Attainment of Thermoregulation as Affected by Environmental Factors1
B. Tzschentke2
Institute of Biology, Humboldt-University of Berlin, 10115, Germany
ABSTRACT The review addresses the development of
thermoregulation in poultry embryos as well as the effect
of acute and chronic changes of environmental factors on
this process and the incubation temperature being the
foremost. In poultry, the early development of adaptive
body functions, like the thermoregulatory system, is characterized by the following peculiarities. First, the development of peripheral as well as central nervous thermoregulatory mechanisms start during the prenatal ontogeny.
However, their maturity is attained during early postnatal development. In the perinatal period, environmental
factors have a high effect on development of temperature
regulation. Second, acute changes in the environmental
conditions induce as a rule first uncoordinated and immediately nonadaptive reactions. Later, the uncoordinated
nonadaptive reactions change into coordinated (adaptive)
reactions. Prenatal environmental influences may have a
training effect on the postnatal efficiency of the thermoregulatory system. Third, functional systems of the organism develop from an open loop system without feedback
control into a closed system controlled by a feedback
mechanism. During this critical period, the actual environment modulates the development of the respective
physiological control systems for the entire life period,
especially by changes in neuroorganization and expression of related effector genes. Knowledge on these mechanisms might be specifically used to generate long-term
adaptation of the organism to the postnatal climatic conditions (perinatal epigenetic temperature adaptation). In
poultry, perinatal epigenetic temperature adaptation was
developed by changes in the incubation temperature.
When a comparison is made in birds, which were incubated at 37.5°C, a low incubation temperature induced
postnatal cold adaptation, and warm incubation temperature induced postnatal heat adaptation. Perinatal epigenetic temperature adaptation exhibited changes in the
neuronal thermosensitivity in the hypothalamus as well
as in the peripheral thermoregulatory mechanisms. These
alterations could be already found at the end of incubation. Further, temperature-experienced embryos have a
lower c-fos expression than in the control after acute
heat stress.
Key words: thermoregulation, environmental factor, poultry embryo, imprinting, critical period
2007 Poultry Science 86:1025–1036
INTRODUCTION
The development of body functions starts early during
the embryonic phase in precocial birds, especially poultry.
This early and very sensitive developmental phase is of
high significance for the adaptability of the organism during later life.
In the hierarchy of regulatory systems, the thermoregulatory system is on a higher level. The goal of temperature
regulation in homoeothermic animals in the postnatal
developmental phase is the maintenance of a stable body
core temperature under most conditions. To realize this,
the thermoregulatory system employs all of the systems
of the body and integrates their activities into appropriate
©2007 Poultry Science Association Inc.
Received November 9, 2006.
Accepted November 19, 2006.
1
Presented as part of the Embryo Symposium: Managing the Embryo
for Performance, July 19, 2006, at the Poultry Science Association Annual
Meeting, Edmonton, Alberta, Canada.
2
Corresponding author: [email protected]
and coordinated reactions. The prenatal development of
thermoregulatory mechanisms in precocial animals is
beneficial for the quick maturation of temperature regulation in the early posthatching phase, which is important
for the performance of the whole organism.
From the results of our investigations and from related
scientific literature, we postulated general rules for the
development of physiological control systems, including
the thermoregulatory system (Nichelmann et al., 1999,
2001; Tzschentke and Basta, 2002; Tzschentke et al., 2004):
I. The development of peripheral as well as central
nervous thermoregulatory mechanisms starts during the prenatal ontogeny.
II. Acute changes in the environmental conditions induce as a rule first uncoordinated and immediately
nonadaptive reactions. Later, the uncoordinated
(immediately) nonadaptive reactions change into
coordinated (adaptive) reactions.
III. During critical developmental phases, a long-term
adaptation to an actual environment occurs via epigenetic adaptation processes.
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TZSCHENTKE
The review addresses the development of thermoregulation in poultry embryos as well as the effect of acute and
chronic changes of environmental factors on this process,
with incubation temperature being the foremost. Further,
the fundamental process of imprinting of physiological
control systems during critical periods of early ontogeny
(Tzschentke and Plagemann, 2006) is described. The
methods developed or adapted and used in my group
are herewith briefly described.
