Journal of Thermal Biology 28 (2003) 217222

Effects of early age feed restriction and heat conditioning on

heterophil/lymphocyte ratios, heat shock protein 70 expression
and body temperature of heat-stressed broiler chickens
I. Zulkiia,*, P.K. Liewa, D.A. Israfb, A.R. Omarc, M. Hair-Bejoc
b

a
Department of Animal Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Department of Biomedical Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c
Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Received 10 July 2002; accepted 11 November 2002

Abstract
We examined the effects of early age feed restriction and heat conditioning on heterophil/lymphocyte ratios (HLR),
heat shock protein (hsp) 70 expression and body temperature of heat-stressed male broiler chickens. On day (d) 1,
chicks were subjected to (1) 60% feed restriction on d 4, 5, and 6 (FR); (2) exposure to 36711C for 1 h from d 1 to 21
(HT); (3) both FR and HT (FRHT); or (4) control. On d 35, all the birds were exposed to 39711C for 6 h. Subjecting
chicks to FR, HT and FRHT reduced HLR response to the heat challenge. The FR and FRHT birds had improved hsp
70 response and the latter were more hyperthermic than controls during the heat exposure.
r 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Heat stress; Early age feed restriction; Heat conditioning; Heterophil/lymphocyte ratio; Heat shock protein 70; Body
temperature; Broiler chickens

1. Introduction
Stress is inevitable to an individual or a population. It
is well documented that many aspects of health,
productivity and well-being of animals may be at some
risk from stress (Siegel, 1995; Zulkii and Siegel, 1995).
One of the methods to minimise the detrimental
consequences of stress responses is to modify the ability
of the animals to cope with stress. There is considerable
evidence that stressful experiences during the neonatal
stage may have a profound impact on various physiological and immunological aspects of a birds response
to subsequent adverse stimuli. Cockerels subjected to
water deprivation between 6 and 10 days of age were
more resistant than controls to Staphylococcus aureus
infection following feed restriction later in life (Gross
*Corresponding author. Tel.: +603-89466908; fax: +60389432954.
E-mail address: zulkii@agri.upm.edu.my (I. Zulkii).

and Siegel, 1982). In another study, Gross and Siegel

(1980) noted that early age overheating or water
restriction improved the antibody response to sheep
erythrocytes in 418-week-old stressed chickens.
In the context of thermal stresses, several studies have
shown the possibility of improving survivability, growth
and feed efciency of heat-stressed chickens by prior
exposure to controlled thermal stressors (Arjona et al.,
1988; Yahav and Hurwitz, 1996; Yahav and Plavnik,
1999; Yalcin et al., 2001). An animal does not always
have to be preconditioned to the same stressor for
habituation to take place. Work by Zulkii et al. (1994a,
b, 2000) indicated that in chicks restricted intake early in
life had smaller increase in heterophil/lymphocyte ratios
(HLR), improved resistance to adenoviral infection,
weight gain and survivability than those fed ad libitum
throughout the experiment in response to heat exposure.
At a molecular level, in poultry, enhanced heat tolerance
as a result of early age feed restriction (Zulkii et al.,
2002) and heat conditioning (Wang and Edens, 1998)

0306-4565/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0306-4565(02)00058-X

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I. Zulkifli et al. / Journal of Thermal Biology 28 (2003) 217222

has been associated with greater heat shock protein

(hsp) 70 response.
When living organisms are exposed to thermal and
non thermal stressors, the synthesis of most proteins is
retarded but a group of highly conserved proteins
known as hsps are rapidly synthesized (Etches et al.,
1995). It is well documented that one of the most
important functions of hsp is to protect organisms from
the toxic effects of heating (Barbe et al., 1988). Hsp may
play roles in protein assembling and disassembling
(Pelham, 1986), protein folding and unfolding (Randall
and Hardy, 1986) and protein translocation (Murukami
et al., 1988). Of the many expressed hsps, those with a
molecular weight of approximately 70 kDa appear to
correlate best with heat tolerance (Craig and Gross,
1991).
There is the question of whether combining both early
age feed restriction and heat conditioning can further
enhance heat tolerance in chickens. To our knowledge,
there is only one report from experiments designed to
study the effects of combining both procedures on heat
tolerance in chickens. Results from earlier studies do not
provide conclusive information (Yahav and Plavnik,
1999) and the effects of combining both procedures on
hsp 70 response have not been determined. Furthermore, the protocol of feed restriction and heat
conditioning practiced by the authors are different from
those of the present study. The aim of our study was to
evaluate the effects of early age feed restriction, heat
conditioning, and the combination of both early age
feed restriction and heat conditioning on HLR, rectal
body temperature and hsp 70 expression in male broiler
chickens exposed to acute heat stress.

