Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan

20/August/2019 Approved: 19/April/2020 ABSTRACT A study was conducted to determine the impact of crating density and transportation distance on losses and physiological response of broilers during the winter season. For this, ROSS-308 broilers were crated at three densities (i.e., 10, 12, and 15 birds/crate) in plastic crates and transported in three distances (i.e., 80, 160, and 240 km) during the winter season at 3.6 – 9.5ºC temperature and 63.3 – 78.8% relative humidity. Results showed that body weight loss increased significantly with the increase in transportation distance and decrease in crating density, whereas dead on arrival, physical injuries and bruises were not different among different transportation distance and crating density treatments. Significant reduction in carcass and breast yield was observed with the increase in transportation distance, whereas an increase in crating density above 12 birds per crate increased breast yield. Birds transported for 240 km had the highest serum catalase activity and the lowest rectal body temperature compared to other treatments. A gradual increase in crating density resulted in the increase in thyroxine concentration and reduction in rectal temperature. Serum glucose, albumin and uric acid concertation increased significantly after 160km of transportation, but serum triglyceride contents decreased. Moreover, an increase in crating density also increased serum glucose and triglyceride. Results suggested that longer transportation distance during the winter season increased body weight shrinkage with a reduction in carcass and breast yield, and the impact of losses and stress was greater in treatments having a lower number of


INTRODUCTION
The journey of broilers from farms to the processing plant, after a relatively comfortable environment of a poultry house, is associated with multi-dimensional stressors (Gregory, 2010). Genetic improvements have enabled broiler to gain maximum weight at a relatively shorter time, but it also increased the sensitivity of the birds particularly for thermal variations (Burkholder et al., 2008). The transportation of broilers has been reported to induce immediate stress in swine and poultry and the reaction of the animal to stress is dependent on the duration and intensity of the stress, their physiological status, and environment after stress (Brown et al., 1998;Fazio & Ferlazzo, 2003). In the case of transportation stress, the intensity of stress depends on environment during transportation, length of transportation journey and duration of the bird being inside the crates (Yalçin et al., 2004). In broiler, heat stress has always been a point of concern for the producers, but considerable data reveals that the cold stress is equally detrimental for the health and welfare of poultry (Aarif et al., 2013;Phuong et al., 2016). Transportation at low environmental temperature can cause hypothermia and can affect performance and welfare of broilers Hussnain F, Mahmud A, Mehmood S, Jaspal MH (Warriss et al., 2005;Qi et al., 2017). The cold stress has been reported to activate the hypothalamic-pituitarythyroid axis of the bird and increases corticosterone and cortisol concentration (Aarif et al., 2013;Slota et al., 2015).

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
The stress associated with transportation can cause dead-on-arrival (DOA) in broilers depending on transportation distance and ambient temperature (Elsayed, 2014). Contrarily, Vošmerová et al. (2010) reported more stress in short distance transported birds due to the lack of post crating recovery period. While Silva et al. (2011) did not find any effect of short distance transportation (up to 57 km) during the winter season on DOA. Not only mortality but an increase in the length of journey has been reported to increase body weight loss along with a significant increase in injuries, broken wings, and condemn carcass percentage and a decrease in the proportion of carcass cut up organs in broilers (Moran & Bilgili, 1995;Carlyle et al., 1997;Bianchi et al., 2005).
Broilers are usually transported to processing plants in loose crates and trucks without any environment control system. Welfare concerns arises with this system as the birds suddenly shifts from the nearly ideal environment of poultry farm to environmental extremes without proper protection (Weeks, 2014). The lack of controlled ventilation in open trucks hinders air flow and lead to building different temperature zones within a truck (Knezacek et al., 2010). Crating density can by hypothesized as the major factor determining the microclimate of the vehicle and its effect varies with the length of journey in different seasons (Whiting et al., 2007). Data regarding the effect of crating density on pre-slaughter losses is scarce. A few studies conducted regarding the effect of crating density has been associated with the changes in physiological response (Delezie et al., 2007;Vieira et al., 2013). So, crating density, in countries where winter temperature is low, need to be optimized. Since transportation stress cannot be eliminated in poultry production, therefore, the study was planned with the objective to assess preslaughter losses and physiological response in broilers at different transportation distances under different crating densities in the winter season.

