The effect of water shower spray on stress physiology and mortality in broiler chickens subjected to road transportation under the hot and humid tropical condition

doi:10.21203/rs.3.rs-1371287/v1

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The effect of water shower spray on stress physiology and mortality in broiler chickens subjected to road transportation under the hot and humid tropical condition

https://doi.org/10.21203/rs.3.rs-1371287/v1

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This study was performed to investigate the effect of water shower spray on rectal temperature (RT), serum levels of corticosterone (CORT), heat shock protein (HSP) 70 and creatine kinase (CK) in broiler chickens road transported at different times of the day. A total of 320 male broiler chickens (35 days of age) were crated and subjected to either water shower spray (WS) for 10 minutes or no water shower (control). The WS and control broilers were then loaded into an open truck and transported either from 0900 h to 1100 h (29.0^oC and 89.0% RH) (AM) or 1900 h to 2100 h (26.0^oC and 94.0% RH) (PM) with an average speed of 80 km/h. The CORT of AM birds were significantly reduced by WS, but the treatment had negligible influence on the PM group. Transportation during AM significantly elevated RT, CORT and HSP70 compared to the PM group. Water shower spraying of birds before transit significantly reduced RT and HSP70 compared to controls. Neither water shower treatment nor transportation time had a significant on CK and mortality rate of broilers. In conclusion, water shower spray is beneficial to alleviate the physiological stress response in road transported broilers under the hot and humid tropical environment.

broiler chickens

road transportation

water shower spray

physiological stress response

mortality

tropical environment

It is well documented that broiler chickens may be exposed to an array of adverse physical and psychological stimuli during road transportation, including extreme weather conditions, overcrowding, feed and water deprivation, vehicle vibration, noise, motion, and social discomfort, which may result in stress, fear, injuries, or mortality (Broom, 2009). In reviewing the welfare of broilers during transit, Mitchell and Kettlewell (2004) suggested that thermal challenge, in particular heat stress, is the major threat to poultry well-being. The consequences of transporting broilers under conditions of heat stress may include higher mortality rate, decreased meat quality and reduced welfare status (Lara and Rostagno 2013). These problems are speculated to increase in the coming years with global temperature rise.

In order to maintain a comfortable environment for birds in transit, it is a common practice in the commercial boiler operation in Malaysia to shower chickens with water spray just before road transportation. Earlier work showed that water shower spraying of heat-stressed broilers during pre-slaughter handling improved meat quality and reduced pale soft exudative syndrome (Xing et al. 2016). There is, however, a dearth of information on the effect of water shower spray on the physiological stress response in transported broilers under hot and humid tropical conditions. Thus, the study aimed to determine the effect of road transportation with water shower spray on mortality rate, serum levels of corticosterone (CORT), heat shock protein (HSP) 70, creatine kinase (CK), and rectal temperature (RT) in commercial broiler chickens.

Animal, housing and husbandry

A total of 320 one-day-old male broiler chicks (Cobb 500) were obtained from a local hatchery and were randomly assigned in groups of 20 to 16 floor pens with wood shavings deep litter, and cyclic temperature (minimum, 24°C; maximum, 35°C). The RH was between 80 and 90%. The birds were fed a 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 35, respectively. Feed and water were provided ad libitum. Chicks were vaccinated against Newcastle disease via drinking water on d 7 and 21. Lighting provided was 24 h and 16 h during days 1–2 and days 3–35, respectively. A 2 × 2 factorial experiment was conducted with two water shower treatments (water shower spray or no water shower spray) and transportation at two different times of the day (0900 h to 100 h or 1900 h to 2100 h). On day 35, the birds were feed deprived for 8 hours prior to road transportation, and drinking water was always available. Following feed deprivation, the birds were removed from their pens and placed in plastic crates (0.80 × 0.60 × 0.31 m) at 10 birds to each crate (the total number of crates was 32). The chickens were crated according to their home pens. Following crating, an equal number of birds were subjected to either water shower spray for 10 minutes (WS) (16 crates of birds) or no water shower spray (control) (16 crates of birds). The birds from each water shower treatment group were then loaded into an open truck and transported either from 0900 h to 1100 h (AM) (eight crates of WS birds and eight crates of control birds) or 1900 h to 2100 h (PM) (eight crates of WS birds and eight crates of control birds) with an average speed of 80 km/h. The journey covered highways, roads with heavy traffic, and traffic lights. The recorded average outside temperatures and RH% during the AM and PM transportations were 29.0^oC and 89.0%, and 26.0^oC and 94.0%, respectively.