2. Materials and methods

A total of 96 one-day-old male broiler chicks (Cobb)
were obtained from a local hatchery. The chicks were
individually wing-banded and distributed at random in
groups of 812 battery cages with wire oors in an
environmentally controlled chamber. The ambient
temperature of the chamber was set at 32711C and
then gradually decreased until 24711C was reached by
day (d) 21. Chicks were fed commercial broiler starter
(crumble form; 21%CP and 2950 kcal ME/kg) and
nisher (pellet form; 19%CP and 3100 kcal ME/kg)
diets from d 1 to d 21 and d 22 onwards, respectively.
Water was available at all times and continuous lighting
was provided.
Commencing from d 1, equal numbers of chicks were
subjected to one of the following four treatments: (i)
60% feed restriction on d 4, 5, and 6 (FR), (ii) exposure
to 36711C (relative humidity, 5060%) for 1 h from d 1
to 21 (HT), (iii) 60% feed restriction on d 4, 5, and 6 and
exposure to 36711C (relative humidity, 5060%) for 1 h

from d 1 to 21 (FRHT). (iv) ad libitum feeding and no

heat treatment (control). The feed restriction was 60%
of the previous days feed intake of the control group.
During the daily heat conditioning from d 1 to 21, the
HT and FRHT chicks were individually removed from
their cages, placed in plastic crates, and transferred to
another environmentally controlled chamber. Both
environmentally controlled chambers were adjacent to
each other and of similar dimensions and set up. Feed
and water were not provided during the heat conditioning period. Immediately, following the 1 h heat conditioning procedure, the birds were returned to their
home cages. To avoid the confounding effect of being
removed and placed in plastic crates, from d 1 to 21, the
control and the FR birds were also subjected to similar
treatment but they were not moved out from the
chamber. All birds were vaccinated against Newcastle
disease via intraoccular route on d 7 and 21.
On d 35, all the chicks were removed from their cages,
transferred to another environmentally controlled room
(with deep litter oor) and exposed to 39711C and 50
60% relative humidity for 6 h. Feed and water were
provided ad libitum throughout the heat episode. Prior
to heat challenge (d 35), rectal body temperature was
taken from 8 birds per treatment group using a digital
thermometer. The probe was inserted about 3 cm into
the cloaca for about 1 min. Immediately following
recording of rectal temperature, blood samples (0.3 ml)
were obtained for heterophil (H) and lymphocyte (L)
counts. Blood samples for H and L were collected in
tubes containing EDTA as anticoagulant. Blood smears
were prepared using MayGrunwaldGiemsa
.
stain; H
and L were counted to a total of 60 cells (Gross and
Siegel, 1983). The HLR is considered to be a reliable
stress indicator in birds (Gross and Siegel, 1983;
Maxwell, 1993).
Immediately after blood collection, four birds from
each treatment group were randomly chosen, killed by
cervical dislocation, and the brain samples were
removed for analysis of hsp 70 (Zulkii et al., 2002).
The samples were frozen quickly in liquid nitrogen and
kept at 701C for further analysis. Similar procedures
(recording of rectal temperature, bleeding and collection
of brain samples) were repeated following 3 and 6 h of
heat exposure with a different sample of birds.
The brain samples for detection of hsp 70 (0.5 g) were
homogenised in an Ultra-Turrax homogenizer, using
5 ml chilled TrisHCl buffer (20 mM Tris pH 7.5, 0.75 M
NaCl, 2 mM 2-mescaptoethanol) and centrifuged at
23,000g for 30 min at 41C. The protein concentration of
the supernatants were quantied by the Bicinchoninic
Acid Protein Assay Kit Procedure No. TRPO-562
(Sigma Chemical Co, St. Louis, MO) with bovine serum
albumin as a standard. Thirty mg total protein were
loaded and separated on 1.5  80  100 mm3 12%
polyacrylamide gels containing SDS (Laemmli, 1970)