Birds and their treatments
The experiment was conducted at the Department of Poultry Production, University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan. In this experiment, ROSS-308 broilers (straight run, body weight 1900 -2050g, age: 35 days) were picked at random from a commercial broiler farm after 4 hours of feed withdrawal before catching. The birds were separated into nine treatment groups (03 distances × 03 crating densities). Transportation distance was categorized as short (80 km), medium (160 km), and long (240 km) under three crating densities: Low = 10 birds (0.050 m 2 / bird), Medium = 12 birds (0.042 m 2 / bird), and high = 15 birds (0.033 m 2 / bird) per crate. To reduce the effect of the truck microenvironment, each treatment was replicated 10 times with individually tagged birds in each crate placed at various locations in the truck. The birds were transported in commercial trucks (capacity 144 crates) carrying different crating densities in loose plastic crates (Engi Plastic Industries Pvt. Ltd., Sundar, Pakistan) having the dimensions of 0.91 m (length) × 0.55 m (width) × 0.31 m (height) at an average speed of 45-50 km/h during late nightearly morning. The experimental birds were weighed and tagged on shanks before being placed in the crates. During growing phase, the birds were placed on litter floor (rice husk) in environmentally controlled commercial broiler farms (0.06 m 2 /bird) under standard temperature and relative humidity conditions. The birds were fed with ad-libitum diets (strain specific broiler ration) and fresh water and light was available for 24 h. All the procedures and bird handling protocols were used according to guidelines and standards after approval from the Ethical Review Committee of UVAS, Lahore (Approval No: DR/916-2017).

Climatic indices
The experiment was conducted during the winter season and the birds were transported in the vicinity of the districts of Kasur and Lahore, Punjab, Pakistan. The temperature and relative humidity were recorded with the help of a digital weather tracker (Kestrel 4500 NV, Nielsen-Kellerman, USA). The range of temperature and relative humidity measured at various distances and time ranged from 3.6 -9.5ºC and 63.3 -78.8%, respectively. During the journey, there were patches of mild to severe fog at several places. The journeys to be traveled were of 240 km, 160 km, and 80 km and started at 12:25 AM, 02:45 AM, and 04:30 AM, respectively, and all the trucks reached the slaughtering facility at almost 07:00 AM.

Transportation losses
Body weight loss (%) during the transportation was measured as the difference in bird weight at Hussnain F, Mahmud A, Mehmood S, Jaspal MH

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
the farm (g) and final body weight (g) measured at the ultimate destination. The dead-on-arrival (DOA) percentage for each group was calculated by dividing the number of birds found dead by the total of birds in the experimental crates. To calculate physical injuries (%), every single bird from each crate was observed and physical injuries (if any) were recorded and the proportion was calculated from total birds. Similarly, bruises on wings, breast, and legs (if any) were visibly observed and their proportion with the total number of birds slaughtered per replicate was calculated.
On arrival at the slaughtering facility, 3 tagged birds from each replicate were randomly picked and slaughtered manually with a knife. After bleeding, the birds were defeathered and eviscerated. Dressing (%) was calculated as hot carcass with neck and without giblets. The percentage of abdominal fat pad was calculated as weight abdominal fat relative to live body weight (g) while breast (%) was calculated relative to carcass weight.

