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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
Department of Animal Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
1 Corresponding author: zulkifli{at}agri.upm.edu.my
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transport stress • heat shock protein 70 • housing • feed restriction • broiler chicken
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rearing experience is known to influence the physiology and behavior of an animal in response to noxious stimuli (Lay, 2000). A greater complexity of rearing conditions may attenuate reactions to subsequent environmental challenges. Environmental complexities can be considered as an enrichment or extra stimulation in the house environment of poultry that may enhance ability to cope with novelties and stressful situations (Fraser and Broom, 1997). The majority of enrichment studies in poultry have focused on underlying fearfulness. Birds housed in different systems have been reported to exhibit different levels of underlying fearfulness (Jones and Faure, 1981; Kujiyat et al., 1983). Scott et al. (1998) reported that free-ranged hens were less fearful than those that were caged. Thus, it is reasonable to hypothesize that housing birds in a more complex environment can dampen physiological stress responses to crating and transportation.
Stress, which occurs early in life, when many systems of the chicks are still developing, may have a long-lasting effect and could improve their ability to cope with stressors later in life (Gross and Siegel, 1993; Zulkifli and Siegel, 1995). For example, exposure of 5-d-old broiler chickens to elevated temperatures improved survivability in an otherwise lethal heat challenge at the age of 42 d (Arjona et al., 1988; Yahav and Hurwitz, 1996). An animal does not always have to be preconditioned to the same stressors for habituation to take place. Zulkifli (2003) showed that early age feed restriction reduced heterophil:lymphocyte ratios of broiler chickens subjected to road transportation later in life.
When living organisms are exposed to thermal stresses, the synthesis of most proteins is retarded, but a group of highly conserved proteins known as heat shock proteins (hsp) is rapidly synthesized. In a heat-shocked cell, the hsp may bind to heat-sensitive proteins and protect them from degradation, or may prevent damaged proteins from immediately precipitating and permanently affecting cell viability (Etches et al., 1995). It has been documented that stressors other than thermal stressors, for example, exposure to heavy metals, toxins, oxidants, bacterial and viral infections (Morimoto, 1993), and feed restriction (Zulkifli et al., 2000) may also elicit hsp response. To the best of our knowledge, the effects of road transportation and environmental enrichment on hsp 70 expression have not been studied in poultry.
In this study, the effects of 2 types of housing system and early age stress on blood parameters and hsp 70 reaction in broiler chickens subjected to road transportation during a sunny afternoon were determined. The 2 housing systems were a conventional open-sided house with cyclic ambient temperature (birds allowed to see the outside environment) and an environmentally controlled house (windowless).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 432 one-day-old female commercial broiler chicks (Cobb) were obtained from a local hatchery. On d 1, the birds were wing-banded. Two hundred sixteen chicks were placed at random in groups of 6 into 36 cages in 3-tiered batteries with wire floors in windowless environmentally controlled chambers (12 cages per chamber; 2.3 x 9.1 x 3.8 m; CH). Floor space allowed was 923 cm2 per bird. Ambient temperature on d 1 was set at 32°C and gradually reduced to 23°C by d 21. The RH ranged between 65 to 75%. The remaining 216 chicks were placed in similar battery cages and housed in conventional open-sided houses (12 cages per house; OH) with cyclic temperatures (minimum, 24°C; maximum, 34°C). The RH ranged between 80 to 90%. An equal number of chicks for each housing system was subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5, and 6 (FR). Food restriction was 60% of food consumption of the AL group on the previous day. All birds were fed standard broiler starter crumble (2,950 kcal of ME/kg; 21% CP) and finisher pellet (3,050 kcal; 19% CP) diets from d 1 to 21 and 22 to 42, respectively. Water was available at all times. The chicks were reared under continuous lighting. Chicks were vaccinated against Newcastle disease via intraocular route on d 7 and 21.