I. Zulkifli et al. / Journal of Thermal Biology 28 (2003) 217222

using the Hoefer Mini Gel apparatus. Gels were

electrophoresed at 150 V until the tracking dye reached
the base of the gel. The fractionated proteins were
visualised by Coomasie blue staining or transferred to
polyvinyledine diuoride (PVDF) membranes (MSI,
USA) (Towbin et al., 1979). After electrophoretic
transfer, the PVDF membranes were stained with
0.5 g/l Ponceau S in 10 g/l acetic acid solution to
visualise and mark the positions of the proteins used
as molecular weight standards. After washing the
Ponceau S with distilled water, the non-specic binding
sites were blocked using 10 ml of cold blocking buffer
containing 10% non-fat milk and 0.05% sodium azide
for 30 min. The membranes were incubated overnight
(41C) with 5 ml of blocking buffer containing antiserum
(mouse anti-chicken hsp 70; Sigma Chemical Co, St.
Louis, MO) against hsp 70 in a 1:1000 dilution.
Following overnight incubation, the blots were washed
four times (5 min each) with 10 ml cold blocking buffer.
The blots were then reacted with goat anti-mouse
secondary antibody conjugated to alkaline phosphatase
(Sigma Chemical Co, St. Louis, MO) for 1 h. After
rinsing with cold phosphate buffered saline (PBS) the
colour reaction on the PVDF membrane was developed
using commercially prepared BCIP/NBT (Sigma Chemical Co, St. Louis, MO). Relative density of the hsp 70
was determined using a densitometer (AlphaImagerTM,
Alpha Innotech Corporation, USA) with the aid of
AphaEaseTM (Alpha Innotech Corporation, USA) software.
Data were analysed by two-way ANOVA and
Duncans multiple range test when appropriate
(Pp0.05, ANOVA) using SAS (SASs, 1991). The data
were tested for the effects of treatment, stage of heat
exposure and their interactions. When interactions
between main effects were signicant, comparisons were
made within each experimental variable. All values are
expressed as means7standard error of the mean and the
level of signicance is Pp0.05.

3. Results
There was a signicant treatment by stage of heat
exposure interaction for HLR data (Table 1). The early
age feed restriction and heat conditioning had no effect
on HLR prior to heat challenge. However, after 3 h of
heat exposure there was a marked elevation in HLR of
FR, HT and controls chickens but not those of FRHT.
Following 6 h of heat challenge, the controls birds had
greater HLR than their FRHT, FR and HT counterparts. The three latter groups did not differ signicantly.
Representative hsp 70 responses in the brains of
broiler chickens following heat exposure are shown in
Fig. 1. Prior to the onset of heat challenge, irrespective
of early age treatment, densitometric analysis indicated

219

Table 1
Mean (7SEM) heterophil/lymphocyte ratios where stage of
heat exposurea by treatmentb interactions were signicant
Treatment

Control
FR
HT
FRHT

Stage of heat exposure (h)

0

0.3470.03z
0.3470.04y
0.2270.02z
0.3270.03y

0.6270.09aby
0.7170.10ax
0.4270.03bcxy
0.4070.03cy

1.0970.12ax
0.7370.07bx
0.7670.14bx
0.5770.06bx

On d 35, all chicks were exposed to 39711C and 50%

relative humidity for 6 h.
b
Control: ad libitum feeding and no heat treatment; FR:
60% feed restriction on d 4, 5 and 6; HT: exposure to 36711C
for 1 h/day from d 1 to d 21; FRHT: combination of FR and
HT.
ac
Means within a column with no common superscript differ at
Pp0.05.
xz
Means within a row with no common superscript differ at
Pp0.05.

that all birds had similar hsp 70 density in the brain

samples (Table 2). Except for the control birds, there
was no signicant increase in hsp 70 response following
3 h of heat challenge, thus resulting a signicant
(Pp0.05) treatment by stage of heat exposure interaction for hsp 70 density. Although a marked elevation in
hsp 70 response was noted in all the birds following 6 h
of heat challenge, the mean hsp 70 densities of FRHT
and FR birds was greater than those of controls. The
magnitude of hsp 70 response in HT birds was not
signicantly (P>0.05) different from the other three
groups.
Irrespective of treatment group, all the birds had
similar rectal body temperature following 0 and 3 h of
heat exposure (Table 3). However, after 6 h of heat
exposure, the mean rectal body temperatures of FRHT
birds was higher than their control counterparts. The
body temperatures of FR and HT birds were not
signicantly (P>0.05) different from those of FRHT
and control groups.

4. Discussion
The increase in HLR following heat exposure is
expected because heat-elicited alteration in blood
leucocytes proportion has been well documented (Zulkii et al., 1994a, b, 1999, 2000). The present ndings are
consistent with those of Zulkii et al. (1999) that high
ambient temperature elevated HLR within 3 h. According to Chancellor and Glick (1960), heat stress resulted
an initial decrease in H and increase of L after exposure
for 1530 min. However, 2 h later the cellular numbers
were reversed.