Physiological Response
On arrival at the slaughtering facility, rectal body temperature was recorded randomly from each treatment with the help of digital thermometer (NB-100, Ossmax, Germany). The tagged birds were separated from the other birds weighed and slaughtered manually. To reduce the effect of stay before slaughtering, all the birds were divided into 6 slaughtering stations and all treatments were slaughtered at same time randomly. The blood was collected from the jugular vein of 10 birds from each treatment in plain vacutainers. Vacutainers were then centrifuged for 10 minutes at 3000 rpm and serum was carefully separated with a sterilized pipette into labeled Eppendorf tubes. Serum samples were stored at -20 ºC for later blood biochemical analysis. The samples were maintained at 3-8 ºC while handling.
The estimation of serum triiodothyronine (T3), and thyroxine (T4) were made using the enzyme immune assay test kit of BioCheck, Inc. (Catalog #: BC-1005 and BC-1007). Whilst, the Cortisol was measured with the help of ELISA Kit of Calbiotech Inc. (Catalog #CO103S). The catalase activity was estimated using the methodology of Hadwan & Abed (2016). Briefly, 4 test tubes (sample, control test, standard and blank) were prepared. The sample tube contained 100 µl serum and 1000 µl hydrogen peroxide (20mM), the control test tube contained 100 µl serum and 1000 µl distilled water, the standard tube contained 100 µl distilled water and 1000 µl hydrogen peroxide, and the blank contained 1100 µl distilled water. All these tubes were vortexed and incubated at 37 ºC for 3 min, after that 4000 µl ammonium molybdate (32.4mmol/l) was added to stop the reaction. Later changes in absorbance were recorded at 374 nm against the reagent blank.

Statistical analysis
The data collected were analyzed by two-way analysis of variance (ANOVA) technique under Completely Randomized Design assuming transportation distance and crating density as the main effect. The significant means were separated using Tukey's HSD test (Tukey, 1953), considering significance level at p ≤ 0.05, with the help of SAS 9.4 (SAS Institute, 2013).

RESULTS AND DISCUSSION
Transportation of animals is a necessary step in meat processing value chain. Along with other preslaughter handling factors, however, transportation can impact bird welfare and, in some cases, lead to economic losses (Vieira et al., 2011). There was a significant increase in BWL (%) with the increase in transportation distance ( Table 1). The average BWL (%) in broilers traveled for 80, 160, and 240 km was 1.99, 2.55, and 3.65%, respectively. Longer transportation times are associated with longer fasting periods in broilers (Savenije et al., 2002;Delezie et al., 2007) and higher exposure to stressful conditions especially in terms of thermal variations, crating, acceleration, vibration, noise, feed and water deprivation and social disruption (Kannan et al., 1997;Mitchell & Kettlewell, 1998;Fletcher, 2002). Similarly, Bianchi et al. (2005) investigated the effect of broiler transportation for ≤3.5 h, 3.5-5 h, >5 h in Bologna, Italy from September 2002 to May 2003 (321 flocks) and reported 0.24, 0.41, 0.45% BWL. The study reported an average BWL of 321 flocks, so, the BWL in that study was relatively lower than in the present study, moreover, higher BWL in the present study could be associated with the lower temperature than the previous study, poor road conditions, differences in the length of transportation and differences in the Hussnain F, Mahmud A, Mehmood S, Jaspal MH

