J Comp Physiol B (1985) 155:195-200

Journal of
Comparative ~.,~m,o.~i~176176176176
and Environ-
Physiology B mental
Physiology

9 Springer-Verlag 1985

Response of egg temperature, heart rate and blood pressure

in the chick embryo to hypothermal stress
Hiroshi Tazawa and Shoji Nakagawa
Department of Electronic Engineering, Muroran Institute of Technology, Muroran, Japan 050

Accepted May 30, 1984

Summary. Chicken eggs incubated for 12-18 days Introduction

were catheterized via the allantoic artery and tem-
perature was monitored simultaneously using a
probe positioned in the allantoic fluid adjacent to
the embryo. Fluid temperature (referred to as egg
temperature), arterial pressure and heart rate were
measured following abrupt exposure to a lower
environmental temperature (ca., 26~28~ Egg
temperature and heart rate diminished exponenti-
ally: The rate of decline of egg temperature ap-
proximated Newton's law of cooling, the rate coef-
ficient being 0.022 ~ 0.025 ~ 9 ~ throughout
the incubation period from 12 to 18 days. The half
time of temperature response of the egg was
27 ~ 28 min. The response was much slower than
that of fertile unincubated eggs (Kaplan et al.
1978), suggesting that the extraembryonic fluids
act as a thermal buffer in embryonated eggs. The
heart rate response in older embryos (17 ~ 18 days)
changed in the same manner as egg temperature,
while in younger embryos (12~ 16 days) the heart
rate diminished more quickly than the change in
egg temperature. During development the cardiac
pacing of the embryo suggests that it becomes re-
sistive to mild cold stress. The systolic pressure
remained almost unchanged or even increased
during one hour of exposure as the embryo de-
veloped, while the diastolic pressure decreased
steadily after exposure irrespective of develop-
ment. The 18-day-old embryos retained the systol-
ic pressure unaltered during 3-hour exposure. In
embryos 3 ~ 4 days prior to hatching the function-
al capacity of the heart apparently allows con-
tinued pumping even after prolonged exposure to
low environmental temperature.
Symbols and Abbreviations: See definitions at the end of the
Materials and methods section
Developing avian embryos demonstrate little con-
trol of body temperature, relying on external ther-
mal supplies (parent birds or incubator) to main-
tain metabolism. Therefore, an ambient thermal
deviation from an optimum incubation tem-
perature constitutes a stress on embryos, disturb-
ing body temperature, metabolic rate, blood acid-
base status and cardiac output (Tazawa 1973;
Tazawa and Mochizuki 1978). The chorioallantois
spreading out under the eggshell, on the other
hand, serves as a depository for excreted waste
products as well as an important respiratory or-
gan. The fluid in the allantoic lumen, together with
the amniotic fluid, surrounds the embryo during
later stages of development. These fluids might
constitute a thermal buffer which mitigates body
temperature changes following an alteration in en-
vironmental temperature.
The role of the amniotic and allantoic fluids as
a thermal buffer may be evaluated by comparing
the temperature dynamics of embryonated eggs
with those of fertile unincubated eggs during cold
exposure. The mitigation of detrimental effects of
cold stress on embryonic cardiac function during
late stages of development may be examined by
recording the heart rate and blood pressure simul-
taneously with egg temperature. If temperature
effects become less detrimental during develop-
ment, this would indicate that the embryo is begin-
ning to acquire a functional capacity for resisting
environmental temperature change.
In chick embryos the response of heart rate to
a lower ambient temperature has been measured
by many investigators. Earlier literature has been
reviewed by Romanoff (1960), and more recently
Girard (1973) used an elaborate microcatheteriza-
tion technique to establish a relationship between
196 H. Tazawa and S. Nakagawa: Response of chick embryo to cold stress
environmental temperature and heart rate. How-
ever, this technique required that some of the egg-
shell was removed and mineral oil or water applied
to the surface of the membrane, which would in-
terfere with normal respiratory conditions.
Therefore, the initial heart rate at an optimum
temperature was found to be much lower than
that measured with a ballistocardiograph or elec-
trocardiograph (Cain et al. 1967; Laughlin et al.
1976). Because the eggshell provides the diffusive
pathway equivalent to that in pulmonary ventila-
tion (Rahn and Paganelli 1982), the modification
of eggshell surface must be restricted to as small an
area as possible in order not to compromise em-
bryonic respiratory properties.
The present study was therefore designed to
measure the response of egg temperature and heart