MATERIALS AND METHODS
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 d, and those of the chicken need 21 d. The eggs
were incubated at 37.5°C and at a RH 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 this period, the eggs were subjected
to automatic turning. In the hatcher, the eggs were incubated at 37.5°C and at a RH of 90%. For experiments on
chronic influence of changes in incubation temperature
on the development of thermoregulation, 1 group of eggs
was incubated at 34.5°C (cold incubated group) and another group at 38.5°C (warm incubated group). After
hatch, the birds were kept during the first 10 d 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 to 45°C). Food and water were given
ad libitum.
Measurement of Body Temperature
Before internal pipping, embryonic temperature was
measured in the allantoic fluid (Taf) near the embryo
using a miniature thermistor probe (Figure 1). On the
blunt side of the egg, a hole (2 × 2 mm) was drilled
into the eggshell without damaging larger blood vessels.
Through this hole, a thermocouple was inserted into the
Taf to a depth of 2.5 cm between the chorioallantoic membrane and the embryo. 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 to 2 cm. Details of the method
are described by Holland et al. (1998) and Nichelmann
and Tzschentke (2003). Allantoic fluid as well as Tc were
measured continuously during the entire period of experiments. In some embryos, both temperatures were measured simultaneously. There were only minor differences
between Taf and Tc (ranging from 0.0 to 0.2°C) at constant
incubation temperature. Under changing incubation temperature, the difference rose to a range of 0.1 to 0.4°C
(Holland et al., 1998). After posthatch, the Tc was gener-
Figure 1. Localization 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, and tube with pressure differential sensor for recording
of breathing activity in the avian egg.
ally used for measurement of deep body temperature
(Tzschentke and Nichelmann, 1999).
Measurement of Heat Production
Assuming a respiratory quotient of 0.72 (Decuypere,
1984), which corresponds to a caloric heat equivalent of
J/mL of O2, heat production (HP) was calculated from
O2 consumption, egg weight, and caloric heat equivalent,
taking into account the actual gas flow. Oxygen consumption was continuously measured using an O2 analyzer
based on the paramagnetic principle. For these measurements, single eggs were placed into the metabolic chambers, and 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, O2 consumption and
the ambient temperature (as well as Taf) were recorded
for 1 h. For acute temperature stimulation of HP, the
ambient temperature was increased (39.5°C) or decreased
(34.5 to 31.5°C) from the normal level (37.5°C) for 3 h
using another water bath. Detailed information on the
methodology is published in Nichelmann et al. (1998,
2001) and Nichelmann and Tzschentke (2002, 2003). Posthatching HP was obtained by a similar method (Tzschentke et al., 1996; Tzschentke and Nichelmann, 1999).
Recording of Respiratory Rate
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 in a tube, which was
inserted in the air cell (Figure 1). The recordings gave
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EMBRYO SYMPOSIUM
information about the respiratory rate, the relative tidal
volume, and relative respiratory minute volume
(Nichelmann and Tzschentke, 2003).
Recording of the Blood Flow
in the Chorioallantoic Membrane
Blood flow in the chorioallantoic membrane was measured by MBF3 laser Doppler instrument. The laser Doppler probe was placed directly on the egg membrane (Figure 1). Before positioning the probe, the egg was candled
to find an area with many small blood vessels. Then a 5
× 5 mm piece of the eggshell was removed. The following
parameters could be measured: mean red blood cell flux,
the red blood cell concentration, and the mean red blood
cell speed. Detailed information is furnished in
Nichelmann and Tzschentke (2003).
Figure 2. Development of heat production and body temperature
(temperature of allantoic fluid) in Muscovy duck embryos from d 20
until d 34 of incubation at an incubation temperature of 37.5°C (modified
from Janke et al., 2002).
