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Poult Sci 2009. 88:471-476. doi:10.3382/ps.2008-00287
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions

I. Zulkifli*,1, A. Al-Aqil*, A. R. Omar{dagger}, A. Q. Sazili* and M. A. Rajion{ddagger}

* Department of Animal Science, {dagger} Department of Veterinary Pathology and Microbiology, and {ddagger} Department of Veterinary Pre-Clinical Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

1 Corresponding author: zulkifli{at}agri.upm.edu.my


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two hundred thirty-five 1-d-old broiler chickens showing short or long tonic immobility responses were classified as low fear (LF) or high fear (HF) responders, respectively. On d 41, they were subjected to either crating or heat challenge (34 ± 1°C) for 3 h and its effect on plasma corticosterone concentration, heterophil/lymphocyte ratios, and heat shock protein (HSP) 70 expression in brain tissue were determined. Crating and heat exposure elevated heterophil/lymphocyte ratios in both LF and HF birds. Circulating corticosterone, however, was greater in HF than LF birds after crating and heat challenge. Although differences between fear responder group for HSP 70 were negligible before heat challenge, after 3 h of heat exposure, the response was greater for the HF than the LF group. Both LF and HF showed similar increases in HSP 70 after crating.

Key Words: heat shock protein 70 • corticosterone • heterophil:lymphocyte ratio • tonic immobility • chicken


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heightened fearfulness and expression of fear responses can seriously harm an animal’s welfare and productivity. The negative relationships between underlying fearfulness and performance may be associated with chronic elicitation of the hypothalamic-pituitary-adrenal axis response. Although fear and stress are not synonymous, fear encompasses adrenergic, dopamergic, and cholinergic systems, which play a pivotal role in the physiological stress response in poultry (Jones, 1986). Broom et al. (1986) reported that hens that showed the greatest avoidance of the experimenter had the greatest plasma corticosterone (CORT) concentrations after catching and transport. Similarly, hens with a history of laying eggs with certain shell defects, a stress-related phenomenon, were more fearful than normal layers (Hughes et al., 1986).

Beuving et al. (1989) studied adrenocortical and heterophil/lymphocyte ratio (HLR) reactions to thwarting of feeding in low-fear and high-fear hens, and although there were no significant differences between groups, the latter exhibited a consistent tendency toward greater stress responses. Subsequent reports to define the precise mechanisms underpinning the relationship between fearfulness and adrenocortical activation are mainly in genetic lines of quail selected for short and long tonic immobility (TI) duration (Mills and Faure, 1991) or low stress and high stress (Satterlee and Johnson, 1981). In his review, Jones (1996) concluded that the long TI duration quail had greater CORT reaction to various stressors and the high stress quail exhibited shorter TI duration. On the contrary, Mills et al. (1997) and Hazard et al. (2005) subjected quail that have been selected for short and long TI duration to mechanical restraint, a powerful stressor, and the former demonstrated greater adrenocortical reactions. Despite the substantial previous work on the relationship between fear responses and adrenocortical responsiveness in quail, the mechanisms underpinning the differences between lines in physiological and behavioral responsiveness have yet to be determined.

Fear may be regarded as an adaptive psychophysiological response to perceived danger (Jones, 1996). In rodents, psychological stress involving exposure to the emotional responses of foot-shocked rats increased heat shock protein (HSP) 70 expression in the blood vessels (Isosaki and Nkashima, 1998). When organisms are exposed to thermal and nonthermal stressors, for example exposure to heavy metals, toxins, bacterial and viral infections (Morimoto, 1993), and feed deprivation (Zulkifli et al., 2002, 2003), the synthesis of most proteins is reduced, whereas a group of highly conserved proteins known as HSP 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 effect of heating (Barbe et al., 1988). Thus, it is reasonable to hypothesize that HSP 70 may play an important role in determining the stress responsiveness in birds differing in TI duration. In this study, broiler chickens showing short or long TI were classified as low fear (LF) or high fear (HF) responders, respectively, and were subjected to either heat challenge or crating. The effects of those stressors on CORT, HLR, and HSP 70 density were determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Welfare

The study was undertaken following the guidelines of the Research Policy of the Universiti Putra Malaysia on animal ethics.