rate and blood pressure of developing embryos to
hypothermal exposure under conditions of normal
diffusive gas exchange, and to estimate quan-
titatively the temperature and heart rate responses.
The results indicate a role of extraembryonic fluids
as a thermal buffer and a functional capacity of
cardiac pumping against mild cold stress which is
established in embryos 3 ~ 4 days prior to hatch-
ing.
Materials and methods
White Leghorn eggs were incubated at 38 ~ for 12 ~ 18 days;
they were turned twice daily. A catheter comprising a 26-gauge
hypodermic needle and thin polyethylene tubing was implanted
in the allantoic artery through a small hole in the eggshell and
chorioallantoic membrane as stated previously (Tazawa et al.
1980). At the same time a thermistor temperature probe (model
No. 520, Yellow Springs Instrument, USA) was positioned in
the allantoic fluid adjacent to the embryo. The hole was sealed
with vinyl tape and epoxy glue. The area was small enough not
to impede embryonic respiration. The implanting procedure
was carried out within 5 rain, and the treated egg was kept in
the incubator prior to measurements after 2 ~ 3 h.
For measurements the egg was transferred from the in-
cubator to a laboratory bench. The polyethylene tubing from
the egg was connected to pressure transducer (model P23ID,
Statham, USA) placed at the same height as the egg. The pres-
sure waves were recorded with a rectilinear pen recorder
(Biophysiograph model 110, San-ei, Tokyo, Japan) as described
previously (Tazawa 1981a,b). The heart rate was calculated
from the duration of five pulses of the pressure wave and the
paper speed of the recorder (6 cm/s). The allantoic fluid tem-
perature (referred to as egg temperature) was monitored with
the YSI telethermometer and the output was recorded on an
analog pen recorder.
Variables indicating the time course of changes in egg tem-
perature were calculated using the following symbols:
To initial egg temperature before exposure
Tlo egg temperature at 10 min of exposure
Taso egg temperature at 180 min of exposure
Tj~ temperature of incubator
T~q equilibrium temperature
Tox temperature of exposed environment
ATi initial temperature difference between egg and in-
cubator before exposure, T o - Tin
ATf final temperature difference between egg and exposed
environment after 3-h exposure, Tls 0 - Tex
ATlso drop of egg temperature during 3-h exposure,
To -- T18o.
The equilibrium temperature (Teq) was taken as the ex-
posed environmental temperature (To~) plus initial temperature
difference between egg and incubator, T~q= T~x+ A Tj.
The temperature difference between pre-exposed egg (To)
and equilibrium (Teq) was noted as T o e " To eq = T o - T~q
%T~o is the rate of temperature decrease m percent per
10min of exposure, calculated as %T,o=[(To-Tlo)/
( T o - T~ s o)]- 100.
tj/2 is the half time (in rain) when temperature has dropped
to half the AT~so.
The egg temperature at time t of exposure (T(t)) was as-
sumed to follow Newton's law of cooling as follows;
-dT(t)/dt = k.[r(t)- req] (1)
where k is a cooling rate coefficient. Putting the measured egg
temperature at time 0 (To at t=O) into Eq. (1), we obtain
T(t) = T~q + ro,eq'exp@kt ). (2)
k was calculated as k=O.693/q/2 by substituting the egg tem-
perature at half time into Eq. (2). The inverse of k is a thermal
time constant.
For changes in heart rate, the similar definition was made
as follows,
HRo heart rate before exposure to cold stress
HRlo heart rate at 10 min of exposure
heart rate at 3 hours of exposure
HR,s0
%HR~0 rate of heart rate decrease in percent during 10 min
of exposure, [(HR0-HR10)/(HRo-HRlso)]-100;
and
tl/2* half time for a fall in heart rate during 3-hour ex-
posure.
Results
Preliminary experiments were performed to deter-
mine the allantoic fluid temperature during em-
bryonic development from day 2 to day 20 (Fig. 1).
AT is the temperature difference between the allan-
toic fluid and incubator. Up to 12 days of incuba-
tion, the egg temperature was lower than Tin. After
12 days, at which time the chorioallantois covered
the whole embryo, the egg temperature exceeded
Tin. Because in the preliminary experiments the
egg was only subjected to implantation of the ther-
mal probe, the temperature measured was also
used as the control to assess the influence of
catheter implantation on the excess temperature of
the egg implanted with both thermal probe and
catheter.
The allantoic artery was catheterized in em-
bryos ranging from 12 to 18 days of age. Record-
ings of arterial pressure of 16-day-old embryos
during 3-h exposure to environmental temperature
of 27.2~ are shown in Fig. 2. Immediately after
the egg had been transferred to the low tem-
perature environment, the manometric transducer
H. Tazawa and S. Nakagawa: Response of chick embryo to cold stress 197

