and cardiovascular diseases as well as behavioural disorders during later life in mammals including man (Plagemann, 2004) as well as in birds (Schwabl, 1996, 1997; Ruitenbeek et al., 2000). On the other hand, knowledge and better understanding of these mechanisms might be specifically used to induce long-term adaptation of an organism, for instance, to postnatal climatic conditions (epigenetic temperature adaptation; Nichelmann et al., 1994, 1999; Tzschentke and Basta, 2002; Tzschentke et al., 2004). In chicken and other precocial birds epigenetic temperature adaptation can be induced by changes in incubation temperature at the end of embryonic development (Decuypere, 1984; Minne and Decuypere, 1984; Nichelmann et al., 1994; Tzschentke and Nichelmann, 1997; Tzschentke and Basta,
2002; Loh et al., 2004) as well as by thermal conditioning during the first days after hatching (Yahav and Plavnik, 1999; Yahav, 2000). Altogether, prenatal temperature experiences induce postnatal warm or cold adaptation (Tzschentke et al., 2004) (Fig. 11). On the first day of post-hatching Muscovy ducklings incubated at lower temperatures than normal, for instance, have a 56% higher heat production and a higher deep body temperature under cold load as compared to controls (1 h at 10 ◦ C). Cold-incubated birds are able to control their actual deep body temperature at this set-point, in contrary to those incubated at 37.5 ◦ C, which display a lower heat production (Nichelmann et al., 1994; Tzschentke et al., 2004). Further, Muscovy ducklings incubated at a low temperature preferred a significantly lower temperature than birds incubated at the normal incubation temperature during the first 10 days of post-hatching. This supports the hypothesis that avian prenatal cold experience leads to a downward shift of the thermoregulatory set-point (Tzschentke and Nichelmann, 1999).
Fig. 10 – Basic concept of induction of epigenetic perinatal malprogramming or epigenetic adaptation processes, such as epigenetic temperature adaptation by environmental factors during ‘critical periods’ of early development.
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Fig. 11 – Epigenetic temperature adaptation in Muscovy duck embryos. Embryos were incubated from day 28 of incubation until hatching in either warmer or colder temperatures than the usual 37.5 ◦ C. A: Changes in the neuronal hypothalamic thermosensitivity at day 10 post-hatching induced by changes in incubation temperature (significant differences, *p < 0.05, 2 -test). For characterization of the neuronal hypothalamic thermosensitivity the proportion of warm sensitive, cold sensitive and temperature insensitive neurons in the PO/AH was determined in relation to all neurons (n = 80 neurons) investigated in the respective incubation groups. B: HP and C: colonic temperature of hatchlings from cold (34.5 ◦ C) and normal (37.5 ◦ C) incubated eggs after 1 h exposure to 10 ◦ C (significant differences, *p < 0.05, t-test).
On the other hand, the preferred ambient temperature in 1- to 10-day-old turkeys is higher after a prenatal heat load (38.5 ◦ C) than in birds incubated at the normal temperature (37.5 ◦ C). This indicates an elevation of the thermoregulatory set-point after prenatal heat load. In our experiments changes in the neuronal thermosensitivity of the hypothalamic control centre of the thermoregulatory system reflect the changes in peripheral thermoregulatory mechanisms after prenatal temperature experiences. Prenatal cold experience increases on day 10 of post-hatching the neuronal hypothalamic warm sensitivity and prenatal heat experiences increase neuronal hypothalamic cold sensitivity. In differentially incubated birds the change in the levels of HP and in neuronal hypothalamic thermosensitivity (Loh et al., 2004) and in neuronal hypothalamic c-fos expression (Janke and Tzschentke, 2006) occur already before hatching. But these changes are proximate non-adaptive during this developmental period. It is interesting to note, that the embryonic body temperature is more influenced by chronic changes in incubation temperature than the HP. If the HP is measured at temperatures at which the embryos were adapted, the HP in cold-incubated (34.5 ◦ C) embryos, for instance, was not lower than in the control, which was incubated at 37.5 ◦ C. On other hand the body temperature was 3 ◦ C lower in the cold-incubated than in the control group (Loh et al., 2004). But
this lower body temperature could be a prerequisite for a lower thermoregulatory set-point during the post-hatching period. In experiments with warm-incubated chicken and Muscovy duck embryos (38.5 ◦ C during the last third of incubation) Taf was approximately 1 ◦ C higher than in the control. This could be a prerequisite for a higher thermoregulatory set-point during the entire life (Loh et al., 2004).
4.
Conclusions
In poultry embryos autonomic and central nervous thermoregulatory mechanisms are developed. Latest at the end of incubation poultry embryos have all prerequisites to react to changes in incubation temperature. During this developmental period some physiological mechanisms show to certain degree similar characteristics to postnatal birds. Regarding the autonomic thermoregulatory mechanisms we could come to this conclusion only by simultaneous recordings of the respective mechanism and the embryonic body temperature. The most sensitive parameter for characterization of the developmental level of embryonic thermoregulation after acute changes in incubation temperature is the deep body temperature (colonic temperature). But its measurement is limited to the post-internal pipping period. During earlier developmen-
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tal stages the internal egg temperature (Taf ) can be used as a very good measure of the embryonic body temperature under constant incubation temperatures. However, for characterization of the thermoregulatory ability under acute temperature changes Taf measurements are limited. During critical developmental periods epigenetic adaptation processes can induce a long-term adaptation to the actual environment. Perinatal epigenetic temperature adaptation could be a tool to adapt poultry embryos or hatchlings to later climatic conditions. For the specific use of perinatal epigenetic temperature adaptation in practice more investigations on the basic mechanisms of ‘imprinting’ of physiological control systems and on the problem of ‘critical periods’ are necessary. Environmental manipulation of immature physiological mechanisms could be used for characterization of ‘critical periods’ of the respective system. Monitoring of changes in the reactions of thermoregulatory mechanisms on the applied changes in incubation temperature during different perinatal time windows could help to limit ‘critical periods’ in the development of the thermoregulatory system. For detection of immediate and long-term effects of perinatal epigenetic temperature adaptation (‘imprinting’ of the thermoregulatory system) changes in plasticity of the central controller of thermoregulation in the hypothalamus are important. Recordings of changes in neuronal hypothalamic thermosensitivity as well as in neuronal response on temperature stress are useful tools and have to be verified by identification of the respective effector genes and epigenetic changes in its expression.
Acknowledgements Research projects carried out in our working group “Perinatal Adapation” were supported by grants of the Deutsche Forschungsgemeinschaft (Ni 336/3-1; TZ 6/2-1, 6/2-2, 6/6-4, 6/10, JA 1440/1-1).
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