Chickens and Husbandry

A total of 216 one-day-old male broiler chicks (Cobb x Cobb) were obtained from a commercial hatchery. On d 1, the chicks were wing-banded and housed in groups of 6 in 36 battery cages with wire floors in an environmentally controlled chamber (2.3 m x 9.1 m x 3.8 m). Floor space allowed was 923 cm2 per bird. An ambient temperature of 32 ± 1°C on d 1 was gradually reduced to 23 ± 1°C by d 21. The chicks were administered (intraocularly) live Newcastle disease vaccine (Nobilis ND Clone 30, Intervet International, Boxmeer, the Netherlands) on d 7 and 21. Feed and water were provided for ad libitum consumption. Birds were fed standard broiler starter crumble (2,950 kcal of ME/kg; 21% CP) and finisher pellet (3,100 kcal of ME/kg; 19.5% CP) from d 1 to 20 and d 21 to 42, respectively. Lighting was continuous and the intensities were 40 and 20 lx from d 1 to 2, and d 3 onwards, respectively.

TI Test

A total of 200 birds (16 birds died from d 1 to 34) were tested for TI on d 35. Each individual was gently caught with both hands, held in an inverted manner, and carried to a separate room (with an ambient temperature of 23°C; no visual contact with other birds) for TI measurements. A modification of the procedure described by Benoff and Siegel (1976) was used. Tonic immobility was induced as soon as the bird arrived in the separate room by gently restraining it on its right side and wings for 15 s. The experimenter then retreated approximately 1 m and remained within the sight of the bird but made no unnecessary noise or movement. Direct eye contact between the observer and the chicken was avoided because it may prolong TI duration (Jones, 1986). A stopwatch was started to record latencies until the bird righted itself. If the bird righted itself in less than 10 s, the restraining procedure was repeated. If TI was not induced after 3 attempts, the duration of TI was considered 0 s. The maximum duration of TI allowed was 600 s. After the measuring of TI, the bird was spray-painted on the back for identification and returned to its home flock. It was assumed that the catching and returning of birds did not disturb the other members of the flock (Lagadic et al., 1990).

The birds were ranked from low to high (1 to 200) according to the duration of TI (Beuving et al., 1989). Those 40 birds showing the shortest TI duration and the 40 scoring the longest durations were classified as LF or HF fear responders, respectively. The remaining 120 birds scoring intermediate TI durations were excluded from the study but remained in their home flock.

Crating

On d 42 (at 0730 h), 20 birds from each fear responder group were randomly selected, removed from their flock, carried by the legs in an inverted manner, and placed in 2 plastic crates (0.80 m x 0.60 m x 0.31 m) with 10 birds to each crate. The crates were left stationery for 3 h in the environmentally controlled chamber (crates were placed in a single stack). The birds were returned to their home flock after the procedure.

Heat Challenge

On d 42 (at 1600 h), the remaining 20 birds (those that were not crated) from each fear responder group were exposed to 34 ± 1°C for 3 h. To avoid any form of social disruption, birds that were crated earlier and returned to their home cages after the procedure were also exposed to the heat challenge. Time required for the ambient temperature to increase from 24 to 34°C was approximately 30 min. Feed and water were not provided during the heat exposure period.

Blood and Brain Samples

Before (0 h) and 3 h after crating or heating, blood samples (0.3 mL) were obtained with EDTA as the anticoagulant from the brachial vein of 10 birds from each fear responder group for measuring CORT and number of heterophils and lymphocytes. Individuals bled at 0 h were not sampled at 3 h. Samples for CORT assay were centrifuged and stored at –20°C until assayed, using a sensitive and highly specific RIA kit (MP Biomedicals, Irvine, CA; Severson et al., 1978). Blood smears were prepared using May-Grunwald-Giemsa stain, and heterophils and lymphocytes were counted to a total of 60 cells (Gross and Siegel, 1983). Immediately after blood sampling, 5 birds from each fear responder group were randomly chosen and killed (by cervical dislocation), and the entire brain samples were removed, frozen quickly in liquid nitrogen, and stored at –70°C until further analysis for HSP 70 density.