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I. Zulkifli et al. / Journal of Thermal Biology 28 (2003) 217222

Table 2
Mean (7SEM) heat shock protein 70 densities where stage of
heat exposurea by treatmentb interactions were signicant
Treatment Stage of heat exposure (h)

Control
FR
HT
FRHT

259807763.0y
279827938.0y
2525271008.9y
2525271008.9y

3230572244.5x
3162273116.5xy
2934772502.5xy
3094073504.7y

3298771503.3bx
3997973222.2ax
3523771610.9abx
4128571402.1ax

On d 35, all chicks were exposed to 39711C and 50%

relative humidity for 6 h.
b
Control: ad libitum feeding and no heat treatment; FR:
60% feed restriction on d 4, 5 and 6; HT: exposure to 36711C
for 1 h/day from d 1 to d 21; FRHT: combination of FR and
HT.
a, b
Means within a column with no common superscript differ
at Pp0.05.
xz
Means within a row with no common superscript differ at
Pp0.05.

Table 3
Mean (7SEM) rectal temperatures where stage of heat
exposurea by treatmentb interactions were signicant
Treatment

Stage of heat exposure (h)

0

Control
FR
HT
FRHT

3
y

41.670.12
41.570.14y
41.570.10y
41.670.18z

6
y

43.470.16
43.870.22y
43.870.19y
43.770.13y

43.770.17bx
44.270.30abx
44.170.21abx
44.570.13ax

Fig. 1. PVDF membrane representation of heat shock protein

70 expression in the brain of control (lane 1), FR (lane 2), HT
(lane 3), and FRHT (lane 4) chickens following 0 h (top), 3 h
(middle), and 6 h (bottom) of heat exposure (39711C) on d 35.
Numbers on the left margin are location and size (KDa) of
molecular weight markers. Control: ad libitum feeding and no
heat treatment; FR: 60% feed restriction on d 4, 5 and 6; HT:
exposure to 36711C for 1 h/day from d 1 to d 21; FRHT:
combination of FR and HT.

In the current experiment we showed that subjecting

birds to FR and HT reduced HLR response to high
ambient temperature, suggesting better ability to cope
with heat stress. This conrms previous observations
that the response of poultry to high ambient temperatures can be modied by early age exposure to thermal
and non-thermal stressors. (Arjona et al., 1988, 1990;
Yahav and Hurwitz, 1996; Zulkii et al., 1994a, b,

On d 35, all chicks were exposed to 39711C and 50%

relative humidity for 6 h.
b
Control: ad libitum feeding and no heat treatment; FR:
60% feed restriction on d 4, 5 and 6; HT: exposure to 36711C
for 1 h/day from d 1 to d 21; FRHT: combination of FR and
HT.
a, b
Means within a column with no common superscript differ
at Pp0.05.
xz
Means within a row with no common superscript differ at
Pp0.05.

2000). In contrast to our observations and those of

Zulkii et al. (1994a, b, 2000), Plavnik and Yahav (1998)
reported that early age feed restriction had no effect on
birds response to high ambient temperature later in life.
The restriction level in the study of Plavnik and Yahav
(1998) was more severe than the restriction level applied
in our experiment, which may explain the discrepancies
in ndings. Zulkii et al. (2000) compared three different
levels of early age feed restriction and concluded that the
magnitude of stress perceived early in life may inuence
the ability of broiler chickens to withstand high
temperatures at market age.