Road Transportation
On d 42, all of the birds from each housing system-feeding regimen subgroup were road-transported at 1200 h. The birds were removed from their cages and placed in plastic crates (0.80 x 0.60 x 0.31 m) at 10 birds to each crate. The crates were loaded to an open truck and transported for 6 h with an average speed of 80 km/h. The journey covered highways, roads with heavy traffic, and traffic lights. At the time of transportation, the ambient temperature was 34 to 36°C.
Blood and Brain Samples
Before (0 h) and after 2, 4, and 6 h of transit, 10 birds from each housing system-feeding regimen group were chosen at random and blood samples (3 mL) were obtained via the wing vein for plasma corticosterone concentration (CORT) assay, heterophil and lymphocyte counts (EDTA, anticoagulant), and serum levels of glucose, cholesterol, sodium, potassium, and chloride. Each bird was caught and sampled, one immediately after another. This procedure should not influence circulating levels of CORT (Lagadic et al., 1990). Blood samples for hormone assay were centrifuged and stored at –20°C until assayed. The CORT was measured by RIA using the ImmuChem Double Antibody125/RIA kit (MP Biomedical, 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). Blood samples for serum levels of glucose, cholesterol, sodium, potassium, and chloride determination were centrifuged, serum separated, and stored at –20°C. Analyses for serum glucose, cholesterol, sodium, potassium, and chloride were conducted with an automated clinical chemistry analyzer (Hitachi 902, Tokyo, Japan) and by using a standard diagnostic kit by Roche Diagnostic GmbH (Mannheim, Germany). Calibration and quality control had been done before this analysis. Immediately after blood collection, 5 birds (those that were used for blood sampling) from each housing system-feeding regimen subgroup were randomly chosen, 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 (Zulkifli et al., 2002).
SDS-PAGE and Immunoblot Analysis
Brain samples (0.5 g) were homogenized in an Ultra-Turrax homogenizer (IKA Works Inc., Wilmington, NC), 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 12% polyacrylamide gels containing SDS (Laemmli, 1970) using the Hoefer Mini Gel apparatus (Harvard Apparatus, Holliston, MA). 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 polyvinylidine 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 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (Sigma Chemical Co.). Relative density of the hsp 70 was determined using a densitometer (UVP, Cambridge, UK) with UVP Gel Base Pro program.
Statistical Analyses
Data were analyzed as a 2 x 2 x 4 factorial design using the GLM procedure of SAS software and multiple means were separated by Duncan’s multiple range test (SAS Institute, 1991). The statistical model included the effects of housing system (OH and CH), feeding regimen (AL and FR), transportation time (0, 2, 4, and 6 h), and their interactions. Data for CORT were normalized using square root transformation. When interactions between main effects were significant, comparisons were made within each experimental variable. Results were considered statistically significant at P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analyses of variance to examine the effects of housing system and duration of transportation on heterophil:lymphocyte ratio (HLR) data showed a significant interaction between the main effects (Table 1
). The interaction was observed because significant effect of housing system was only noted at 6 h of road transportation. After 6 h of transportation, the CH birds had a significantly greater HLR response than their OH counterparts. The HLR of both OH and CH birds increased significantly after 4 h of transportation. There was a significant housing system x feeding regimen interaction for HLR (Table 2). The interaction was observed because the significant effect of housing system was only noted for the AL. The CH birds exhibited a significantly greater increase in HLR than those of OH-AL group.
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Interaction of housing system x duration of transportation was significant for CORT (Table 3). The interaction resulted from a significant effect of housing system on CORT after 4 h of transportation only. The CORT of the CH birds were significantly higher than those of OH after 4 h of transportation. The CORT of both OH and CH birds increased significantly with duration of transportation. A significant feeding regimen x duration of transportation interaction for CORT was found (Table 4). The FR birds showed significantly lower CORT than their AL counterparts after 2 and 4 h of transportation.
