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Hepatic concentration of heat shock protein

70 kD (Hsp70) in broilers subjected to
different thermal treatments
P.E. N. GIVISIEZ , J.A. FERRO , M.I.T. FERRO , S.N. KRONKA , DECUYPERE &
MACARI
Version of record first published: 28 Jun 2010.

To cite this article: P.E. N. GIVISIEZ , J.A. FERRO , M.I.T. FERRO , S.N. KRONKA , DECUYPERE & MACARI
(1999): Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal
treatments, British Poultry Science, 40:2, 292-296

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 British Poultry Science (1999) 40: 292–296

Hepatic concentration of heat shock protein 70 kD (Hsp70) in

broilers subjected to different thermal treatments
P. E. N. GIVISIEZ, J. A. FERRO1 , M. I. T. FERRO1 , S. N. KRONKA2 , E. DECUYPERE3 AND M.
MACARI
Departments of Animal Morphology and Physiology, 1 Technology and 2 Statistics, UNESP, Brazil and 3 Department of
Animal Science, KU Leuven, Belgium

Abstract 1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was
studied in broiler chickens.
2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups
were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35°C for
4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room
temperature of 35°C.
3. From 39 to 42 old, the birds experienced acute heat stress at 41°C. Resistance to heat stress was evaluated
Downloaded by [Ohio State University Libraries] at 22:20 20 April 2013

by the time taken for rectal temperature to increase by 3°C above the pre-treatment value. Livers were
collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with mono-
clonal anti-Hsp70 antibody.
4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat
stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed
between Hsp70 concentration and the time taken for a 3°C increase in rectal temperature (r= 0·42; P<0·01).
5. Exposing birds to episodes of heat stress (35°C) during rearing may improve their resistance to acute heat
stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after
exposure to more severe heat stress (41°C).

INTRODUCTION al., 1994). It has been suggested that they might be

Heat stress causes serious losses in poultry produc-
tion because it increases mortality and reduces
performance in broilers and laying hens (Teeter et
al., 1985). Birds exposed to high ambient tempera-
tures divert part of their production energy to
maintain internal thermal homeostasis, and show
physiological responses such as panting. A decrease
in food intake and growth have been reported and
food conversion efficiency is significantly reduced
(Bonnet et al., 1997). Greater heat resistance has
been achieved by exposing birds to cycling tempera-
tures (May et al., 1987; Yahav et al., 1996) or neonatal
heat stress (Arjona et al., 1988, 1990; Yahav and
Hurwitz, 1996). Molecular mechanisms that may
explain this improvement in thermotolerance have
not yet been explained (Gabriel et al., 1996).
It is already known that heat and other stres-
sors (for example, anoxia and heavy metal ions)
rapidly induce the synthesis of heat shock proteins
(Hsp) (Lindquist and Craig, 1988). In a normal
environment, Hsps are synthesised at low rates by
the cells and their functions are related to intracel-
lular protein synthesis and transportation, folding
and assembly of protein complexes, or in the regula-
tion of the activity of key proteins involved in some
cellular processes (Baler et al., 1992; Morimoto et
involved in cellular protection in adverse situations,
and a relationship has been proposed between the
development of thermotolerance and Hsp synthesis,
especially in those with molecular weight of approxi-
mately 70 kD (Hsp70) (Lindquist and Craig, 1988;
Parsell and Lindquist, 1994). During heat stress,
Hsp70 locates in the nucleus, binding to the RNA
synthesis machinery and preventing synthesis and
aggregation of abnormal protein (Lee, 1992). In
birds, Hsps of molecular weights between 22 and
108 kD have been reported, in situations of in vitro
and in vivo heat stress, in reticulocytes (Banerji et al.,
1986), lymphocytes (Morimoto and Fodor, 1984),
and in lymphoblastoid and myeloid lineage cells
(Banerji et al., 1986; Miller and Qureshi, 1992).
Yahav et al. (1997) showed that the conditioning of
chicks at 36°C when they were 5-d-old improved
thermotolerance at 42 d, but that the concentrations
of Hsp70 in heart and lung tissues were lower than in
controls, suggesting that Hsp70 is not a part of the
long-term mechanism evoked by the early age
conditioning. On the other hand, Hsp70 synthesis is
broilers is thermo- and time-dependent during heat
stress (Gabriel et al., 1996). The aim of this study was
to establish a relationship between hepatic Hsp70
concentrations and thermotolerance in broilers
subjected to different heat stress treatments.