Blood samples and rectal temperature

Following transit, the crates were immediately unloaded from the truck, and the number of dead birds was recorded. Four birds from each crate were randomly selected for blood sampling and rectal temperature recording. Different birds were used for each procedure. Blood samples (5 ml) were obtained via the wing vein to determine CORT, HSP70 and creatine kinase. Each bird was caught and sampled, one immediately after another. The catching and bleeding procedure did not exceed 1 min and should not influence CORT (Lagadic et al. 1990; Romero and Reed 2005). Blood samples for hormone assay were centrifuged and stored at − 20°C until assayed. Immediately following blood sampling, rectal body temperature was measured by a digital thermometer (± 0.1°C; Huger Electronics GmbH, Baden-Württemberg, Germany) with an insertion probe and recorded when the reading was stable for 15 s.

Corticosterone assay

According to the manufacturer recommendations, CORT was determined using commercially available high sensitivity EIA kits (AC-15F1, IDS, Boldon, UK). Cross-reactivity of the CORT antiserum was less than 6.7% and 7.8%, respectively, and the detection limit was 27 ng/mL.

Heat shock protein 70 assay

The HSP70 was determined using a commercial ELISA kit specific to chicken (Shanghai Sunred Biological Technology, Shanghai, China) according to manufacturer recommendations. All samples were run in the same assay to prevent inter-assay variability.

Creatine kinase assay

Analysis for CK was conducted on an automated spectrophotometer (Ultraspec 300, Cobas-Mira, Roche Diagnostic System, Basel, Switzerland) using a standard diagnostic kit.

Statistical Analysis

The experiment was conducted as a completely randomised design with four treatments arranged factorially and main effects (water shower treatment and transportation time), and their interactions were analysed by ANOVA using the GLM procedure of SAS Institute (2003). When interactions between main effects were significant, comparisons were made within each experimental variable. Statistical significance was assumed at P ≤ 0.05.

There were no shower treatment x transportation time interactions for all the parameters measured except for CORT (Table 1). The WS significantly reduced the CORT of AM birds but had negligible influence on the PM group (Table 2). Irrespective of water shower treatment, the AM broilers had significantly higher CORT than the PM group. Rectal temperature and HSP70 were significantly affected by water shower treatment and transportation time. The AM treatment significantly elevated RT and HSP70 of broilers compared to their PM counterparts. Water showering of birds before transit significantly reduced RT and HSP70 compared to controls. Neither shower treatment nor transportation had a significant on CK. No mortality was recorded in the study.

Table 1

Effect of water shower treatment and transportation time on rectal temperature (RT), and serum levels of corticosterone (CORT), heat shock protein (HSP)70 and creatine kinase (CK).
Group	Variable
	RT (̊C)	CORT	HSP70	CK
	RT (̊C)	(ng/mL)	(pg/mL)	(U/L)
Water shower treatment (WST)
Non-showered	41.17 ± 0.08	4.16 + 0.53	824 ± 135	16462 ± 1155
Showered	40.83 ± 0.09	2.83 + 0.22	603.9 ± 95	19430 ± 1066
Transportation time (TT)
AM	41.18 ± 0.09	4.56 + 0.40	1142 ± 120	18277 ± 1219
PM	40.67 ± 0.09	2.10 + 0.18	405 ± 40	16956 ± 1126
Source of Variation
WST	0.0043	0.0015	0.017	0.0785
TT	0.0037	< 0.0001	< .0001	0.2910
WST x TT	0.8720	0.0176	0.1443	0.7703
AM: transportation from 0900 h to 1100 h; PM: transportation from 1900 h to 2100 h

Table 2

Mean serum corticosterone concentrations (ng/mL) where the water shower treatment and transportation time interactions were significant
Transportation time	Water shower treatment
Transportation time	Showered	Non-showered	P-value
AM	3.50 ± 0.27	5.88 ± 0.67	0.0016
PM	1.92 ± 0.15	2.29 ± 0.33	0.3261
P-value	< 0.0001	< .0001

AM: transportation from 0900 h to 1100 h; PM: transportation from 1900 h to 2100 h

In tropical countries like Malaysia, heat stress during transport is a primary factor that may compromise farm animals' health, well-being, and product quality. In order to evaluate the well-being of the animals during transit, it is necessary to make measurements in a quantifiable and repeatable manner (Knowles and Warriss 2000). Physiological variables have been commonly used to determine the stress experienced by animals during transit. In the present study, we measured RT, CORT, HSP70 and CK, to assess the effect of water showers on broilers transported at different times of the day.