SDS-PAGE and Immunoblot Analysis

Brain samples (0.5 g) were homogenized in an Ultra-Turrax homogenizer (IKA Ultra-Turrax, Staufen, Germany, using 5 mL of chilled Tris-HCl buffer (20 mM Tris pH 7.5, 0.75 M NaCl, 2 mM 2-mercaptoethanol), and were centrifuged at 23,000 x g for 30 min at 4°C. The protein concentration of the supernatants was quantified by the bicinchoninic acid Protein Assay Kit Procedure No. TRPO-562 (Sigma Chemical Co., St. Louis, MO; Brown et al., 1989) with BSA as the standard. Thirty micrograms of total protein was loaded and separated on 1.5 x 80 x 100 mm of 12% polyacrylamide gels containing SDS (Laemmli, 1970) using the Hoefer Mini Gel apparatus (Hoefer Scientific Instrument, San Francisco, CA). Gels were electrophoresed at 150 V until the tracking dye reached the base of the gel. The fractionated proteins were visualized by Coomassie Blue staining or transferred to polyvinylidene difluoride (PVDF) membranes (MSI, Westborough, MA; Towbin et al., 1979). After electrophoretic transfer, the PVDF membranes were stained with 0.5 g/L of Ponceau S in 10 g/L of acetic acid solution to visualize and mark the positions of the proteins used as molecular weight standards. After washing the Ponceau S with distilled water, the nonspecific binding sites were blocked using 10 mL of cold blocking buffer containing 10% nonfat milk and 0.05% sodium azide for 30 min. The membranes were incubated overnight (4°C) with 5 mL of blocking buffer containing antiserum (mouse anti-chicken HSP 70; Sigma Chemical Co.) against HSP 70 in a 1:1,000 dilution. After overnight incubation, the blots were washed 4 times (5 min each) with 10 mL of cold blocking buffer. The blots were then reacted with goat anti-mouse secondary antibody conjugated to alkaline phosphatase (Sigma Chemical Co.) for 1 h. After rinsing with cold PBS, the color reaction on the PVDF membrane was developed using commercially prepared BCIP/NBT (Sigma Chemical Co.). Relative density of the HSP 70 was determined using a densitometer (UVP, Cambridge, UK) with the UVP Gel Base Pro program.

Statistical Analysis

Data were analyzed using the GLM procedure of SAS (SAS Institute, 1991). A 2-way ANOVA was used to analyze the TI duration. The CORT, HLR, and HSP 70 density data were subjected to 2-way ANOVA with the fear responder group, duration of stress (crating or heating), and the interactions between them as main effects. When interactions were significant, separate ANOVA were conducted within each main effect. Before analysis, CORT were transformed to square roots. Untransformed means are presented in the tables. All statements of significance were based on P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TI Duration

There was a significant difference (P < 0.0001) in TI duration between the LF (16.4 s ± 2.00) and HF (373.9 s ± 15.19) groups.

Effects of Crating

There was no significant fear responder group x duration interaction for HLR and HSP 70 densities. The mean HLR and HSP 70 densities of LF and HF birds were not significantly different (Figure 1Go). Irrespective of fear responder group, 3 h of crating significantly elevated HLR and HSP 70 density (Figure 2Go). Interaction of fear responder group x duration of crating interaction was significant for CORT (Table 1Go). Before crating, both LF and HF birds had similar CORT with the latter having a greater response than the former after 3 h of crating.


Figure 1
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  		Figure 1. The effect of crating on heterophil/lymphocyte ratios (top) and heat shock protein 70 densities (bottom) by fear responder group. Means are not significantly different (P > 0.05). LF = low fear group; HF = high fear group.

 

Figure 2
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  		Figure 2. The effect of crating on heterophil/lymphocyte ratios (top) and heat shock protein 70 densities (bottom) by duration. a,bMeans with no common letters differ (P < 0.05).

 

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  		Table 1. Mean (±SEM) plasma corticosterone concentrations (ng/mL) in which fear responder group x duration of crating interactions were significant
 
Effects of Heat Challenge

There was no significant fear response group x duration of heat exposure interaction for HLR. Although the HLR of HF and LF birds were not significantly different (Figure 3Go), 3 h of heat challenge significantly elevated HLR of both groups (Figure 4Go). The fear responder group x duration of heat exposure interaction was significant for CORT (Table 2Go). Although there was no significant difference at 0 h, after 3 h of heat exposure, the HF birds had significantly greater CORT than the LF ones. Comparisons between LF and HF birds revealed a significant fear responder group x duration of heat exposure interaction for HSP 70 density (Table 3Go). Although a marked elevation in HSP 70 response was noted in both LF and HF chicks after 3 h of heat challenge, the mean HSP 70 densities of the latter were significantly greater.


Figure 3
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  		Figure 3. The effect of heat exposure on heterophil/lymphocyte ratios by fear responder group. Means are not significantly different (P > 0.05). LF = low fear group; HF = high fear group.

 

Figure 4
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  		Figure 4. The effect of heat exposure on heterophil/lymphocyte ratios by duration. a,bMeans with no common letters differ (P < 0.05).