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Comparisons between OH and CH revealed a significant housing system x duration of transportation interaction for hsp 70 densities (Table 5). The interaction was found because a significant effect of housing system was only noted at 4 h of transportation. The mean hsp 70 densities of the OH birds were significantly higher than those of CH after 4 h of transportation. The hsp 70 densities of both OH and CH birds increased significantly with duration of transportation. There were significant housing system x feeding regimen interactions for hsp 70 densities (Table 6). The interactions were observed because the significant effect of feeding regimen was only noted among the AL birds but not for FR. Among the AL birds, the OH chicks had significantly greater hsp 70 responses than those of CH.
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There was no significant duration of transportation x feeding regimen interactions for serum levels of cholesterol, glucose, potassium, and serum chloride (Table 7). The serum cholesterol, glucose, and chloride concentrations after 6 h of transportation were significantly higher compared with 0, 2, and 4 h of road transportation. Housing systems had no significant effect on serum levels of cholesterol, potassium, and chloride. However, serum levels of glucose were higher for OH birds than those of their CH counterparts. The effects of feeding regimen on serum levels of cholesterol, glucose, potassium, and chloride were not significant.
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). The interaction was observed because the significant effect of housing system was only noted at 2 h of transportation. The serum sodium levels of the OH birds were significantly higher than those of CH after 2 h of transportation. The serum sodium levels of both OH and CH birds increased significantly with duration of transportation. Feeding regimen had no significant effect on serum sodium concentration (AL, 156.31 ± 0.94; FR, 156.90 ± 0.75).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Habituation, a learning process, involves the waning of the response of an individual to a constant or repeated stimulus (Fraser and Broom, 1997). According to Broom and Johnson (1993), such a waning process could be attributed to a simple gating process, which involves reducing the efficacy of synoptic transmission in the nervous system. The greater HLR and CORT reactions with time suggest that the broilers failed to habituate to the stress after 6 h of transit. Similarly, Ehinger and Gschwindt (1981) reported that concentration of blood sugar, plasma proteins, and plasma cholesterol, indicators of acute stress of broiler chickens transported for 6 h, increased with transit time. The present findings provide additional support to the thesis that habituation occurs more readily to some types of stress than to others (Broom and Johnson, 1993). For example, Zulkifli (1999) reported that the effect of adapting to 60% (of ad libitum) feed restriction dissipated between 16 to 21 d; a similar finding was not noted for water restriction.
The present results, as measured by CORT and HLR, clearly showed that the OH birds were better able to cope with transport stress compared with their CH counterparts. There appears to be no obvious explanation for the greater magnitude of distress experienced by the CH birds after transportation and only a speculative one can be offered at this time. Unlike animals in the wild, animals in captivity tend to live in highly predictable and structured environments, where they are challenged infrequently or not at all. Earlier studies have shown that lack of environmental challenge may compromise animal welfare (Wemesfelder and Birke, 1997). Some environmental challenges are considered as biologically essential for maintaining normal biological functions (Zulkifli and Siegel, 1995). In the present study, we compared the effects of 2 different housing systems, varied in the level of environmental stimulation, on reaction to road transportation. During the rearing period, the OH birds had experienced a greater variety of visual and auditory stimuli than their CH counterparts. The visual and auditory stimuli can be considered as a form of environmental enrichment. Previous studies indicated that chickens exposed to outdoor environment (Grigor et al., 1995; Sanotra et al., 1998; Scott et al., 1998) or even video stimulation (Jones, 1996) were less fearful than those deprived of extra stimulation in the home environment. Fear is a potent stress elicitor in poultry (Jones, 1996). According to Wemesfelder and Birke (1997), environmental challenge may be considered as an integral part of behavioral development and well-being. Successful enrichment may increase the behavioral repertoire, reduce the occurrence of abnormal and undesirable behaviors, and enable animals to cope with challenges in a normal fashion (Chamove and Anderson, 1989).