Before transit, the recorded average ambient temperatures were outside the thermal comfort for broiler chickens, which were 11°C-25.1°C (Luthra, 2017). As expected, the AM transport of broilers resulted in higher RT than those of PM. Thus, it is common in Malaysia to transport broilers during the cooler part of the day. However, for long-distance transportation, the transit may be extended until the warmer part of the day. This may result in physiological stress and higher dead-on-arrival incidence. The present findings showed that water shower spray effectively reduced the RT of broilers during transit. The benefits of water shower spray are primarily due to the birds' surface wetting and subsequent evaporation by body heat, thus increasing the heat loss (Wolfenson et al. 2001). Mutaf et al. (2009) reported that sprinkling water intermittently onto the head and appendages of caged laying hens significantly reduced their body core temperatures. According to Webb and King (1984), the thermal resistance of plumage of chickens was significantly reduced when feathers were wet.

Stress responses are elicited with activation of the hypothalamic-pituitary-adrenal axis, increased secretion of adrenocorticotropic hormone and corticosterone release by the adrenal gland (Scanes, 2016). Hence, circulating corticosterone concentration has been widely used as a physiological indicator of stress associated with road transportation (Al-Aqil and Zulkifli 2009; Al-Aqil et al. 2013; Zhang et al. 2014; Bello et al. 2018) in poultry. Stress attributed to transport is multifactorial, including several potentially traumatic events (handling, crating, loading, and the transport itself). We noted a significant water shower treatment x time of transportation interaction for CORT. Irrespective of water shower treatment, transporting birds during AM, as measured by CORT, was more stressful than the PM group. The elevated CORT in AM birds could be associated with the higher RT compared to their PM counterparts. It is interesting to note that WS had a negligible influence on CORT in the PM birds. Hence, it appears that water showers are not necessary for birds transported at PM. On the contrary, the CORT results suggested that the practice was beneficial to dampen physiological stress in broilers during AM transit.

Heat shock proteins are molecular chaperones with multiple physiological roles. The expression of heat shock proteins in response to stress protects against the initial insult, augment recovery, and produce a state of resistance to subsequent stresses (Kregel, 2002). The HSP will bind with heat-sensitive proteins in a heat-shocked cell and protect them from degradation or prevent damaged proteins from immediately precipitating and permanently affecting cells viability (Etches et al. 2008). HSPs are rapidly synthesised when poultry are exposed to thermal (Zulkifli et al. 2002) and nonthermal (Zulkifli et al. 2009) stressors. Our results confirmed earlier reports (Yu et al. 2007; Al-Aqil and Zulkifli 2009; Aqil et al. 2013; Li et al. 2021) that road transportation may elicit HSP reaction in chickens. The noted significantly higher HSP70 in the AM broilers than those of PM suggested that the former were more physiologically distressed. The present results showed that the water showers reduced RT and concomitantly dampened the HSP70 response in transported birds. The synthesis of heat HSP70 is temperature-dependent, and its response is considered a cellular thermometer (Somero, 2020).

Increased creatine kinase activity indicated skeletal muscle damage due to disruptions in muscle cell membrane function and permeability (Mitchell and Sandercock 1995). Earlier studies demonstrated an increase in CK following road transportation in broilers (Mitchell and Kettlewell 1998b; Al-Aqil and Zulkifli 2009; Zhang et al. 2009). In the present study, both WS and transport time had a negligible effect on CK. There is a possibility that the duration of transport in the present study was not long enough to alter CK. Previous work (Mitchell and Kettlewell 1998b; Al-Aqil and Zulkifli 2009; Zhang et al. 2009) on transportation and CK in broilers involved more than 3 hours of transit.

Mitchell and Kettlewell (1994) concluded that the consequences of the various potential stressors associated with road transportation in poultry may range from mild discomfort, distress and aversion to death. In the present work, as measured by CORT and HSP70, the broilers were physiologically stressed by road transportation, but there was no recorded transport-induced mortality. Warriss et al. (2005) showed a significant positive relationship between ambient temperature and mortality of transported broilers in the United Kingdom. The authors indicated progressively higher broiler mortality rates in transit when the maximum daily temperature rose above 17^oC. In this study, the highest recorded ambient temperature during transit was 30^oC, with a 79% RH. On a cautionary note, however, transport-induced mortality may also be attributed to various factors such as transit duration, crating density, live weight of birds, housing system under which the birds were raised, catching method, the health status of birds, and ventilation within the vehicle (Chauvin et al. 2011; Caffrey et al. 2017; Jacobs et al. 2017).