Correspondence to: M. Macari, Department of Animal Morphology and Physiology, Faculty of Agricultural and Veterinary Sciences,
UNESP, Campus of Jaboticabal Rod. Carlos Tonanni, km 5–14.870–000, Jaboticabal SP, Brazil. Email: macari@fcav.unesp.br.
0007–1668/99/020292–05 © 1999, British Poultry Science Ltd
 THERMOTOLERANCE AND HEAT SHOCK PROTEIN
MATERIALS AND METHODS
Bird management
Birds were maintained in a climatic chamber
measuring 3·0×4·0×3·0 m, in cages of 0·5×0·6×
0·5 m at a density of 2 birds/cage. The temperature
of the chamber was adjusted to thermoneutrality,
decreasing from 33°C during week 1 to 24°C during
week 6, at a rate of approximately 1·8°C per week.
Four groups of 12 birds each were kept at thermone-
utral temperature and submitted to 4 h episodes of
heat stress at 35°C, 0, 1, 2 or 3 times per week,
from week 1 to week 6 (groups TN-0, TN-1, TN-2
and TN-3, respectively). Another group (HT-35)
was reared in a climatic chamber measuring
1·85×2·30×1·85 m, using cages and stocking density
similar to those described above, at a constant
ambient temperature of 35°C. All groups received
water and food ad libitum. Food was supplied in mash
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form containing 13·39 MJ ME/kg and crude protein
content 220 g/kg in the 1st phase and 200 g/kg in
the 2nd phase.
Acute heat stress
Between 39 and 42 d of age, birds were exposed to
41°C and 70% to 80% RH to evaluate their resist-
ance to acute heat stress. Birds from the groups that
were intermittently heat stressed were maintained
at thermoneutrality (24°C) for 4 d before being
subjected to acute heat stress. From the 12 birds in
each group, 3 birds were slaughtered as controls
and the other 9 birds were subjected to acute heat
stress. Rectal temperature was measured at 15-min-
intervals using a telethermometer (Yellow Spring
Instruments, Model 46 TUC) and a probe inserted
5 cm into the rectum (Probe YSI n·702). When it
increased by 3°C above pre-treatment values, birds
were slaughtered as soon as possible. Liver samples
from slaughtered birds were immediately frozen in
liquid nitrogen and stored at –70°C until Hsp70
analysis was performed. The time taken for rectal
temperature to increase by 3°C (D T3°C) was
recorded to evaluate resistance to acute heat stress.
Protein determination and electrophoresis
Liver samples (1 g) were homogenised in 50-ml poly-
propylene centrifuge tubes, using 10 ml lysis
buffer (20 mM Tris-HCl, pH 7·5; 9 g/l NaCl; 2 mM
b -mercaptoethanol). Samples were homogenised 3
times (30 s) using an Ultra-Turrax homogeniser, at
20 000 rpm and ice-bath intervals of 30 s. Lysate
was centrifuged at 31,000 g for 30 min at 4°C. The
supernatant was transferred to 15-ml polypropylene
tubes and was manually homogensied 10 times,
using a Potter-Elvehjem homogeniser. Two 300-µl
aliquots were separated, for total protein determina-
tion and electrophoresis. Three hundred µl of 2×
concentrated sample buffer (125 mM Tris-HCl, pH