Recordings of Neuronal Activity
Extracellular recordings of single-cell activity were carried out in brain slices (400 ␮m) from neurons of the
preoptical 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 the 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 posthatching 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 posthatching
(Tzschentke and Nichelmann, 1999). 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. The temperature response curve of
each neuron was evaluated by relating firing rate to slice
temperature and fitted to a piecewise, rectilinear, or both
regression function (Vieht, 1989). The thermosensitivity
of a neuron was defined by a temperature coefficient of
greater than or equal to 0.6 impulse/s per degree Celsius
(change in temperature) for warm-sensitive (WS) neurons
and less than or equal to −0.6 impulse/s per degree Celsius for cold-sensitive (CS) neurons. All other neurons
are named as temperature insensitive (TI) according to
this definition (Nakashima et al., 1987). For characterization 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). Further details of the methodology are described in Tzschentke and Basta (2002) and
Tzschentke et al. (2004).
Investigation of c-fos Expression
On the last day of incubation, acute heat stress (42.5°C)
for 90 min was applied before starting the experiment.
Then, the extracted embryos were anesthetized and trans-
cardial 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
detected by immunohistochemical method. Analysis was
made by light microscopy and digital photography (magnification of 50-fold). The c-fos-positive neurons were
counted in a standardized 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).
Determination of Preferred Ambient
Temperature
Preferred ambient temperature was determined in a
temperature gradient tunnel temperated between 10 and
45°C. Groups of 5 birds were kept for 10 d in the temperature gradient immediately after hatching. During this period, the chosen ambient temperatures were observed for
9 h every 10 min every day (Tzschentke and
Nichelmann, 1999).
RESULTS AND DISCUSSION
Development of Peripheral as well
as Central Nervous Thermoregulatory
Mechanisms in Poultry Embryos
The development of peripheral as well as central nervous thermoregulatory mechanisms in precocial birds
starts during the prenatal ontogeny. However, their maturity is attained during early postnatal development
(Nichelmann and Tzschentke, 2002).
Development of Endothermic Reactions. The development of HP and body temperature (Taf) under normal
incubation temperature (37.5°C) follows an exponential
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TZSCHENTKE
Figure 3. Course of body temperature (temperature of allantoic fluid)
and heat production before and during 3 h of cooling in a single Muscovy
duck embryo on d 34 of incubation (Nichelmann and Tzschentke, 1999).
Figure 4. Influence of increase in incubation temperature [ambient
temperature (Ta)] on the course of body temperature [colonic temperature (Tc)] and blood flow (measured in arbitrary units of flux) in the
chorioallantoic membrane in a single chicken embryo after internal
pipping (modified from Holland et al., 1998).
function (Nichelmann et al., 1998; Janke et al., 2002). Initially a continuous increase in HP is observed. In precocial
birds 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 the lungs (Tazawa and Whittow, 2000).
From internal pipping until hatch, HP increases a lot. A
similar developmental pattern of HP has been found in
all precocial and altricial bird species investigated (Prinzinger and Dietz, 1995). It is interesting to note that Taf
follows the developmental pattern of HP (Figure 2). The
relationship between HP and Taf could be described by
a highly significant linear correlation (Janke et al., 2002).
The subject of investigations was if and at which stage
of embryonic development poultry embryos change HP
during acute alterations of ambient (incubation) temperature. In our investigations, we already found endothermic
reactions before internal pipping in Muscovy duck and
chicken embryos (Nichelmann et al., 1998). During an
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
(Figure 3). 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). An endothermic counterreaction occurs, which can be determined using the Q10
method (Nichelmann et al., 1998). With increasing embryonic age, the cold load induced decrease in HP diminishes. Near hatching time, with decreasing Taf, in some
embryos, a short-term increase in HP was observed. However, poultry embryos are not able to keep body temperature constant under cold load. The phase of full-blown
homeothermy starts during the first days of posthatching
(Tazawa and Rahn, 1987; Nichelmann and Tzschentke,
2002; Tazawa et al., 2004).
