computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171

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Monitoring the development of thermoregulation in poultry
embryos and its inuence 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
Keywords:
Thermoregulation
Incubation temperature
Poultry embryo
Epigenetic adaptation
Critical period
a b s t r a c t
The general purpose of this review is to show the stage of the development of peripheral
and central nervous thermoregulatory mechanisms in poultry embryos at the end of
incubation, and the impact of long-term changes in incubation temperature. Methods
are described which (a) allow continuous measurement of peripheral thermoregulatory
mechanisms simultaneously with the body temperature of the embryo, and (b) can be used
for identication 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 feed-
back control into closed system controlled by feedback mechanism. Acute changes in the
environmental conditions (e.g. incubation temperature) induce as a rule, initially uncoor-
dinated 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 manipulationof immature physio-
logical 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 epige-
netic 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 veried by identication of the respective
effector genes and epigenetic changes in its expression.
2008 Elsevier B.V. All rights reserved.

Tel.: +49 30 2093 6276; fax: +49 30 2093 6008.

E-mail address: barbara.tzschentke@rz.hu-berlin.de.
0168-1699/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.compag.2008.05.003
62 computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171
1. Introduction
In the hierarchy of the regulatory systems the thermoreg-
ulatory 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 circula-
tion, respiration, metabolism) and integrates their activities
into appropriate and coordinated reactions for control of body
temperature. In poultry the development of thermoregula-
tory 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 mat-
uration 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 environ-
ment of the developing organism(Tzschentke andPlagemann,
2006). In the course of the early development physio-
logical control systems, like the thermoregulatory system,
develop from open loop system without feedback control
into closed control system regulated by feedback mecha-
nisms (D orner, 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 long-
lasting perinatal epigenetic programming or malprogramming
(Tzschentke and Plagemann, 2006). On one hand subopti-
mum 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 specically used to induce long-term adaptation of
the organism, for instance, to the postnatal climatic condi-
tions (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 poul-
try.
Prerequisites for the proper management of incubation cli-
mate are acquiring knowledge of the physiological needs of
poultry embryos andthe critical periods as well as systematic
investigations on long-term effects of chronic environmental
changes.
This review summarizes investigations, which were car-
ried out in my working group Perinatal Adaptation. It is
focused on the development of thermoregulation in poul-
try embryos as well as the impact of chronic changes of
incubation temperature. These investigations require meth-
ods, which allow continuous measurement of peripheral
thermoregulatory mechanisms simultaneously with the body
temperature of the embryo. Further, methods for identica-
tion of changes in the sensitivity of the central controller of
body temperature due to the development as well as pre-
natal temperature experiences and of critical periods in
the development of the respective body function are neces-
sary.
The methods developed or adapted and used in our group
are herewith briey 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 secondhalf of incubation. Eggs of the Mus-
covy 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 incuba-
tion 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 inuence of chronic changes in incubation
temperature on the development of thermoregulatory mech-
anisms.
For experiments on chronic inuence of changes in incu-
bation 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 rst 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 (1045

C). Food and water were given ad libitum.

2.2. Simultaneous measurement of body temperature
and O
2
-consumption in poultry embryos
2.2.1. Body temperature
Embryonic body temperature was measured in the allantoic
uid (T
af
) near the embryo using a miniature thermistor probe
(Testo GmbH & Co., Lenzkirchen, Germany). On the blunt
side of the egg a square hole (2mm2mm) was drilled into
the eggshell without damaging larger blood vessels. Through
this hole a thermocouple was inserted into the allantoic
uid 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 (T
c
).
After locating the tail feathers, the cloaca was easily iden-
tied and the thermistor probe was inserted to a depth of
12cm. T
af
as well as T
c
was measured continuously dur-
ing the entire period of experiments. A comparison between
T
af
and T
c
in single embryos showed only minor differ-
ences between T
af
and T
c
(ranging between 0.0 and 0.2

C)
at constant incubation temperature. Under changing incuba-
tion temperature the difference rose to a range of 0.10.4

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 O
2
-consumption
Oxygen consumption was continuously measured using an
oxygen analyser (Magnos 4, Hartmann & Braun, Frank-
furt/Main, Germany) based on the paramagnetic principle.
computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171 63
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 (vol-
ume 290ml) were watertight and consist of acrylic glass. The
respective ambient (incubation) temperature was regulated
by a temperature controlled water bath. After a minimum
of 2h of acclimation of the embryos to the respective ambi-
ent temperature, oxygen consumption simultaneously with
ambient temperature and T
af
were recorded for 1h. Oxygen
consumption(vol%) was measured as difference betweenoxy-
gen concentration of the air inowand outowof the climatic
chambers taking into account the actual gas ow (l/h). The
inuence 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 19.7J 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
(34.531.5

C) of ambient (incubation) temperature from the

normal level (37.5

C) for 3h using different water baths.