 

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  		Table 2. Mean (±SEM) plasma corticosterone concentrations (ng/mL) in which fear responder group x duration of heat exposure interactions were significant
 

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  		Table 3. Mean (±SEM) heat shock protein 70 densities in which fear responder group x duration of heat exposure interactions were significant
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present findings confirmed earlier reports that heat challenge (Zulkifli et al., 1999, 2003; Mahmoud et al., 2004) and crating (Kannan and Mench, 1996; Zulkifli et al., 2001) may elevate CORT and HLR in chickens. However, the effect of heat and crating on CORT has not been consistent. McFarlane and Curtis (1989) reported that exposing 7-d-old chicks to environmental heat stress for 7 d increased HLR but not CORT. Edens and Siegel (1975) indicated that increases in CORT attributable to heat stress were maintained for only 70 min. Although birds subjected to crating only, as practiced in the present study, are not exposed to potential stressors such as acceleration, vibration, and motion, Knowles and Broom (1990) indicated that the physiological stress reactions of birds subjected to crating only, or both crating and transportation, were not significantly different. On the contrary, Zulkifli et al. (2001) reported that broilers subjected to crating and transportation had greater HLR than those that were crated and left stationery. Although there is no clear explanation for the phenomenon, the discrepancies could be associated with several factors including timing of blood sampling after onset of exposure to a stressor (Siegel, 1995).

Our findings are consistent with those of Zulkifli et al. (1999, 2003) that high ambient temperatures raised HLR within 3 h. Mitchell et al. (1992) reported a rapid HLR response in chickens after 3 h of crating and road transportation. Here, we report that 3 h of crating alone elevated HLR.

The relationship between fearfulness and adrenocortical responsiveness to stressors in quails has been inconsistent (Jones, 1996). We observed significant fear responder group x duration of stress interactions for CORT and provide evidence that the LF and HF birds responded differently to heat challenge and crating. Before heat challenge and crating, CORT was similar for both fear responder groups. However, the HF birds were more distressed than their LF counterparts after 3 h of heat exposure and crating. One might have expected that HF birds are only more susceptible to crating, a fear-elicited stressor, and not to thermal insults. Previous studies, however, suggested a possible link between thermoregulation and underlying fearfulness. According to Brown and Nestor (1974), turkeys selected for low adrenocortical response to cold stress exhibited reduced excitability. Furthermore, Campo and Carnicer (1994) showed that heat stress prolonged TI duration in chickens. Hence, it appears that response to thermal stressors is associated with fear-related behavior.

We are unaware of prior reports on an association between HSP 70 expression and underlying fearfulness in avian species. Although the crating procedure was more stressful to the HF than LF chickens, the HSP 70 densities of both groups were not different. On the contrary, the heat challenge resulted in significantly greater CORT and HSP 70 density in HF than LF birds. There appears to be no definite explanation for the discrepancies in HSP 70 expression in response to crating and heat challenge, and only a speculative one can be offered at this stage. Based on CORT and HLR, crating is a putatively milder stressor than heat exposure and thus it is possible that the magnitude of stress experienced was insufficient to evoke a variation in HSP 70 reaction between the 2 fear responder groups. There is considerable evidence that the synthesis of HSP 70 is temperature-dependent (Wang and Edens, 1998; Zulkifli et al., 2003), and thus HSP 70 response is considered 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 specific for the heat shock gene (Etches et al., 1995). Hence, it is not unexpected that the HF birds that were more heat stressed, as measured by CORT, showed greater HSP 70 expression.

Corticosterone and HLR are commonly used physiological indices of stress after catching and crating of chickens (McFarlane and Curtis, 1989; Jones et al., 1991; Zulkifli et al., 2001). Our findings that HSP 70 density may be used as a biological index of stress attributed to crating is consistent with the report in which road transportation increased HSP 70 expression in heart and kidney tissues of pigs (Yu et al., 2007).

In conclusion, the HF chickens showed greater adrenocortical responsiveness to both crating and heat challenge than those of LF. Subjecting birds to crating or heat stress resulted in the induction of HSP 70. Although as measured by CORT, HF birds were more stressed than LF ones after crating and heat challenge, the latter showed greater HSP 70 expression after heat exposure, a more powerful stressor. Hence, HSP 70 reaction in chickens may be dependent on the nature of the stressor and the magnitude of its response. It remains to be investigated whether the increase in HSP 70 induction among the heat-stressed HF birds actually protects them against the adverse effects of high ambient temperatures. Earlier work in our laboratory (Liew et al., 2003) showed that greater HSP 70 response is beneficial in enhancing resistance to viral infection in heat-stressed chickens. Under conditions of this experiment, induction of HSP 70 appears to be associated in the physiological stress responsiveness of chickens varying in the magnitude of underlying fearfulness.


   ACKNOWLEDGMENTS
 
The research was supported by King Faisal University, Al-Hassa, Saudi Arabia. We are thankful to P. B. Siegel (Virginia Polytechnic Institute and State University, Blacksburg) for critical reading of the manuscript.

Received for publication July 16, 2008. Accepted for publication November 12, 2008.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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