I. Zulkifli et al. / Journal of Thermal Biology 28 (2003) 217222

The HLR data do not clearly suggest that the FRHT

combination can further reduce physiological response
to heat stress. The HLR of FRHT, FR and HT birds
were not signicantly different throughout the heat
exposure period. Nevertheless, it is interesting to note
that although the FR, HT and control birds produced a
signicant rise in HLR after 3 h of heat treatment, a
HLR response in the FRHT birds was only noted after
6 h of heat challenge.
This experiment conrmed previous ndings (Zulkii
et al., 2002) that exposing broiler chickens to high
temperatures increased the induction of hsp 70 in the
brain. Higher hsp 70 expression in the livers (Gabriel
et al., 1996), blood leucocytes (Wang and Edens, 1998),
lungs and hearts (Yahav et al., 1997) of hyperthermic
chickens has also been reported. It is well documented
that hyperthermia may increase the activity of the heat
shock transcription factor, which enhances hsp 70
mRNA synthesis and consequently hsp 70 concentration
(Craig and Gross, 1991). Results of the present study are
in agreement with those of Zulkii et al. (2002) that
subjecting chicks to feed restriction at d 4, 5 and 6 of age
enhanced hsp 70 expression in heat-stressed broilers.
Following 6 h of heat challenge at d 35 of age, the mean
hsp 70 densities of FR birds were higher than controls. It
is interesting to note that the early age feed restriction
had the potential to improve hsp 70 synthesis 4 wk
following the cessation of feed restriction. It has become
increasingly clear that stressful experiences during the
neonatal stage can elicit hsp mRNA transcription but
the RNA may have been sequestered and not
translated until exposure to heat stress later in life
(Craig, 1985).
Wang and Edens (1998) found that heat conditioning,
via a daily 1 h exposure to 411C, enhanced in vitro hsp
expression in leucocytes of poults heat conditioned for 1,
3 or 5 week. On the contrary, our results suggest that
conditioning of chicks by exposure to moderate heat
stress has no profound impact on induction of hsp 70
synthesis as compared to those of controls. The contradiction between these experiments could be attributed to
differences in poultry species, samples collected for hsp
70 analysis and protocol of heat challenge. The study by
Wang and Edens (1998) involved in vitro heat challenge
of blood leucocytes in turkeys.
Because HT alone failed to improve hsp 70 response
as compared to the control group, it is not unexpected
that heat conditioning as an additional treatment to
early age feed restriction (FRHT) will not further
improve the synthesis of hsp 70 in heat-stressed
chickens. Thus, it appears that FR alone own is
sufcient to enhance hsp 70 activity during heat stress.
Previous studies have shown that acclimatised birds
were able to maintain body temperatures at a point that
was lower than that of non-acclimated birds following
heat exposure (May et al., 1987; Yahav and Hurwitz,

221

1996; Yahav and Plavnik, 1999). On the contrary,

Arjona et al. (1990) and Yalcin et al. (2001) failed to
note a signicant difference in the body temperature of
acclimated and non-acclimated birds. In this experiment, following 6 h of heat treatment, although the
FRHT birds were less stressed (as measured by HLR)
than controls, the former were more hyperthermic.
Considering the superior hsp 70 response of FRHT
birds and the positive correlation between the rate of hsp
70 synthesis and body temperature (Gabriel et al., 1996),
higher body temperature of FRHT birds was expected.
There is considerable evidence that the synthesis of hsp
70 is temperature dependent (Wang and Edens, 1993,
1998), and thus hsp 70 response is considered as a
cellular thermometer (Craig and Gross, 1991). Studies in
Escherichia coli and cultured Drosophila cells have
shown that cells responded to temperature increase by
increasing either the amount or the activity of a
transcription factor that is specic for the heat shock
gene (Craig and Gross, 1991). In view of this, there is a
possibility that acclimatisation or habituation characterised by the ability to maintain lower body temperature under heat stress condition (May et al., 1987; Yahav
and Hurwitz, 1996; Yahav and Plavnik, 1999) may not
be associated with enhanced hsp response. This notion is
strengthened by the ndings of Yahav et al. (1997) that
early age conditioning by exposure of chicks to heat for
24 h at 5 days of age induced a long term mechanism
that acted to reduced hyperthermia during heat challenge and thus reduce the synthesis of hsp. It is apparent
that expression of hsp may not be the only factor
required for the development of improved heat tolerance
in chickens (Wang and Edens, 1998).
There are conicting results in the literature on the
body temperature that will evoke signicant expression
of hsp in chickens. Yahav et al. (1997) noted that
moderate hyperthermia of 44.51C or less did not lead to
profound induction of hsp, whereas hyperthermia of
45.41C or higher is followed by elicitation of hsp 90, hsp
70 and hsp 27. On the other hand, Gabriel et al. (1996)
reported that the hsp 70 response peaked when the body
temperature of broiler reached 43.51C. Working with
turkeys, Wang and Edens (1998) reported that the
synthesis of hsps was very low and difcult to detect in
several turkeys in which the body temperature did not
reach 431C. In the present study, signicant induction of
hsp 70 was detected at hyperthermia of 43.71C or higher.
Although there is no clear explanation for these
discrepancies, the hsp 70 response is evoked after a shift
to a lethal temperature, in which case, non-hsp-synthesis
is shut off and cells produce hsps at maximal rates as
long as protein synthesis continue (Craig and Gross,
1991).
We conclude from the present experiments that FR
on its own can dampen HLR response and improve
hsp 70 expression in broiler chickens subjected to acute

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I. Zulkifli et al. / Journal of Thermal Biology 28 (2003) 217222

heat challenge later in life. As measured by HLR

and hsp 70 density, the FRHT combination did not
further improve heat tolerance. It is also apparent
that FR has advantages over HT in altering the birds
ability to elicit hsp 70 activity in response to high
temperatures. An important outcome of this study
is the conrmation that induction of hsp 70 synthesis
in chickens is directly correlated to the extent of
hyperthermia.

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