Changes in HP were analysed in relation to simultaneously
recorded body temperature (T
af
). As no net-increase inHP with
decreasing body temperature was observed in embryos it was
possible to estimate thermoregulatory counter reactions dur-
ing cooling using the Q
10
method. A quadratic regression was
usedto describe the relationshipbetweenHP andT
af
. Fromthe
regression line for each individual embryo Q
10
was calculated
using the vant Hoff formula (Precht et al., 1973):
Q
10
=(HP
T2
/HP
T1
)
10/(T2T1)
(T1>T2) in steps of 0.1

C
changes in the T
af
(Nichelmann et al., 1998).
2.3. Simultaneous measurement of body temperature
and respiration parameters as well as body temperature
and blood ow
2.3.1. Recording of respiratory parameters
Breathing by lung occurs after internal pipping. The breathing
activity induces pressure uctuations in the air cell of the egg.
These pressure uctuations can be registered using a Statham
element (Hugo Sachs Electronic KG, March, Germany) ina tube
which is inserted in the air cell (Fig. 2). The recordings furnish
information about the respiratory rate, the relative tidal vol-
ume and relative respiratory minute volume (Nichelmann and
Tzschentke, 2003) in relation to changes in the body tempera-
ture.
2.3.2. Recording of the blood ow in the chorioallantoic
membrane
Blood ow 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 nd an area with a large number of
small blood vessels. Thena 5mm5mmpiece of the egg shell
was removed. The following parameters could be measured:
mean red blood cell ow rate (ux), the red blood cell concen-
tration and the mean red blood cell speed. These parameters
were measured simultaneously with the embryonic temper-
ature. 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 (400m) 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 articial cerebrospinal uid. 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 rst days

post-hatching (Tzschentke and Nichelmann, 1999). Neuronal
activity and slice temperature were recorded by conventional
electrophysiological equipment andstoredona personal com-
puter using a 1401 interface (Cambridge Electronic Design)
(CED) and the CED software Spike 2. To identify the ther-
mosensitivity of single neurons, the bath temperature was
sinusoidally changed to a maximum of 3

C and a velocity of
about 0.02

Cs
1
. Temperature sensitivity was calculated by a
64 computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171
Fig. 2 Methods for recording of body temperature and different physiological parameters simultaneously. The focus in this
gure is on localisation of thermistor probes for measurement of the temperature of the allantoic uid (T
af
) and colonic
temperature (T
c
), laser-Doppler-probe for estimation of the blood ow 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 r-
ing rate to slice temperature and tted to a piecewise and/or
rectilinear regression function (Vieht, 1989). The thermosen-
sitivity of a neuron was dened by a temperature coefcient
of greater than or equal to 0.6imps
1
C
1
for warm-sensitive
(WS) neurons and less than or equal to 0.6imps
1
C
1
for
cold-sensitive (CS) neurons. All other neurons are named
as temperature insensitive (TI) according to this denition
(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 ther-
mosensitivity the proportion of WS, CS and TI neurons in
the PO/AH was determined in relation to all neurons inves-
tigated (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 organ-
ism. 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 inves-
tigations, on the last day of incubation acute heat stress
(42.5