Altogether, in comparison with the heat loss mechanisms, efficiency of thermoregulatory HP is very low in
embryos. 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 remarkable tolerance of precocial bird embryos to low body temperatures seems to be
a part of their thermal strategy to save energy. An increase
in high-energy, costly thermoregulatory HP to keep body
temperature constant is, to a degree, not necessary for
the survival of the bird embryos (Nichelmann and
Tzschentke, 2001, 2002; Tzschentke, 2003).
Changes in the Blood Flow of the Chorioallantoic
Membrane. During the last third of incubation time, poultry embryos are able to react on changes in incubation
temperature with changes in the blood flow of the chorioallantoic membrane (Figure 4). At end of the plateau
phase, blood flow increases with increasing incubation
temperature or decreases with decreasing ambient temperature. In chicken embryos, for instance, the body core
temperature remains constant for more than 40 min after
the beginning of the increase in ambient temperature by
activating this heat loss mechanism (Nichelmann and
Tzschentke, 2003).
Changes in Respiration. First, rhythmic contractions
of the respiratory muscles start before internal pipping.
These movements are without ventilation of the lung.
One goal of this movement is to consolidate the morphology and function of the respiratory tract (Tazawa, 1987;
Murzenok et al., 1997). After internal pipping, lung ventilation occurs. In Muscovy duck embryos between internal
and external pipping, panting reactions were found when
body core temperature increased. Like in adult birds
(Arad and Marder, 1983), 2 phases of panting occurred in
Muscovy duck embryos, too. At body core temperatures
from 38.5 to 40.5°C, respiratory rate increased and the
tidal volume decreased, and above 40.5°C, the second
phase of panting started, which was characterized by a
decrease in respiratory rate and an increase in tidal volume (Figure 5).
Communication with the Environment. After internal
pipping, acoustic responses can be used by the bird em-
EMBRYO SYMPOSIUM
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Figure 6. 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. E = embryonic day;
d = day posthatching.
Figure 5. Influence of colonic temperature (body temperature) on
respiratory rate, tidal volume, and relative respiratory minute volume
in a single Muscovy duck embryo on d 34 of incubation (Nichelmann
and Tzschentke, 1999). Respiratory rate is given in number/minute;
tidal volume and relative respiratory minute volume is given in arbitrary units.
bryo for protection of endothermic reactions (Nichelmann
and Tzschentke, 1997). In a cold environment, the distress
call rate increases. After rewarming, this rate decreases
again. It is found in embryos of geese (Nichelmann and
Tzschentke, 1997) and ring-billed gulls (Larus delawarensis;
Evans et al., 1994). Under natural conditions, cold-induced vocalization is a signal of the need of the offspring
for warmth from the incubating parents.
Another possibility for communication between the
embryo and the incubating parents seem to be NO emission (Ifergan and Ar, 1999). Nitric oxide emission was
found mainly via the chorioallantoic membrane in a study
made on 18 bird species under cold conditions by Ar et
al. (2004). Nitric oxide emission was increased under cold
load. Ar et al. (2004) speculated that NO emission from
eggs might carry a “message” from the embryo to the
incubating parents and vice versa. It was suggested from
the same group to use NO emission as a general marker
for embryonic stress (Samuni and Ar, 2006).
Development of Central Nervous Thermoregulatory
Mechanisms. Just like peripheral mechanisms of thermoregulation, central nervous thermoregulatory mechanisms are developed early and might show the same
fundamental characteristics in the prenatal condition as
experienced in the postnatal. During investigation in
Muscovy duck embryos, Tzschentke and Basta (2000)
found thermosensitive PO-AH neurons on E22 and E23
that showed characteristics similar to posthatching
(Tzschentke and Basta, 2000), growing (unpublished
data), and adult birds (Nakashima et al. 1987) as well
as mammals (Schmid and Pierau, 1993). From d 28 of
incubation until hatching, 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 (Figure 6; Tzschentke and Basta, 2000,
Tzschentke et al., 2004).
