Transportation welfare under commercial conditions: A multifactorial assessment of stress physiology, meat quality, and microbial load in broiler chickens

Abstract

Chickens are often transported for a long distance under overcrowded conditions for human consumption, which affects their welfare, health, and even meat quality. The objective of this study was to investigate the interaction effects of transport distance (60 vs. 80 km), stocking density (0.65, 0.86, and 1.30 ft²/bird), and lairage duration (0 vs. 3 hours) on physiological, haemato-biochemical, microbial, carcass, and meat quality characteristics. A total of 324 broilers was used in a 2 × 3 × 2 factorial arrangement, with truck as the experimental unit, and linear mixed models were employed for data analysis. Higher transportation distance and more space allowance were responsible for more body weight loss during transportation (p < 0.05). Significant distance × density interactions were found for white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), packed cell volume (PCV), corticosterone, and aspartate aminotransferase (AST) (p < 0.05). Birds transported over 80 km had higher WBC, heterophil to lymphocyte ratio (H: L), lactate dehydrogenase (LDH), and glucose compared to those birds transported over 60 km (p < 0.05). The interactions between distance and lairage on triglyceride and AST indicated partial physiological recovery after 3-hour lairage (p < 0.05). The density-lairage interactions for corticosterone, triglyceride, and RBC were significant (p < 0.05). Prolonged transportation distance increased pathogenic bacterial load (log₁₀ CFU/g) and had a negative effect on meat quality characteristics, including cooking loss of thigh muscle and early postmortem pH of breast muscle (p < 0.05). Overall, extended transportation increased physiological stress and microbes, whereas increased space and lairage reduced many of the adverse effects. The study demonstrated the need to improve transportation techniques to enhance broiler welfare and meat quality.

Introduction

Broilers are typically farmed in scattered locations and then transported to centralized slaughter and processing facilities, covering different distances (dos Santos et al., 2020). On-farm slaughter and direct carcass transport have the advantage of providing an alternative to live animal transport, which might ease welfare problems. However, these methods are still in an experimental phase and need to be fully validated and invested in to be used on a large scale (Samoylov et al., 2023). The traditional transportation of poultry is a critical process at which animal welfare, product quality, economic efficiency, and environmental sustainability converge (Siddharth et al., 2024; Vaarst et al., 2015). It is therefore important to optimize the design and logistics of the transportation process and regional production systems to develop a poultry system that is both humane and sustainable.

During the process of loading, road transportation, and subsequent unloading, broilers are subjected to a complex combination of neuroendocrine, metabolic, and thermal stressors. Such stressors have a direct influence on the welfare, mortality rate, and quality of broiler meat (Vošmerová et al., 2010; Zheng et al., 2020a). Pre-slaughter stress accelerates muscle glycogen depletion and AMPK-mediated glycolysis, resulting in a rapid decline in meat pH, protein denaturation, and reduced water-holding capacity (WHC). Therefore, physicochemical properties of meat are useful indicators of transport-associate stress and meat quality deterioration in broilers (Liu et al., 2025); (Wang et al., 2017). Transportation stress influences gut health because it compromises the gut barrier, causes an imbalance of microbes in the gut, and allows for the establishment of opportunistic pathogens. This can result in the shedding of bacteria in the gut, which has food safety and bird health implications. It is important to consider both the beneficial and pathogenic bacteria to have a complete picture of the impact of transport stress (Patra and Kar, 2021; Obianwuna et al., 2023).

Among the factors affecting the intensity of stress, the transportation distance or duration is recognized as a surrogate for the cumulative exposure to stress. Many physiological and performance parameters deteriorate progressively as the distance increases (dos Santos et al., 2020). Similarly, the stocking density during transportation is a significant factor affecting the birds’ thermal load, as at high stocking densities, the birds act as a “thermal mass,” generating more metabolic heat per unit surface area, which reduces the rate of heat dissipation and increases rectal temperature and panting during heat stress (Son et al., 2022). Lairage, the period before processing, allows the birds to have a brief period of rest, which helps to restore homeostasis, control the level of acute stress, and promote muscle-to-meat conversion (Warriss et al., 1999). However, the success of the lairage period largely depends on the transport conditions.