6·8; 200 ml/l glycerol; 40 g/l SDS; 0·02 ml/l
293
bromophenol blue) and 40 µl b -mercaptoethanol
were added to the electrophoresis samples. Samples
were boiled for 2 min and stored at –20°C until
electrophoresis. The protein concentration of super-
natant aliquots was determined in quintuplicate,
according to the method described by Hartree
(1972). The standard curve was produced with
bovine serum albumin (BSA Sigma) in triplicate
samples of 0, 20, 40, 60, 80 and 100 µg. Thirty µg
total protein were loaded and separated on 9% poly-
acrylamide gels containing SDS (Laemmli, 1970),
using the Mini-Protean II apparatus (Bio-Rad) at a
constant voltage (200 V). Before loading, the samples
stored at-20°C were reboiled for 2 min and a sample
of supernatant from a control bird from the TN-0
group was loaded on all gels, as a reference standard.
A pre-stained molecular weight standard (Gibco-
BRL) was used in all gels.
Western Blot Analysis
After fractionation through SDS-polyacrylamide
gels, the proteins were electrophoretically transferred
to nitrocellulose membranes using the procedure of
Towbin et al. (1979). Transference was performed
for 12 h at 4°C at constant voltage (66 V), using a
mini trans-blot cell (Bio-Rad). The membranes were
stained with 0·5 g/l Ponceau S in 10 g/l acetic acid,
for 3 min, to evaluate the transference. After several
washings with deionised water, non-specific interac-
tion sites were blocked using 10 ml of cold TBS
buffer (10 mM Tris-HCl pH 8·0; 150 mM NaCl)
containing 50 g/l non-fat dried milk and 0·2 g/l
Tween-20, for 1 h at room temperature, in a shaker
(100 rpm, approximately). The membranes were
then incubated with 10 µl monoclonal anti-Hsp70
antibody (H-5157, Sigma) in 10 ml of cold TBS-milk
solution (1:1000 dilution) containing 0·2 g/l Tween-20,
for 1 h at room temperature in a shaker. Four wash-
ings of 5 min each using 10 ml TBST (10 mM Tris-
HCl, pH8·0; 150 mM NaCl; 0·5 g/l Tween-20) and
a 10 min washing using 10 ml of cold TBS buffer
were performed. The membranes were incubated
with 2 µl secondary anti-mouse antibody conjugated
to alkaline phosphatase (A-5153, Sigma) diluted in
10 ml of cold TBS-milk solution (1:5 000 dilution),
for 1 h at room temperature with constant shaking.
After rinsing with cold TBST and TBS as described
above, the colour was developed for 2 min with 33 µl
nitro-blue tetrazolium chloride solution (50 g/l in
dimethylformamide) and 66 µl 5-bromo-4-chloro-3-
indolylphosphate p-toluidine (50 g/l in dimethylfor-
mamide 700 g/l) added to 10 ml AP buffer (100 mM
Tris-HCl, pH9·5; 100 mM NaCl; 5 mM MgCl2 ). The
reaction was blocked by addition of a solution of
trichloroacetic acid (30 g/l). The membrane was
washed with deionised water and dried at room
temperature, protected from light. The colour signal
of the bands corresponding to Hsp70 was analysed
by a densitometer at 525 nm (Shimadzu CS-9301)
using reflection mode and zigzag scanning.
 294 P. E. N . GIVISIEZ ET AL.