In contrast to the growing and adult ducks, the neuronal hypothalamic thermosensitivity in embryos and
ducklings during the first days of posthatching is characterized by a higher neuronal cold sensitivity. A qualitative
change occurs from d 5 to 10 in the neuronal thermosensitivity of the PO-AH from the “juvenile” to the “adult”
type (Tzschentke and Basta, 2000), which is characterized
by a high warm sensitivity and a low cold sensitivity
(Nakashima et al., 1987). A similar developmental pattern
is also found in chicken during early postnatal development (Sallagundala et al., 2006).
In Muscovy duck embryos as well as in ducklings during the first 10 d of life (Tzschentke et al., 2000), thermosensitivity of PO-AH neurons can be modulated by the
neuropeptide bombesin. Bombesin is one of the most investigated neuropeptides known to influence thermoregulation in ectothermic and endothermic vertebrates
(Brown et al., 1977; Janský et al., 1987; Schmid et al., 1993;
Kozlovskii and Pastukhov, 1995; Leger and Mathieson,
1997). Further, temperature guardian neurons, which are
temperature sensitive over a small temperature range not
more than 1°C during the applied sinusoidal temperature
range, have been found in Muscovy duck embryos on
E28 (Tzschentke and Basta, 2000; Tzschentke et al., 2004).
Such neurons were first described from my group in 10-
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TZSCHENTKE
d-old Muscovy ducklings (Basta et al., 1997). Temperature
guardian neurons are exclusively sensitive to extreme
low or high brain temperatures and may activate more
effective thermoregulatory mechanisms if the normal regulatory range is exceeded.
Altogether, during late prenatal development, poultry
embryos have all prerequisites (autonomic, behavioral,
and central nervous mechanisms) to react on changes
in incubation temperature. In bird embryos, especially
studied in the Muscovy duck, mechanisms for central
nervous control of temperature regulation are well developed. For early consolidation and maturation of body
functions, sensory inputs are necessary. Environmental
influences (e.g., temperature, light, acoustic signals) can
stimulate this process (training effect). The typical reaction pattern of body functions, especially thermoregulatory mechanisms, on external stimuli during early ontogeny has been explained in detail in the following section.
Typical Reaction Pattern of Physiological
Mechanisms on Acute and Chronic
Environmental Stimulation during the
Perinatal Period
Environmental manipulations during the prenatal or
early postnatal phase first lead to uncoordinated and almost nonadaptive reactions of the respective physiological control systems. The theory is that during early ontogenesis of body functions, it seems not to be important
for the organism that a distinct adaptable reaction on
various environmental influences occurs, but rather that
any reaction occurs seems to be important for the adaptability during later life (training effect). These proximate
nonadaptive reactions become coordinated and adaptive
during later development, probably with closing of the
regulatory system. For instance, experiments at the end
of incubation time in chicken embryos have revealed first
proximate nonadaptive and later adaptive reactions with
respect to the influence of cooling and warming on blood
flow in the vessels of the chorioallantoic membrane
(Nichelmann and Tzschentke, 2003). In chicken embryos,
the blood flow increased or decreased while warming or
cooling on E15 until E19 (proximate nonadaptive). 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 (Figure 7). Similar changes in the
blood flow during cooling or warming have also been
found in Muscovy duck embryos at the end of incubation
(Tzschentke, 2002). Also in other systems (e.g. heart frequency; Höchel et al., 2002) and on cellular levels in the
brain (e.g. neuronal hypothalamic thermosensitivity,
mentioned in next paragraph), first proximate nonadaptive reaction on acute or chronic environmental stimulation was found during the perinatal period. As shown in
Figure 7, in chicken embryos, E19 seems to be the critical
day related to changes from proximate nonadaptive into
adaptive reactions in the blood flow in the chorioallantoic
membrane under both environmental conditions (cooling
as well as warming). In Muscovy ducklings, this change
after cooling and warming seems to occur on different
days of incubation. It is similar with the early development of the primate visual system, in which for each
specific visual function, different and partially overlapping critical periods were found (Harwerth et al., 1986).
However, this characteristic reaction pattern could be
a physiological tool, that helps to characterize a critical
period of the respective system during early development, whereas regulatory systems develop from an open
loop system without feedback into a closed control system
with feedback (Tzschentke and Plagemann, 2006).