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
crating densities in both studies. BWL (%) remained comparable (p>0.05) in birds transported at low and medium crating density, while a significant decrease in BWL (%) was observed in birds transported at high crating densities (15 birds/crate) compared to other treatments. Higher BWL (%) in birds crated at lower crating densities (i.e., 10 birds per crate) could be attributed to relatively higher struggle and enhanced metabolic activity to cope with lower environmental temperature (Dadgar et al., 2012). Other groups, however, have demonstrated that more birds in a crate generated enough metabolic heat that they survived better in Canadian winter temperatures of -5, -10, and -15 o C (Strawford et al., 2011).
No differences in dead on arrival (DOA) and bruising percentage (breast and wing bruises) were observed among different transportation distance and crating density treatments (Table 1). These findings suggested that the stress associated with winter transportation was bearable by the birds. The birds transported for 240 km or those transported at low crating densities (10 birds per crate) showed 0.94 and 1% DOA, respectively; but the differences were not significant (p>0.05) among different treatments (Table 1). However, these little margins can result in significant variations in terms of profit margin. In accordance, Silva et al. (2011) reported no effect of 57 km transportation on DOA (%) during the winter season in Londrina, Brazil, while Chauvin et al. (2011) also reported no effect of transportation on mortality rates in France at an average 13 ºC temperature. High mortality in birds transported at low crating density suggested that the DOA during the winter season was more associated with the chilling effect in birds, produced due to hypothermia, in trucks having lower number of birds per crate (Watts et al., 2011). Similarly, another study reported that mortality at very low ambient temperature (below -14 ºC) was higher if the birds were placed at low (<40 kg/m 2 ) crating density as compared to the birds at higher (40-45 kg/m 2 ) crating density in Canada (Caffrey et al., 2017). Fewer birds per crate translate to more space for birds, which could result in relatively higher chances of bruising and injuries during jerks and jolts of transportation (Elrom, 2000). The bruising (%) in the present study remained comparable to that of Jacobs et al. (2017), who reported 3.65 % bruised breast and wings and 1.38 % bruised leg in birds after transportation. However, some studies also reported bruises % ranges from 0.022 to 25% in broilers (Farsaie et al., 1983;Ekstrand, 1998).
Broilers transported for 80 km had the highest carcass and breast (%) while significant reduction was observed with each level increase in transportation distance (Table 1). Similarly, breast (%) was increased significantly with the increase in crating density but abdominal fat (%) was neither affected (p>0.05) by different transportation distance nor by different crating densities (Table 1). A higher dressed carcass (%) in birds transported for short distances and subsequent decrease with the increase in transportation distance could be due to a reduction in the share of viscera and other inedible portions for 80 km traveling (Table  1). Kim et al. (2007) reported a significant decrease in the share of small intestine after 3 hours of feed withdrawal. Similarly, in the 80 km treatments, to cope with the cold environment the metabolism gets faster and resulted in faster emptying of the intestine as compared to the rest of the treatments; thereby, increasing the relative share of the dressed carcass. While

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
the further reduction in carcass (%) with an increase in distance could possibly be attributed to shrinkage of the carcass as the birds were feed 4 hours before catching and up till slaughtering the total withdrawal time reached to 8 -11.5 hours. In accordance, Kim et al. (2007) also reported a significant decrease in carcass yield after 6 hours of the feed withdrawal period. Similarly, Mazanowski (1997) also reported a significant decrease in carcass weight when weight loss during transportation exceeds 3% and weight loss in the present study for broiler transported for 160 and 240 km were 2.55, and 3.65%, respectively. Although the percentage of different losses during transportation was less; these small values can combine to cause severe economic losses to the processor as well as severely affect animal welfare (Ali et al., 2008;Chauvin et al., 2011). Data regarding the effect of crating density on carcass characteristics is scarce and no study has been reported on this aspect.
Rectal body temperature was found to be higher in birds transported for 80 and 160 km while a significant decrease was observed when transportation distance increased up to 240 km ( Table 2). The finding could be associated with relative decrease in the rate of metabolism with the length of the journey and the increase in fasting period (Delezie et al., 2007). Broilers with slower metabolism were unable to regulate their body temperature, particularly those subjected to prolonged exposure (as in longer transportation) to a low ambient temperature of up to -18 o C (Watts et al., 2011). Similarly, Hunter et al. (1999) reported 2.1ºC and 10ºC drop in rectal temperature of the birds exposed to 12ºC and from 0 to 4ºC ambient temperature during transportation. Accordingly, Dadgar et al. (2012) reported a decrease in body temperature in birds exposed to low ambient temperature (i.e. -15, -12, -9, and +22 ºC). Results also showed that the crating of broilers at high crating density (15 birds per crate) resulted in higher rectal temperature compared to those transported at low and medium crating densities (Table 2). Similar findings were also previously reported by Delezie et al. (2007), but the study was conducted during the hot season and no such study was previously conducted during the winter season.
Results regarding physiological response showed that broiler transportation during the winter season did not affect (p>0.05) serum triiodothyronine (T3) and serum thyroxine (T4) contents (Table 2). Whereas, a significant (p<0.05) increase in serum catalase concentration was observed in the broilers transported for 240 km as compared to other treatments (i.e., 80 and 160 km). Similarly, serum T3 and catalase contents were not affected by different crating densities, but serum T4 concentration decreased significantly (p<0.05) with the increase in crating density (Table 2). Catalase is an antioxidant enzyme, and its activity is a measure to assess the oxidative stress on the living organism (Cadenas & Davies, 2000). Higher catalase activity in longer transported broilers could be due to increase in production of free radicals in birds subjected to longer stress periods (Ismail et al., 2013). No study has been conducted till date regarding serum catalase activity in response to transportation distance and crating densities. Similarly, Doktor & Połtowicz (2009) reported no effect of broiler transportation on T3 concentration compared to non-transported birds. Different crating densities had significant differences in T4 contents, while T3 contents and catalase activity Values are least square mean ± standard error T3: Triiodothyronine, T4: Thyroxine, temp: temperature Crating density: Low = 10 birds/crate (0.050 m 2 / bird), 12 birds/crate (0.042 m 2 / bird), and 15 birds/crate (0.033 m 2 / bird).