Under the conditions of the present study, road transportation of broilers, from 0900 h to 1100 h (at an average speed of 80km/h), increased RT, CORT, and HSP70 compared to those transported from 1900 h to 2100 h. The present findings could potentially be used to inform management decisions to reduce stress in transit broilers. Taken together, our results imply that spraying broilers with water before transit is beneficial to alleviate the stress attributed to road transportation under the hot and humid tropical environment. Hence, the water shower spray practice is beneficial for animal welfare, product quality and economic reasons. Furthermore, by reducing stress, such practice may reduce the susceptibility of broilers to the colonisation of foodborne pathogens. Clinically healthy birds carrying Salmonella and other pathogens may increase their shedding of the organisms if environmental stressors upset the equilibrium of their intestinal flora (Iannetti et al. 2020). Further studies are needed to assess the effects of water shower spray treatment and transportation time on mortality and physiological stress in transported broilers under the commercial setting.

Statements and Declarations

Author contribution: ZZA, NFAS, SKR, and ZI conceived and designed research. ZZA and NFAS conducted experiments. ZZA, NFAS, and EAA performed the statistical analysis and analyzed data. ZZA and NFAS prepared the manuscript. All authors reviewed manuscript upon submission.

Funding This work was supported by Malaysia Research University Network.
Competing interests The authors declare no competing interests.
Data availability Data are available from the corresponding author on request.
Ethics approval All experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Research Policy at Universiti Putra Malaysia (UPM) (Approval number: UPM/IACUC/AUP-R056/2020).
Consent to participate Not applicable. 
Consent for publication Not applicable. 