was connected to the catheter (0 rain in the left

upper panel). The systolic pressure remained high
during at least 1 h of exposure, while the diastolic
Incubation day pressure decreased with time. In Fig. 3, the arterial
Fig. 1. Changes in temperature difference between the allantoic pressure before and after a 3-h exposure is plotted
fluid and incubator during embryonic development. Note in- against developmental age. Five embryos were ex-
crease on day 12
amined for each day. Because the rectangle in-
dicates the pulse pressure, the upper end corre-
sponds to the systolic pressure averaged for 5 em-
bryos and the lower end, the diastolic pressure.
Time courses of the changes in blood pressure
(BP), egg temperature and heart rate of 13- and
17-day-old embryos are shown in Fig. 4 and
- - ~ - 7 = ~-~T- -= -- Fig. 5, respectively. The temperature to which the
.---r ~ 1 i f i84 .2.~-
__5m,6t,,, I, eggs were exposed (T~) is shown by the solid line
i' in the middle panels. The solid curves of the mid-
dle panels indicate the theoretical cooling rate cal-
culated from Newton's law (Eq. (2)). The changes
in heart rate vs. egg temperature of the same em-
bryos are depicted in Figs. 6 and 7, respectively.
These two embryos are representative for ages
from 12 to 16 days and for ages of 17~ 18 days,
respectively.
Variables relating to the time course of egg
~ - T ' T ~ - = U "==- temperature and heart rate are summarized in
_~ 2.2 . . . . . . . I. . ! i-,-ri 7.i i ill
i ii~l [lli
t~ 771
i i !:
J
i! L!Li_ Jl i ~J~J! Table 1.

Fig. 2. Consecutive recordings for allantoic arterial pressure
after exposure to lowered environmental temperature (27.2 ~
for 3 h. Age of embryo: day 16. The figures under each trace
indicate heart rate (beats/min), blood pressure (systolic/diastolic
in mmHg) and egg temperature (~
25'
Discussion
E x c e s s temperature. The temperature difference
across the shell is reversed at about day 12 of
incubation (Fig. 1 and ATi in Table 1). Subsequent-
ly, the temperature in the egg becomes gradually
higher than the environmental temperature. This
temperature increase is related to the metabolic
heat produced by the incubated egg. The evapora-

Ii I1
tive heat loss exceeds metabolic heat production
~20 before day 12. Afterwards, when the chorioallan-
E toic membrane completely surrounds the embryo,
metabolic heat production is greater than evapora-

0I0I
{D tive heat loss. The changes in temperature dif-
ference during development from day 12 to 18
|
L
IX
shown by cannulated eggs (Table 1) are identical to
g those of uncannulated eggs (Fig. 1). This indicates
o that the cannulation of the allantoic vessel does not
Ill
disturb metabolic heat production and thermal
G 1; 1; 1; 17 17 conductance across the shell.
Age (days) As the embryo grows and its metabolic rate
Fig. 3. Changes in arterial pressure before and after 3-h exposure increases, the egg temperature after 3 hours of
to lowered environmental temperature during development hypothermal stress shows that the temperature dif-
from day 12 to day 18 of incubation. Five embryos were exam- ference (ATf) between egg and environment is in-
ined for each day. The open rectangles indicate the average pulse
pressure at a temperature of 38 ~ and the shaded rectangles,
creasing (Table 1 and Figs. 4 and 5). ATf increases
those measured after 3-h exposure to lowered temperature. The on average from 0.9 ~ at day 12 to 1.9 ~ at day
vertical lines show one SD 18. ATf, as given by the difference between TaB0
 198 H Tazawa and S. Nakagawa: Response of chick embryo to cold stress

~J
\
r
p.
(0
111
3~
Xxx X
xx~ ~ , , X xXx x,x x,x x,xx x ~v X x• x ,
- --~•215215215215215215215215215
'x

L
~ x HRz~o
2 2
O3

~ )r
L~
Tex:27. O~
Tex=26.0~

"~ ~ 9 .......-,.-..
ZI2

g
. ' . ' - . . . . .... . . . . . . ....