Imprinting of Physiological Control
Systems during the Perinatal Period:
Prenatal Epigenetic Temperature
Adaptation
Basic Concept of Imprinting of Physiological Control Systems. During critical developmental periods, a
long-term adaptation to the actual environment occurs
via epigenetic adaptation processes. Our hypothesis
states that, during the perinatal period, imprinting of
physiological control systems occur that is probably realized by both neural imprinting at the microstructural
level (e.g., in terms of synaptic plasticity) as well as by
lasting environment-induced modification of the genome
(Tzschentke and Plagemann, 2006). Basically, most functional systems of the organism develop from open loop
systems without feedback control into closed control systems regulated by feedback mechanisms. During critical
periods, the actual level at which physiological parameters occur may predetermine 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. Determination of
the set point depends on the environment experienced
by the embryo and fetus during critical periods of development (first described as determination rule by Dörner,
1974). In general, in the etiological concept of epigenetic
perinatal programming of the lifetime function of fundamental regulatory systems, developed by Dörner (1974),
hormones play a decisive role as environment-dependent
organizers of the neuroendocrine immune system, which
finally regulates all fundamental processes of life (Dörner,
1975, 1976). During critical periods, hormones as well as
neurotransmitters and cytotokines (as immune cell hormones) act as critical endogenous effectors that transmit
environmental information (e.g., sensible input) to the
genome. Finally, they thereby also act as epigenetic
factors.
On one hand, this mechanism seems to be a possible
basis for perinatal malprogramming, which 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-
EMBRYO SYMPOSIUM
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Figure 7. Influence of warming (38.5°C) and cooling (35.5°C) on blood flow in the chorioallantoic membrane of 15- to 21-d-old chicken and 26to 34-d-old Muscovy duck embryos. Each column represents the reaction in 1 individual embryo, expressed in flux, which is given in arbitrary
units. The blood flow was measured using the laser Doppler method (modified from Nichelmann and Tzschentke, 1999, 2003).
term adaptation of an organism, for instance, to postnatal
climatic conditions (epigenetic temperature adaptation).
Figure 8 summarizes this conceptional approach.
Epigenetic Temperature Adaptation. Knowledge on
imprinting of physiological control systems might be specifically used to induce long-term adaptation of an organism, for instance, to the postnatal climatic conditions. For
an imprinting like that of the thermoregulatory system,
the term epigenetic (temperature) adaptation was introduced (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). The change in the levels of HP in differentially
incubated birds occur already before hatching (Figure 9;
Loh et al., 2004).
On the last day of incubation, the cold-experienced
chicken embryos had a significant higher HP when measured at 37.5°C. When investigations were made at
34.5°C, cold-incubated embryos did not show lower HP
compared with the control (incubated at 37.5°C), even
though the incubation temperature as well as Taf were at
3°C lower than in the control (Figure 10). The interesting
aspect is that warm incubation seems to stimulate HP
as well. However, to explain changes in HP in poultry
embryos due to prenatal temperature experiences, it
should be taken into consideration that temperature-dependent alterations in physiological processes can be
caused by the Q10 effect related to the Van’t Hoff rule
(Precht et al., 1973), by epigenetic mechanisms, or both. As
described in the previous paragraph, further, proximate
nonadaptive reactions on external stimuli can occur during the early development of body functions.
On the first day posthatching, Muscovy ducklings incubated at lower temperatures than normal, for instance,
having a 56% higher HP and a higher deep body temperature under cold load as compared with controls (1 h at
10°C). Cold-incubated birds are able to control their actual
deep body temperature at their set point, in contrast to
those incubated at 37.5°C, which display a lower HP
(Nichelmann et al., 1994; Tzschentke et al., 2004). Minne
and Decuypere (1984) found that cold-incubated chicken
had a higher HP over a range of ambient temperatures up
to 8 wk of posthatching. The higher HP was accompanied
with higher triiodothyronine and thyroxine levels. In a
temperature gradient, Muscovy ducklings incubated at a
low temperature preferred a significantly lower temperature than birds incubated at the normal incubation temperature during the first 10 d of posthatching. This supports the hypothesis that avian prenatal cold experience
leads to a downward shift of the thermoregulatory set
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Figure 8. Induction of epigenetic perinatal malprogramming or epigenetic adaptation processes such as epigenetic temperature adaptation by
environmental factors during the critical period of early development; basic concept (Tzschentke and Plagemann, 2006).
point (Tzschentke and Nichelmann, 1999). On the other
hand, the preferred ambient temperature in 1- to 10-dold 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.