C) for 90min 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/AHregion of the slices c-fos expression was immunohisto-
chemically detected. Analysis was made by light microscopy
and digital photography (magnication 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 990m 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. Identication of critical periods
Environmental manipulation of immature physiological
mechanisms may be a physiological tool for characterization
of critical periods (Tzschentke and Plagemann, 2006). A typ-
ical 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 fundamen-
tal developmental processes in chicken embryos using the
computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171 65
thermoregulatory systemas an example, which could be sum-
marized 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 ow). 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 devel-
opment poultryembryos have all prerequisites (peripheral and
central nervous mechanisms) to react to changes in incuba-
tion temperature.
3.1.1. Heat production
Long-term recordings of O
2
-consumption indicated that in all
precocial and altricial bird species investigated the develop-
ment 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 approx-
imately 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 (inter-
nal pipping) and starts respiration through lungs (Tazawa and
Whittow, 2000). After internal pipping until hatchthere is a large
increase inHP. Similar developmental patternof HP was found
in our experiments (Nichelmann et al., 1998; Janke et al., 2002).
Even though there is a similar qualitative developmental pat-
tern, 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. differ-
ent high yielded chicken breeds, Janke et al., 2004). If HP is
recorded simultaneously with embryonic body temperature
(T
af
) a strong linear relationship between both parameters
could be observed (Fig. 3). Under constant incubation tem-
peratures this relationship could be described by a highly
signicant linear correlation (Janke et al., 2002).
The relationship between HP and T
af
changed absolutely, if
acute changes in the incubation temperature occurred. With
changing incubationtemperature, e.g. during cooling, the rela-
tionship between HP and T
af
can be described by quadratic
regressions (Nichelmann et al., 1998). During acute decrease
in incubation temperature T
af
also shows a similar decrease
withthe difference inincubationtemperature but T
af
is always
higher than ambient temperature. On the other hand, HP
only decreases moderately (Fig. 4). A drop in body temper-
ature, due to low incubation temperature mostly causes a
decrease of net HP but the decrease is lower as assumed
by the vant Hoff rule (Nichelmann et al., 2001). To come to
this conclusion is only possible after simultaneous record-
ings of HP and embryonic body temperature. Using the Q
10
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 Q
10
method enabled us to calculate
the effect of thermoregulatory heat production with decreas-
ing 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, dimin-
ishes and near hatching time in some embryos a short-term
Fig. 4 Course of body temperature (temperature of
allantoic uid) and heat production before and during 3h
cooling in a single Muscovy duck embryo on day 34 of
incubation (Nichelmann and Tzschentke, 1999).
66 computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171
Fig. 5 Inuence of increase in incubation temperature
(ambient temperature, T
a
) on the course of body
temperature (colonic temperature, T
c
) and blood ow in the
chorioallantoic membrane in a single chicken embryo after
internal pipping (modied 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
efciency 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 showa 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 rst days of post-hatching (Tazawa andRahn,
1987; Nichelmann and Tzschentke, 2002; Tazawa et al., 2004).
3.1.2. Heat loss mechanisms
3.1.2.1. Changes in the blood ow of the chorioallantoic mem-
brane. In poultry embryos non-specic changes in the blood
ow of the chorioallantoic membrane due to changes in incu-
bation temperature could be found already during the last
third of incubation time. But at end of the plateau phase,
blood ow increases with increasing incubation temperature
or decreases withdecreasing ambient temperature. Inchicken
embryos after internal pipping, for instance, the body core tem-
perature remained constant for more than 40min after the
beginning of increase in ambient temperature up to 40.5

C
(Fig. 5) by activating this heat loss mechanism (Nichelmann
andTzschentke, 2003). This result couldbe only foundby mea-
surement of the deep body temperature in the colon of the
embryo. In these experiments, simultaneous measurements
of T
af
were not sensitive enough for the monitoring of the
short-term regulation of the body temperature by changes in
the blood ow 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 devel-
opmental level of the respiration as a heat loss mechanism
Fig. 6 Inuence 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 nmin
1
, tidal volume and
relative respiratory minute volume in arbitrary units.
in thermoregulation can be only investigated by simultane-
ous 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 pant-
ing reactions show similar characteristics to adult birds (two
phases of panting, Arad and Marder, 1983). At body core tem-
peratures 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 respira-
tory rate and an increase in tidal volume (Fig. 6).
At the later stage of incubation heat loss mechanisms
(blood ow, 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 post-
natal birds.
3.1.3. Development of central nervous thermoregulatory
mechanisms
Inpoultry embryos thermoregulationis only possible if central
nervous thermoregulatory mechanisms are developed early.
computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171 67
Fig. 7 Example of a hypothalamic cold sensitive neurone
in a Muscovy duck embryo on day 22 of incubation
(T
C
=thermal coefcient) imp s
1
C
1
, from Tzschentke et
al. (2004).
We could nd thermosensitive neurons in brain slices of
Muscovy duck embryos using the method of extracellular sin-
gle neuron recording. In this species thermosensitive PO/AH
neurons were found on E22 (Fig. 7) and E23 that show charac-
teristics similar topost-hatching (Tzschentke andBasta, 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 signicantly different from that in hatch-
lings (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 rst days of post-hatching is charac-
terised by a high neuronal cold sensitivity (Fig. 8). Aqualitative
change occurs between days 5 and 10 in the neuronal ther-
Fig. 8 Inuence 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
(modied from Tzschentke and Basta, 2000). Asterisks
represent signicance at the level of *p<0.05 (D: embryonic
day, d: day post-hatching).
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. Chickenembryos that were heat stressed(42.5