Time [rain] Time (mln]

Fig, 4. Time course of changes in blood pressure (BP), egg
temperature and heart rate of 13-day-old embryo during 3-h
exposure to lowered environmental temperature (T~=26.0~
shown by solid line). The solid curve indicates the time course
of egg temperature according to Newton's law of cooling
Fig. 5. Time course of changes in blood pressure (BP), egg
temperature and heart rate in a 1?-day-old embryo The ambient
temperature (T,~) was 27.0~ The deviation of the measured
egg temperature response from the calculated curve according
to Newton's law of cooling, and of T~so from To~(ATd is larger
than in the 13-day-old embryo (Fig 4) t-IRla 0 is also higher
than that of the younger embryo

0:3 m" ,"

I

t'N

#
E E
I

=9
.,=. ==
(II 9
iv- 9 r
I-I9,I
Z z (D 9 i=

CI ll-
n- -r"
@.

CI C~

8~7 2'. 2'~ ~'o 3'I 3'2 ~'~ 3'~ 3'~ ~'6 3'7 ~'~ #8
E~9 T e m P e r a t u r e [~ E~g T e m P e r a t u r e [~
Fig. 6. Curvilinear relationship between heart rate and egg tem- Fig. 7. Linear relationship between heart rate and egg tem-
perature in the 13-day-embryo of Fig 4 perature in the 17-day-embryo of Fig 5
H. Tazawa and S. Nakagawa: Response of chick embryo to cold stress 199

Table 1. Variables indicating the time course of egg temperature and heart rate response to lowered environmental temperature

Age Toq To, eq ATi ATf %Tlo tl/2 ]c HR0 HRls0 %HR10 tl//
(days) (~ (~ (~ (~ (%) (rain) (~176 (min -1) (min -1) (%) (rain)

12 25.9i0.4 12.0::t_0.9 ~).2+0.4 0.9=L0.2 25::t-3 284-2 0.022• 2444-19 83::1: 4 294-6 23~5
13 2 7 . 3 i l . 2 ll.l:t-l.1 0.44-0.2 1.1~0.2 24=1-4 284-3 0.0234-0.003 262• 5 844- 7 274-5 23~3
14 27.54-1.2 11.9:t:1.3 0.64-0.3 1.1• 27• 27+3 0.023:k0.002 271• 82• 9 284-2 24:t:2
15 28.5• 10.2~1.2 0.7• 1.14-0.2 25+3 27~1 0.024:t_0.002 2714-12 1004- 6 27_t_2 22:t-2
16 28.34-0.4 10.9:t:0.1 1.3t0.4 1.5• 254-3 27+3 0.025• 272• 92• 284-3 23:t:1
17 28.0::1:0.2 ll.O:t-O.O 1.0::t_0.2 1.74-0.3 255:3 28• 0.023:~ 0.001 2704- 1 974- 4 24i2 28~1
18 28.1• ll.O::LO.O 1.14-0.2 1.9~0.1 26:t:3 284-3 0.023• 269• 95:t: 5 24• 28~:3

Values are mean:t: SD (N= 5). Incubation temperature was 38.0 ~ except 3 eggs on day 14 whose temperature was 38.7 ~