Prerequisites for the changes in the thermoregulatory
set point are the changes that occur in prenatal body
temperature during critical periods of early development
(as mentioned under “Basic Concept of Imprinting of
Physiological Control Systems”). In our experiments, we
found a strong influence of chronic changes in incubation
Figure 9. Heat production of cold-, warm-, and normal-incubated chicken embryos on the last day of incubation investigated at 37.5°C (left
panel) and at different incubation temperatures when the embryos were adapted (right panel). Results presented in boxplots are based on median
and quartiles. Compared with all investigated groups, cold-incubated embryos have a significantly (P < 0.05) higher heat production when
investigated at 37.5°C. The heat production in warm-incubated embryos is significantly (P < 0.05) higher when investigated at the respective
adaptation temperature (modified from Loh et al., 2004); c = cold-incubated/cold temperature (34.5°C); n = normal-incubated/normal temperature
(37.5°C); w = warm-incubated/warm temperature (38.5°C); the first letter indicates the incubation group and the second the incubation temperature
at which the experiments were carried out.
EMBRYO SYMPOSIUM
1033
Figure 10. Influence of incubation temperature on embryonic temperature (temperature of allantoic fluid) in warm- (ww; 38.5°C), cold- (cc;
34.5°C), and normal- (nn; 37.5°C) incubated chicken embryos from d 18 of incubation until hatch (Loh et al., 2004). Results presented in boxplots
are based on median and quartiles (* is an extreme value). Note: In the present experiments, the first day of incubation is d 1 (in practice, it is d
0). In cold-incubated embryos, the development is delayed. These birds hatch 2 d later than the other groups. n = number of birds investigated.
temperature at the end of embryonic development on
body temperature (Taf) of chicken as well as Muscovy
duck embryos (Figure 10). If there occurs a critical period
at the end of the embryonic development of the thermoregulatory system, the lower Taf under cold conditions
and the higher Taf under warm conditions is related with
the lower and higher posthatching set points.
Our basic concept of imprinting of physiological control systems (Tzschentke and Plagemann, 2006) states that
on one hand this process is probably realized via neural
imprinting at the microstructural level (e.g., in terms of
synaptic plasticity). In our experiments, changes in the
neuronal thermosensitivity of the hypothalamic control
center of the thermoregulatory system reflect the changes
in peripheral thermoregulatory mechanisms after prenatal temperature experiences. Prenatal cold experience increases on d 10 of posthatching; the neuronal hypothalamic warm sensitivity and prenatal heat experiences increase neuronal hypothalamic cold sensitivity. Changes
in neuronal hypothalamic thermosensitivity start at the
end of incubation. On the last day of incubation, changes
in neuronal hypothalamic thermosensitivity are not sig-
nificant, but they become significant on d 1, 5, and 10
posthatching. From the last day of incubation until d
5 of posthatching, the changes are independent of the
temperature experienced within the prenatal period
(proximate nonadaptive). On d 10 of life, the changes are
proximate adaptive (Figure 11). During the early ontogeny, such strong changes in the neuronal network that
control specific body functions, like thermoregulation,
could be related to the development of synaptic contacts.
At the beginning of this procedure, the number of synaptic contacts increases, whereas later their number decreases (Brown et al., 2004). For both steps of the development of synaptic contacts, sensory stimulation is essential.
Only those contacts remain that are necessary to maintain
homeostasis under the respective surroundings and that
repeatedly receive sensory input (Bock et al., 2003).