Hsp70 quantification Table 1. Hsp70 concentrations (ng Hsp70/µg total protein) in livers
of broilers from different thermal treatments 4h exposure to 35°C
Hsp70 (H-9776, Sigma) was resuspended in glycerol 0,1,2 or 3 times per week: TN-0, TN-1, TN-2, TN-3 or constant
300 ml/l in a final concentration of 500 ng/µl. Aliq- exposure to 35°C: HT-35 and subjected to acute heat stress (41°C)1
uots containing 50, 100, 200, 400, 600 and 800 ng Liver Hsp70 (ng/µg
of the protein were prepared and loaded on the Factors total protein)
electrophoresis gel as well as the reference standard
which was analysed in triplicate. After electro- Thermal treatments (35°C/4 h) Supernatant
TN-0 2´5244 B
phoresis, Western Blot analysis was performed as TN-1 2´6361 B
described. The bands were analysed by densitom- TN-2 2´2650 B
etry and a standard curve for Hsp70 quantification TN-3 3´3611 A
was obtained by plotting the Hsp70 concentration HT-35 3´1839 A
Acute heat stress (41°C) Supernatant
against the ratio of the density of each concentra- Before 2´8787 A
tion relative to that of the average value for the After 2´7096 A
triplicate reference standard (see Figure). The ratio Sources of variation P(F)
between samples and reference standard in each Treatments (T) P<0´01
membrane was used to determine Hsp70 quantity Acute heat stress (AHS) P<0´16
Interaction (T× AHS) P<0´94
in the supernatant. Data were expressed as ng Standard deviation 0´40
Hsp70/µg total protein.
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Statistical analysis
The body weight before acute heat stress, the initial
rectal temperature and the time (min) taken for rectal
temperature to increase by 3°C (D T3°C) during
acute heat stress for the different thermal treat-
ments were subjected to analysis of variance
(ANOVA) in a completely random design. Hsp70
concentrations were analysed in a 5×2 factorial
arrangement (5, thermal treatments; 2, non-stressed
and stressed birds). Means were verified by Turkey’s
test (Steel and Torrie, 1980) at the 5% level of
significance.
RESULTS
The Figure shows the blots for the Hsp70 standard
curve and the area under the curve obtained by the
densitometer. It also shows the ratio obtained by
dividing the area of each standard value by the
mean area of the reference standard, which was
loaded in triplicate. As an internal standard, 200
and 400 ng of Hsp70 were added to the reference
standard. The correlation coefficient of the curve
was high (r= 0·988), revealing a strong relationship
between the amount of protein used and the signal
obtained in the membrane. This quantification
procedure also revealed that the concentrations of
Hsp70 obtained in the samples were within the linear
part of the standard curve. Hepatic Hsp70 concen-
1
For each independent factor, means in the same column not
sharing a common postscript are statistically different (P<0´05).
trations in supernatant were influenced by thermal
treatment with P<0·01 (Table 1). The birds reared
at constant high temperature (HT-35) had lower
(P<0·01) body weight and higher rectal temperature
(P<0·01) (Table 2). The time taken for rectal
temperature to increase by 3°C (D T3°C) during
acute heat stress did not differ (P<0·24) between
treatments (Table 2), but there was a positive correla-
tion between D T3°C and Hsp70 concentration in
the supernatant (r= 0·42; P<0·01). The treatments
did not affect liver Hsp70 during acute heat stress
(41°C), as can be seen in Table 1, resulting in an
absence of interaction (T×AHS).
DISCUSSION
Greater thermotolerance was associated with an
increased time interval between the start of acute
heat stress (41°C) and a 3°C-increase in rectal
temperature (D T3°C). Although not statistically
different (TN-0<TN-1<TN-2<TN-3<HT-35; Table
2), those broilers submitted to intermittent heat stress
or continuously kept at 35°C increased their thermo-
tolerance when this criterion for thermotolerance
was used.
Thermal treatments affected Hsp70 concentra-
tion in the supernatant of liver samples. In Tables 1
and 2 it is shown that treatments with a greater