Al-Aqil, A., and Zulkifli, I. 2009. Changes in heat shock protein 70 expression and blood characteristics in transported broiler chickens as affected by housing and early age feed restriction. Poultry Science, 88, 1358–1364.
Al-Aqil, A., Zulkifli, I., Hair Bejo, M., Sazili, A. Q., Rajion, M. A. and Somchit, M. N. 2013. Changes in heat shock protein 70, blood parameters, and fear-related behavior in broiler chickens as affected by pleasant and unpleasant human contact. Poultry Science, 92, 33–40.
Bello, U. A., Zulkifli, I., Yong Meng, G., Awad, E. A. and Soleimani Farjam, A. 2018. Gut microbiota and transportation stress response affected by tryptophan supplementation in broiler chickens. Italian Journal of Animal Science, 17, 107–113.
Broom, D. M. 2009. Welfare assessment and welfare problem areas during handling and transport. Livestock Handling and Transport 2nd Edition, CABI Publishing, Wallingford.
Caffrey, N. P., Dohoo, I. R. and Cockram, M. S. 2017. Factors affecting mortality risk during transportation of broiler chickens for slaughter in Atlantic Canada. Preventive Veterinary Medicine, 147,199–208.
Chauvin, C., Hillion, S., Balaine, L., Michel, V., Peraste, J., Petetin, I. and Le Bouquin, S. 2011. Factors associated with mortality of broilers during transport to slaughterhouse. Animal, 5, 287–293.
Etches, R. J., John, T. M., and Verrinder, G. A. M. 2008. Behavioural, physiological, neuroendocrine and molecular responses to heat stress. Poultry Production in Hot Climates: Second Edition, 48–79. Iannetti, L., Neri, D., Santarelli, G. A., Cotturone, G., Vulpiani, M. P., Salini, R. and Messori, S. 2020. Animal welfare and microbiological safety of poultry meat: Impact of different at-farm animal welfare levels on at-slaughterhouse Campylobacter and Salmonella contamination. Food Control, 109, 106921. Jacobs, L., Delezie, E., Duchateau, L., Goethals, K. and Tuyttens, F. A. 2017. Broiler chickens dead on arrival: associated risk factors and welfare indicators. Poultry Science, 96, 259–265.
Knowles, T. G. and Warris, P. D. 2000, Stress Physiology of Animals During Transport, Livestock Handling And Transport. CABI Publishing, Wallingford.
Kregel, K. C. 2002. Invited review: Heat shock proteins: Modifying factors in physiological stress responses and acquired thermotolerance. Journal of Applied Physiology, 92, 2177–2186.
Lagadic, H., Faure, J. M., Mills, A. D. and Williams, J. B. 1990. Effects of blood sampling on plasma concentrations of corticosterone and glucose in laying hens caged in groups. British Poultry Science, 31, 823–829.
Lara, L. J. and Rostagno, M. H. 2013. Impact of heat stress on poultry production. Animals, 3, 356–369.
Li, C., Zhang, R., Wei, H., Wang, Y., Chen, Y., Zhang, H. and Bao, J. 2021. Enriched environment housing improved the laying hen's resistance to transport stress via modulating the heat shock protective response and inflammation. Poultry Science, 100, 100–139.
Luthra, K. 2017. Evaluating Thermal Comfort of Broiler Chickens during Transportation Using Heat Index and Simulated Electronic Chickens. ProQuest Dissertations Publishing, Michigan.
Mitchell, M. A. and Kettlewell, P. J. 2004. Transport and handling. Measuring and auditing broiler welfare, CABI Publishing, Wallingford.
Mitchell, M. A. and Kettlewell, P. J. 1998b. Physiological Stress and Welfare of Broiler Chickens in Transit: Solutions Not Problems! Poultry Science, 77, 1803–1814.
Mitchell, M. A. and Sandercock, D. A. 1995. Creatine kinase isoenzyme profiles in the plasma of the domestic fowl (Gallus domesticus): effects of acute heat stress. Research in Veterinary Science, 59,30–34.
Mitchell, M. A. and Kettlewell, P. J. 1994. Road transportation of broiler chickens: induction of physiological stress. World's Poultry Science Journal, 50, 57–59.
Mutaf, S., Seber, K. N. and Firat, M. Z. 2009 Intermittent partial surface wetting and its effect on body-surface temperatures and egg production of white and brown domestic laying hens in Antalya (Turkey). British Poultry Science, 50, 33–38.
Romero, L. M. and Reed, J. M. 2005. Collecting baseline corticosterone samples in the field: Is under 3 min good enough? Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 140, 73–79.
Scanes, C. G. 2016. Biology of stress in poultry with emphasis on glucocorticoids and the heterophil to lymphocyte ratio. Poultry Science, 95, 2208–2215.
Somero, G. N. 2020. The cellular stress response and temperature: Function, regulation, and evolution. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 333, 379–397.
Warriss, P. D., Pagazaurtundua, A. and Brown, S. N. 2005. Relationship between maximum daily temperature and mortality of broiler chickens during transport and lairage. British Poultry Science, 46,647–651.
Webb, D. R. and King, J. R. 1984. Effects of wetting on insulation of bird and mammal coats. Journal of Thermal Biology, 9, 189–191.
Wolfenson, D., Bachrach, D., Maman, M., Graber, Y. and Rozenboim, L. 2001, Evaporative Cooling of Ventral Regions of the Skin in Heat-Stressed Laying Hens. Poultry Science 80, 958–964.
Xing, T., Li, Y. H., Li, M., Jiang, N. N., Xu, X. L. and Zhou, G. H. 2016. Influence of transport conditions and pre-slaughter water shower spray during summer on protein characteristics and water distribution of broiler breast meat. Animal Science Journal, 87, 1413–1420.
Yu, H., Bao, E. D., Zhao, R. Q. and Lv, Q. X. 2007. Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs. American Journal of Veterinary Research, 68, 1145–1150.
Zhang, L., Li J. L., Gao, T., Lin, M., Wang, X. F., Zhu, X. D. and Zhou, G. H. 2014. Effects of dietary supplementation with creatine monohydrate during the finishing period on growth performance, carcass traits, meat quality and muscle glycolytic potential of broilers subjected to transport stress. Animal, 8, 1955–1962.
Zhang, L., Yue H. Y., Zhang, H. J., Xu, L., Wu, S. G., Yan, H. J. and Qi, G. H. 2009. Transport stress in broilers: I. Blood metabolism, glycolytic potential, and meat quality. Poultry Science, 88, 2033–2041.
Zulkifli, I., Liew, P. K., Israf, D. A., Omar, A. R. and Hair-Bejo, M. 2002. 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. Journal of Thermal Biology, 28, 217–222.
Zulkifli, I., Al-Aqil, A., Omar, A. R., Sazili, A. Q. and Rajion, M. A., 2009. Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions. Poultry Science, 88, 471–476.

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You are reading this latest preprint version

The effect of water shower spray on stress physiology and mortality in broiler chickens subjected to road transportation under the hot and humid tropical condition

Status:

Version 1

Abstract

Introduction

Materials And Methods

Animal, housing and husbandry

Blood samples and rectal temperature

Corticosterone assay

Heat shock protein 70 assay

Creatine kinase assay

Statistical Analysis

Results

Discussion

Statements and Declarations

Declarations

References

Status:

Version 1