High ambient temperature is considered a major factor in the elicitation of physiological stress reactions during road transportation (Mitchell and Kettlewell, 1998). Hence, the dampened physiological stress responses of OH birds to transportation during a sunny afternoon could also be associated with acquisition of a certain degree of acclimatization after the continuous exposure to the cyclic temperatures throughout the rearing period. Hutchinson and Sykes (1953) demonstrated the possibility of acclimatizing chickens to a hot, humid environment by 24 daily 4-h exposures to elevated temperature. The work of Zulkifli et al. (2008), however, does not support the notion because the OH birds also had smaller increases in CORT and HLR after night transportation, which was cooler (ambient temperature of 25°C). Thus, these findings suggest that the exposure of OH birds to extra visual and auditory stimuli during the rearing period is important in altering their physiological response to transport stress.
The present findings confirmed the results of Zulkifli et al. (2002) that FR can alleviate the stress of road transportation in broiler chickens. The FR birds in the present study had smaller increases in HLR and CORT than those of AL in response to the 6-h road transportation. Because during transportation the birds are exposed to an array of potential stressors including high ambient temperature, there is the question of response to which stressor was dampened by FR. There is considerable evidence that FR can reduce the detrimental effects of heat stress on chickens (Zulkifli et al., 1994, 2000; Liew et al., 2003). The present work and that of Zulkifli et al. (2002) involved transportation of birds under ambient temperatures above 30°C. Hence, it is not clear whether FR may aid the birds to cope with other stressors associated with road transportation.
It has been documented that stressors other than thermal stressors, for example, exposure to heavy metals, toxins, oxidants, bacterial and viral infections, and feed restriction (Morimoto, 1993; Zulkifli et al., 2002; Liew et al., 2003), may also elicit hsp response. Although road transportation induced hsp 70 expression in pigs (Yu et al., 2007), such reaction has not been previously reported in poultry. In the present study, the greater hsp 70 expression with transit time clearly suggests that the proteins are involved in the stress caused by transportation in chickens. The significant interaction between housing system and transit time for mean densities of hsp 70 suggests that rearing experience can influence induction of hsp 70.
It appears that the OH birds, which have experienced a greater variety of visual and auditory stimuli than their CH counterparts, were better able to express hsp 70 after transportation. These results showed that environmental stimulation during rearing may improve hsp 70 induction under stressful conditions and concur with studies in rodents that environmental enrichment may modify neurobiological functions (Renner and Rosenzweig, 1987). Heat shock proteins have been proven to play a key role in protecting stressed cells and organisms and preventing or reversing disorders caused by stress (Li and Werb, 1982; Barbe et al., 1988). The protein acts as a molecular chaperone by binding to other cellular proteins, assisting intracellular transport, and folding into the proper secondary structures and thus preventing aggregation of protein during stress (Chirico et al., 1988). The lower HLR and CORT showed by the OH birds in the present study suggests a negative relationship between hsp 70 expression and the magnitude of physiological stress reaction. Hence, there is a possibility that the increase in tissue hsp 70 concentrations may protect muscle damage after extended duration of transportation.
Our results concurred with those of Ehinger and Gschwindt (1981) that stress attributed to road transportation may increase serum levels of glucose and cholesterol in animals. Elevation in CORT after transportation may have elicited gluconeogenesis, in which amino acids are converted to glucose, and therefore blood glucose levels increased (Siegel and van Kampen, 1984; Malheiros et al., 2003). The increase in serum sodium and chloride after transportation could be attributed to thermal stress (Ait-Boulahsen et al., 1989). However, Arad et al. (1983) indicated heat stress had negligible effects on plasma concentrations of ions in normally hydrated chickens.
In conclusion, our results suggested that raising chickens in conventional open-sided houses with cyclic ambient temperatures improved hsp 70 expression and may lead to better ability to cope with the stresses associated with road transportation in hot, humid climates than those under environmentally controlled closed houses. Hence, extra careful attention must be paid to the catching, crating, and transportation procedures of broiler chickens raised a under closed house system to ensure better welfare. Early age feed restriction appears to be beneficial in alleviating transport stress in broiler chickens at market age.
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ACKNOWLEDGMENTS |
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Received for publication December 18, 2008. Accepted for publication March 5, 2009.
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