Cfor
90min) 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, rst uncoordinated and immediately
non-adaptive reactions
To investigate the developmental level of the thermoregula-
tory system(open loop systemwithout feedback mechanisms
or closed system with developed feedback mechanisms)
acute changes in ambient (incubation) temperature have
to be applied. The reactions of thermoregulatory mecha-
nisms on the applied changes in incubation temperature
during different time windows of the perinatal period
show a typical pattern. First uncoordinated and immedi-
ately non-adaptive reactions occur. Later the uncoordinated
(immediately non-adaptive) reactions change into coordi-
nated (adaptive) reactions, probably with closing of the
regulatory system.
First, during the development of physiological control sys-
tems it seems to be unimportant for the organism that a
distinct adaptable reaction of physiological mechanisms on
various environmental inuences occurs, but rather that any
reactionoccurs seems to be important for the adaptability dur-
ing the later life (Nichelmann et al., 1999, 2001; Tzschentke
et al., 2004). These rst reactions seem to be important for
the training of the respective function to develop feedback
mechanisms. For instance, in chicken embryos the blood ow
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 ow
in the chorioallantoic membrane increased during warming
and decreased during cooling, as expected (Fig. 9, Nichelmann
and Tzschentke, 2003). Similar changes in the blood ow dur-
ing cooling or warming were also found in Muscovy duck
embryos at the end of incubation (Tzschentke, 2002). Also
in other systems rst 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 stim-
ulation could be classied; heart rate increase, heart rate
decrease, increase or decrease in heart rate variability with-
out a change in the mean heart rate (H ochel et al., 2002).
Also after chronic changes in the incubation temperature
at the end of embryonic development rst proximate non-
adaptive 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). Obvi-
ously, the development of regulatory systems from an open
loop system without feedback into a closed control sys-
68 computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171
Fig. 9 Inuence of warming (38.5

C) and cooling (35.5

C) on blood ow 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 ow 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 inuences
could be typical for this phase. In conclusion, environmen-
tal manipulation of immature physiological mechanisms may
be a physiological tool for characterization of critical peri-
ods 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 plastic-
ity) as well as by a lasting environment-induced modication
of the genome (Tzschentke and Plagemann, 2006). During
critical periods of development of physiological control sys-
tems (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 con-
trol 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 pos-
sible 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 inbirds (Schwabl, 1996, 1997;
Ruitenbeek et al., 2000). On the other hand, knowledge and
better understanding of these mechanisms might be speci-
cally used to induce long-term adaptation of an organism, for
instance, to postnatal climatic conditions (epigenetic temper-
ature adaptation; Nichelmann et al., 1994, 1999; Tzschentke
and Basta, 2002; Tzschentke et al., 2004).
In chicken and other precocial birds epigenetic tempera-
ture adaptation can be induced by changes in incubation tem-
perature at the end of embryonic development (Decuypere,
1984; Minne and Decuypere, 1984; Nichelmann et al., 1994;
Tzschentke and Nichelmann, 1997; Tzschentke and Basta,
2002; Lohet al., 2004) as well as by thermal conditioning during
the rst 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 rst day of post-hatching Muscovy ducklings incu-
bated at lower temperatures than normal, for instance, have
a 56% higher heat production and a higher deep body tem-
perature under cold load as compared to controls (1h 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 signicantly lower temperature than birds incubated at
the normal incubation temperature during the rst 10 days
of post-hatching. This supports the hypothesis that avian
prenatal cold experience leads to a downward shift of the ther-
moregulatory 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.
computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171 69
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 (signicant 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 1h exposure to 10

C (signicant 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 reect the changes in
peripheral thermoregulatory mechanisms after prenatal tem-
perature 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 embry-
onic body temperature is more inuenced by chronic changes
in incubation temperature than the HP. If the HP is mea-
sured 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) T

af
was approximately 1

C higher than in the control. This could

be a prerequisite for a higher thermoregulatory set-point dur-
ing the entire life (Loh et al., 2004).
4. Conclusions
In poultry embryos autonomic and central nervous ther-
moregulatory mechanisms are developed. Latest at the end of
incubation poultry embryos have all prerequisites to react to
changes in incubation temperature. During this developmen-
tal period some physiological mechanisms show to certain
degree similar characteristics to postnatal birds. Regarding the
autonomic thermoregulatory mechanisms we could come to
this conclusiononly by simultaneous recordings of the respec-
tive mechanism and the embryonic body temperature. The
most sensitive parameter for characterization of the devel-
opmental level of embryonic thermoregulation after acute
changes in incubation temperature is the deep body temper-
ature (colonic temperature). But its measurement is limited
to the post-internal pipping period. During earlier developmen-
70 computers and electroni cs i n agri culture 6 4 ( 2 0 0 8 ) 6171
tal stages the internal egg temperature (T
af
) can be used as a
very good measure of the embryonic body temperature under
constant incubation temperatures. However, for characteriza-
tion of the thermoregulatory ability under acute temperature
changes T
af
measurements are limited.
During critical developmental periods epigenetic adapta-
tionprocesses caninduce a long-termadaptationto the actual
environment. Perinatal epigenetic temperature adaptation
could be a tool to adapt poultry embryos or hatchlings to later
climatic conditions. For the specic 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 neces-
sary. 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 perina-
tal 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 ther-
moregulation 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 veried by identication of the respective
effector genes and epigenetic changes in its expression.
Acknowledgements
Research projects carried out in our working group Perina-
tal 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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