On the other hand, the basic concept of imprinting of
physiological control systems states that this process is
realized by lasting environment-induced modification of
the genome (Tzschentke and Plagemann, 2006). Initial
studies on hypothalamic c-fos expression (Janke and
Tzschentke, 2006) related to prenatal temperature
1034
TZSCHENTKE
Figure 11. Proportion of warm-, cold-, and temperature-insensitive hypothalamic neurons in relation to all neurons investigated in Muscovy
duck embryos and ducklings incubated at 34.5, 37.5 (control group), and 38.5°C [modified from Loh et al., 2004 (embryos) and Tzschentke and
Basta, 2002 (ducklings)]. Total number of investigated neurons in embryos was 81 (cold-incubated group), 74 (control group), and 87 (warmincubated group). In 1-, 5-, and 10-d-old ducklings, the total number was 80 neurons for each incubation temperature. Asterisks represent significance
at the level of *P < 0.05, **P < 0.01, and ***P < 0.001.
changes as well as expression of brain-derived neurotrophic factor (Katz and Meiri, 2006) and R-Ras3 gene
(Labunskay and Mairi, 2006) related to postnatal thermal
conditioning could show changes on the level of gene
regulation. In our experiments (Janke and Tzschentke,
2006), we used the transcription factor and early immediate gene c-fos as stress markers to detect acute heat stress
in chicken embryos on the last day of incubation adapted
to different incubation temperatures (34.5, 37.5, and
38.5°C). Heat stress evoked c-fos expression in the POAH in all embryos but not in the unstressed control. Even
the differences between the groups were not significant,
trends that show that temperature-experienced embryos
have a lower c-fos expression than in the control after
acute heat stress. These proximate nonadaptive changes
(in this study related to the cold-incubated group) are in
accordance with the previously described results from
electrophysiological (Tzschentke and Basta, 2002;
Tzschentke et al., 2004) and metabolic (Loh et al., 2004)
investigations in temperature-experienced poultry embryos. After thermal conditioning on d 3, posthatching
changes in the R-Ras3 gene expression are found in the
PO-AH with a 10-fold peak after 12 h of heat conditioning
and a 4-fold increase after 6 h of cold conditioning (Labunskay and Meiri, 2006). Similar changes were found in the
brain-derived neurotrophic factor induction in both heatand cold-exposed birds. A main question that needs to
be addressed during further investigations is if these
changes in the central nervous network and also in the
periphery are long-lasting (Yahav and Tzschentke, 2006).
SUMMARY AND CONCLUSIONS
At the end of prenatal development, poultry embryos
have all prerequisites (autonomic, behavioral, and central
nervous mechanisms) to react on changes in incubation
temperature, even though the thermoregulatory system
is not mature. The level of full maturity is attained in the
posthatching period. In comparison with the heat loss
mechanisms, in embryos efficiency of thermoregulatory
HP is very low. Mechanisms for central nervous control
of temperature regulation seem to be well developed.
For early consolidation and maturation of body functions, sensory inputs (e.g., environmental influences) are
essential and can stimulate this process (training effect).
The typical reaction pattern of thermoregulatory mechanisms on external stimuli during early ontogeny has been
first an uncoordinated and proximate nonadaptive reaction that changes during later development into a proximate adaptive one. The early training of body functions
(e.g., by environmental stimulation) can improve the development of central as well as peripheral physiological
mechanisms. This might also be a basis for postnatal
health, welfare, and productivity in poultry.
During critical developmental periods, a long-term adaptation to the actual environment occurs via epigenetic
EMBRYO SYMPOSIUM
adaptation processes. During the perinatal period, imprinting of physiological control systems, like the thermoregulatory system, occurs. Imprinting is realized by both
the changes of synaptic plasticity as well as by lasting
environment-induced modification of the genome. 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.
ACKNOWLEDGMENTS
Research projects carried out in my working group,
Perinatal Adapation, were supported by grants of the
Deutsche Forschungsgemeinschaft (Ni 336/3-1; TZ 6/21, 6/2-2, 6/6-4, 6/10).
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