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
were not affected by different crating densities (Table  2). It is well established that thyroid hormones status correlates with body weight and energy expenditure (Fox et al., 2008). These hormones influence key metabolic pathways that control energy balance by regulating energy storage and expenditure (Cheng et al., 2010). The activation of the thyroid will convert T4 into T3 to regulate metabolism (Mullur et al., 2014). In accordance, the transportation of broiler at low crating density resulted in an increase in T4 concentration ( Table 2) to help sustain body temperature during winter transportation.
Significantly higher glucose concentration in birds transported for 240 km compared to rest of the treatments were observed (Table 2). Birds transported for 80 km had higher glucose concentration than those transported for 160 km (Table 2). Blood glucose has been considered as cellular fuel and studies have reported an elevation in plasma glucose concentration with the increase in transportation length or stress (Kent & Ewbank, 1986), primarily due to glycogen breakdown in the liver (Mayes, 1996). Similarly, Yalçin & Güler (2012) also reported a significant increase in blood glucose concentration with the increase in transportation distance (65 -165 km) in a comprehensive study of 27 flocks (eliminating the effect of seasons). In accordance, Scope et al. (2002) also reported a significant increase in blood glucose concentration in racing pigeons after 3 h transportation. An increase in the serum glucose contents was also observed with the increase in number of birds per crate ( Table 2). The increase in blood glucose levels with the increase in crating density could be due to reduced utilization of glucose at higher crating densities (Zhao et al. 2012). The birds kept at higher crating densities have relatively higher microclimate temperature due to the accumulation of metabolic heat, thereby they showed less struggle for maintaining temperature at low ambient temperature i.e., up to -18 o C (Watts et al., 2011). Similarly, some studies also reported a decrease in blood glucose at low ambient temperature (up to -14 ºC) (Dadgar et al., 2012) and during winter season (16-18 ºC) as compared to summer season (28-32 ºC) in Turkey (Yalçin et al., 2004).
No differences (p>0.05) in serum total protein contents could be observed in birds after different transportation distance and under different crating densities (Table 2). In accordance with the present study, Sarkar et al. (2013) also reported no effect of transportation distance on total proteins contents when compared with a non-transported treatment group at 24±0.5 ºC and 62% relative humidity. In a study on pheasants, Suchy et al. (2007) also reported non-significant difference in the total protein contents between low or high crating densities. While Voslarova et al. (2011) also reported no effect of different crating durations on total protein contents. Serum Albumin contents were higher (p<0.05) in birds transported for 240 km as compared to those transported for 80 and 160 km ( Table 2). The increase in albumin contents could be due to dehydration associated with longer journeys (Ghanem et al., 2008). Similarly, other studies (Yalçin et al., 2004;Yalçin & Güler, 2012) have also reported an increase in blood albumin contents after an increase in transportation distance under various temperatures in Turkey.
Serum uric acid contents were significantly higher in birds transported for 240 km compared to those transported for 80 and 160 km. No difference in uric acid contents were observed between 80 and 160 km transportation. Similarly, uric acid contents were not affected in different crating densities (Table 2). Uric acid has been reported as one of the major antioxidants in body; an increase in its concentration can be attributed with stress (Simoyi et al., 2002;Hartman et al., 2006). While Sturkie (2000) suggested that the circulating uric acid is a measure for protein catabolism in birds. As it was already discussed that the low-temperature transportation triggers gluconeogenesis, which converts amino acids to glucose and produces uric acid as a waste product, thus resulting in an increase in the uric acid concertation in longer transported (240 km) broilers (Siegel & Van Kampen, 1984;Malheiros et al., 2003). Reviews regarding the effect of winter transportation on uric acid concentration is scarce. Some studies reported an average increase in uric acid contents in different seasons and transportation under different crating densities was also reported in many studies previously (Yalçin et al., 2004;Delezie et al., 2007). Similarly, Elsayed (2014) also reported a significant increase in uric acid with transportation, but within transportation distances of 15 -150 km the differences remained non-significant. Similarly, no effect of crating and transportation of broiler were observed on uric acid contents (Sarkar et al., 2013).
Serum triglyceride (TGL) contents were found to be higher in birds transported for low and medium distances, whereas significant reduction in TGL levels was observed in longer (240 km) transported birds (Table  2). Moreover, the birds subjected to transportation with the low crating density had low levels of TGL as compared to those transported at higher stocking Hussnain F, Mahmud A, Mehmood S, Jaspal MH