Figure. Blot of the standard curve of Hsp70. Samples in the 1st row are: RS the reference standard; 50–800 are increasing quantities of pure Hsp70; IS-1 and
IS-2 are the internal standards (200 and 400 ng of Hsp70 added to the reference standard). The 2nd row shows the area (arbitrary units) of each respective sample
in the 1st row read by the densitometer. The ratio (3rd row) was obtained by dividing the area of each sample by the mean area of the reference standard.
 THERMOTOLERANCE AND HEAT SHOCK PROTEIN
Table 2. Body weight (g), initial and final rectal temperature (°C), and time taken for a 3°C increase in
rectal temperature during acute heat stress (41°C). Each value is the mean of 9 birds
Treatment Body weight
(g)
TN-0 2268´89 A2
TN-1 1992´22 AB
TN-2 2117´78 AB
TN-3 2125´56 AB
HT-35 1887´78 B
P(F) P<0´01
Standard deviation 206´55
1
D T3°C time taken for rectal temperature to increase by 3°C during acute heat stress (41°C).
2
Means in the same column not sharing a common postscript are statistically different (P<0´05).
quantity of Hsp70 in the supernatant (HT-35 and
TN-3, P<0·01) also tended to have a greater D T3°C.
There were no interaction effects of thermal
treatments and acute heat stress on Hsp70. This
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indicates that thermal history does not influence
Hsp70 if the heat stress is too severe (41°C).
However, the D T3°C was increased by the previous
thermal history of these heat stressed birds. The
other possibility is that time for a changed expres-
sion in Hsp70 after acute heat stress is too short to
reveal any differences as a function of previous
thermal history. The fact that no differences were
found before and after acute heat stress support this
suggestion. A similar result was found by Hallberg
and Hallberg (1996), showing that Hsps are not
responsible for the higher resistance of Saccharomyces
cerevisiae cells submitted to 43°C after a previous 1-h
incubation at 37°C, although these cells synthetise
more Hsps due to the previous heat stress treat-
ment. Therefore, firm conclusions can not yet be
formulated about the effect of previous thermal
history on Hsp70 content after acute heat stress.
Some authors have suggested that there is a
relationship between thermotolerance and Hsp
synthesis rate, but not with actual concentrations in
cells. (Lee and Dewey, 1987; Laszlo, 1988). Turkey
leukocytes collected from animals experiencing heat
stress have shown greater resistance to heat stress
than cells stressed in vitro (Wang and Edens, 1994).
Evidence suggests that Hsp70 is only one of the factors
involved in cellular protection, and that Hsp70 expres-
sion alone is not enough to increase cellular resistance
to heat. Supporting this, Nagao et al. (1990) have shown
that cells incubated at high temperatures (severe heat
stress), given a recovery period and then subjected to
mild heat stress did not increase Hsp synthesis in
response to the mild heat stress. On the other hand, if
the temperature of the 2nd heat stress was greater
than that of the 1st one, protection did occur. It is as
if the thermotolerant tissue needed to pass a new
temperature setpoint before it could respond, this
process being characterised as thermotolerance
induced by pre-treatment.
Another thermotolerance process occurs when
cells are subjected to continuous heat stress, when
the survival curve shows 2 phases. In the 1st phase,
295
Rectal temperature D T3°C1
(°C) (min)
Initial Final
41´32 AB 44´72 58´33
41´19 B 44´48 63´33
41´28 B 44´43 70´00
41´29 B 44´52 75´00
41´74 A 45´06 90´00
P<0´01 ± P<0´24
0´34 ± 30´42
mortality is high; it then decreases in the 2nd phase,
showing a positive correlation with Hsp70 and
Hsp28 relocalisation. Both proteins migrated to the
nucleus in the 1st phase and were redistributed in
the cytoplasm in the 2nd, which coincided with the
beginning of normal cellular function (Burdon,
1986; Lee, 1992).
Until now, the expression of Hsp after heat
stress and subsequent thermotolerance has mainly
been described for cellular systems. Our paper
describes the effect of heat stress at the organism
level on the content of Hsp70 and its possible
involvement in heat acclimation. It was shown that
intermittent heat stress could increase Hsp70 in liver
cells together with a degree of thermotolerance, as
measured by the time needed for a 3°C increase in
body temperature after acute heat treatment.
Although the correlation between Hsp70 and
D T3°C is significant, the fact that this correlation is
only 0·42 may be linked to the possibility that the
correlation between D T3°C and the increase in liver
temperature (as the tissue in which Hsp is measured)
is not perfect. It was assumed that the increase in
measured body temperature also reflected a similar
or comparable increase in liver temperature. To what
extent the effects observed in the current study are
causally linked remains to be elucidated, as well as
the exact mechanism by which the treatment
increases Hsp70.
ACKNOWLEDGEMENTS
The authors thank Dr Sara Weytjens for critically
reading the methodology, and M.M. Silva, E.R. Secato
and J.R. Guerreiro for their excellent technical assist-
ance. This work was supported by grants from
Conselho Nacional de Desenvolvimento Cientifico
e Tecnológico (CNPq) and Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), which
the authors gratefully acknowledge.
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