and Tex, is large in a 17-day embryo compared to
a 13-day embryo (Figs. 4 and 5).
Response of egg temperature. When eggs incubated
for more than half of the total incubation period
are exposed to lowered ambient temperature, the
egg temperature at 10min (%Tlo in Table 1) is
decreased by a quarter of the total temperature
drop induced during 3 h (AT18o). At 27-28 rain of
exposure, the egg temperature reaches 50% of
AT~8o (refer to tl/2 in Table 1). The rates of tem-
perature response indicated by % T l o and tl/2
(Table 1) are identical for all developmental stages
from 12 to 18 days. These incubated eggs have a
cooling rate significantly slower than that of fertile
unincubated eggs. In the study of Kaplan et al.
(1978) the half time of temperature response in the
center of unincubated eggs was only 18 min and the
temperature at a depth of 1 cm from the shell de-
creased by 50% of the total temperature drop in
only 5 min. The latter position corresponds to that
of the embryo. The main differences of embryon-
ated eggs from fertile unincubated eggs in relation
to heat transfer are presence of heat production,
amniotic and allantoic fluids and blood circulation.
While the extraembryonic circulation functions to
accelerate the heat loss, the heat production may
affect the temperature response while the extraem-
bryonic fluids act as a buffer against environmental
temperature change. The temperature response (as
seen in the cooling curve) which is slow compared
with unincubated eggs is observed in embryos with
differing excess temperatures. Hence, the slower
temperature response might be attributable to the
extraembryonic fluids acting as a thermal buffer.
The rate coefficient of Newton's law of cooling
(k in ~ 9 ~ is also almost constant through-
out the developmental stages from 12 to 18 days
(Table 1). In incubated eggs, k may be influenced
by (I) thermal conduction through the allantoic
fluid and the shell, (2) convective heat transfer
between the embryo and extraembryonic fluids due
to the circulating blood and (3) the degree of
evaporative heat loss. The thermal probe was posi-
tioned in the allantoic fluid adjacent to the embryo.
As the embryos developed, the probe moved closer
to the shell. In addition, the chorioallantoic blood
flow increases with embryonic development (Taza-
wa and Mochizuki 1976; Bissonnette and Metcalfe
1978). In theory these factors may alter the mag-
nitude of k, but so far no specific changes in k have
been observed during development. However, the
thermal response curve shows an upward deviation
from the theoretical curve calculated from New-
ton's law of cooling (Figs. 4 and 5). The deviation
tends to increase with embryonic development. In
addition, the difference between egg temperature
and Tex after 3 h of exposure (ATf) increases with
development as discussed in the previous section.
All this indicates that the embryo tends to maintain
temperature during exposure as it develops and, to
a slight degree, reduce temperature variation due to
ambient temperature changes. This could be regar-
ded as evidence of the beginnings of thermoregula-
tion.
Response of the heart rate. The heart rate also
responds in an exponential fashion to an abrupt
lowering of Tin. After 10 rain, the heart rate has
decreased by about 30% in embryos of 12-16 days
(referred to as younger embryos) and by about one
quarter in embryos of 17-18 days (referred to as
older embryos). This corresponds to half-times of
22 ~ 24 rain in the former, and 28 min in the latter
(Table 1). The half-time of the change in older
embryos is the same as that of the change in egg
temperature, resulting in the linear relationship
between egg temperature and heart rate as shown
in Fig. 7, while in younger embryos the relationship
is curvilinear (Fig. 6). The rate of the cardiac
pacing for unit change in egg temperature averages
17.5 (+ 1.0 SD) beats/min~ in older embryos. In
younger embryos, correlating with the curvilinear
relationship, the heart rate falls more rapidly in the
200 H. Tazawa and S. Nakagawa: Response of chick embryo to cold stress
higher temperature range. The cardiac pacing
resists the mild cold stress in embryos approaching
hatching. This is also shown by the HRlso value
which becomes larger during development (Table
1 and Figs. 4 and 5).
Response of blood pressure. The response of blood
pressure, particularly systolic pressure, to cold
stress changes with development. In younger em-
bryos, both the systolic and diastolic pressures be-
gin to decrease immediately after exposure. This
pattern of the change was observed in 4, 2, and 1
embryos for days 12, 13 and 14, respectively. As
embryos develop, the systolic pressure remains un-
changed during at least 1 h of exposure and after-
wards decreases slightly (Figs. 4 and 5). Figure 3
shows that both the systolic and diastolic pressures
decrease in embryos aged from 12 to 17 days after
a 3-h exposure to low temperature. However, two
out of five embryos of 17 days age showed a slight
increase in the systolic pressure under 3-hour cold
stress, and the averaged systolic pressure difference
before and after 3-hour exposure to low tem-
perature is statistically insignificant (P > 0.3). Fur-
thermore, 18-day old embryos retain the systolic
pressure unaltered even after low temperature ex-
posure (Fig. 3). The resistance of cardiac function
to prolonged lowered environmental temperature
arises around days 17-18 of incubation.
A change in the cardiac output during low tem-
perature exposure has been reported for 16-day-old
embryos based on a model analysis (Tazawa and
Mochizuki 1978). The cardiac output was esti-
mated to decrease to half the output of normother-
mic embryos during a 2-h exposure to 30 ~ while
the drop of blood pressure is not more than one-
third the normothermic value on day 16 (Fig. 3).
Thus, the total peripheral resistance must increase
under cold stress. However, the drop of cardiac
output is probably mainly due to the decrease in the
heart rate as measured in the present study. Cardiac
function may be unaffected by prolonged hypother-
real exposure in older embryos.
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