Effect of Broiler Crating Density and Transportation Distance on Preslaughter Losses and Physiological Response During the Winter Season in Punjab, Pakistan
densities (Table 2). Probably, winter transportation triggers metabolic heat production to maintain body temperature and results in hypoglycemia and decrease in TGL contents with the increase in transportation distance (Strawford et al., 2011;Vošmerová et al., 2010;Elsayed, 2014). Moreover, the length of feed withdrawal due to an increase in the length of the journey also caused negative energy balance and stress in birds during transportation that triggers the glycogen breakdown (Nijdam et al., 2005). In accordance with the present study, Vošmerová et al. (2010) also reported a significant increase in TGL contents from 10 to 70 km but after that, the TGL contents decreased significantly. The differences in both studies could be attributed to the use of three temperatures in previous studies while in the present study the birds were transported at low temperatures only. Similarly, studies conducted in India (Sarkar et al., 2013) and Egypt (Elsayed, 2014) also reported a significant decrease in TGL contents in broiler subjected to transportation as compared to the control group. Moreover, Voslarova et al. (2011) also reported a significant decrease in triglyceride contents with the increase in crating time up to 2 h. However, the study was conducted at relatively higher temperatures than the present study. Most of the studies related to transportation distance and its effect on blood biochemical profile were conducted in countries with mild or hot temperature whilst the data regarding effect of different transportation distance and crating densities on blood biochemical profile during winter season is scarce.
The economic analysis showed transportation of 10 birds per crate for 240 km resulted in the maximum losses followed by those transported for 240 and 160 km with 12 and 10 birds per crate respectively (Table 3). Transportation for 80 km distance showed the least losses compare to those transported for 160 and 240 km. Regarding crating densities, placing 15 birds per crate for 160 and 240 transportation distance showed lower losses compared to placing 10 and 12 birds per crate for the same distance. However, in broiler transported for 80 km, keeping 10 birds per crate resulted in the least losses while losses increased with the increase in the number of birds per crate, but the difference between 12 and 15 birds per crate was marginal (Table 3). The table also showed that, excluding carcass (%), birds transported for 80 km with 15 birds per crate crating density showed the least losses during the winter season. These findings can be important from a middleman or transporter perspective, who only transport broilers from farm to processing plants in Pakistan.

CONCLUSION
The current study demonstrated that transportation of broiler is associated with economic losses due to live body weight shrinkage, mortality and bruising of the carcass. 80 km transportation with 15 birds per crate resulted in lower live losses, however, distance up to 160 km during the winter season also showed comparable results. Transportation of broilers also resulted in a disturbance in blood metabolites levels, but it did not result in irregularity of body physiological homeostasis till 160 km of transportation.