The transport distance, stocking density, and lairage duration all affect the microclimate of the crate or holding barn. This microclimate then influences the poultry's mortality rates, weight loss, physiological stress, and meat defects (dos Santos et al., 2020). The majority of research done so far has been based on single-factor experiments and laboratory settings. This research gap limited the applicability to real-world poultry production systems.

These issues have been addressed in this study by arranging a 2 × 3 × 2 factorial study to examine the main and interaction effects of transport distance, stocking density, and lairage duration on physiological and haemato-biochemical stress indicators, cecal microbial load, and meat quality across different farms. The study was designed considering the poultry transportation condition in Bangladesh. By integrating welfare, metabolic, microbial, and meat quality data, a comprehensive picture of the impact of pre-slaughter handling will be developed. We propose the hypothesis that increased transport distance, stocking density, and no lairage time will have additive and synergistic effects in increasing stress, altering the gut microbiome, and deteriorating meat quality.

These findings of the study will be used to develop transport management strategies based on scientific evidence, providing critical limits to ensure the welfare of the birds, minimize losses, and maintain meat quality in commercial poultry operations.

Materials and methods

Ethical approval

All procedures involving live birds were reviewed and approved by the Ethical Standard of Research Committee of the Sylhet Agricultural University Research System. Approval No (ARP2026062)

Experiment design and animal management

A total of 324 commercial broilers (Cobb 500) at the age of 35-36 days, were used in the study, and the experiment was conducted using a 2 × 3 × 2 factorial arrangement within a hierarchical experimental structure, consisting of two transport distances (60 and 80 km), three transport stocking densities (0.65, 0.86, and 1.30 ft² per bird), and two lairage durations (0 and 3 h). Here, all stocking densities followed the minimum space allowance requirement per bird considering the regulation of (Commission., 2004), and lairage duration means holding time in an area where poultry was rested prior to slaughter.

For each distance, six independent trucks were used. The trucks used for 60 km and 80 km distances were loaded and transported separately. When the birds arrived at the processing plant, the birds from three trucks of each distance were subjected to haemato-biochemical and meat-related tests immediately after arrival (0 h lairage), and the birds from the other three trucks of each distance were subjected to the tests after 3 h of lairage.

Each truck was equipped with nine transport crates. For each truck, three crates were allocated to each of the stocking density treatments (0.65, 0.86, and 1.30 ft²/bird), resulting in 27 birds per truck. The hierarchy of observations was bird-nested within crate, crate-nested within truck, and truck-nested within distance. The truck was the experimental unit for the distance and lairage treatments, the crate was the experimental unit for stocking density, and the birds were subsamples within crates. Furthermore, thirty birds were selected from one of the farms for the non-transported control group treatment. As the present study followed a multifactorial experimental design, the complete treatment combinations (density and lairage) were not applicable under the 0 km non-transported condition. It was only used to obtain baseline information for similar rearing conditions (Supplementary Table S1). All the birds were randomly selected from commercial broiler sheds with uniform management practices.

Transportation and lairage protocol

All birds experienced a 6-hour feed withdrawal before loading, as it reduces intestinal content and carcass contamination and keeps the meat quality acceptable (Xue et al., 2020). The transportation trial was conducted during the summer season in the afternoon under commercial conditions. Transportation of the broilers was done in a standardized, mechanically ventilated poultry transport vehicle, where the average speed maintained throughout the transport was 30 km/h, consistent with previous summer transportation (Yu, Chathuranga, et al., 2024). The internal ambient conditions in the transport vehicle were continuously monitored throughout all transportation using a calibrated data logger, where the mean temperature and relative humidity levels were 85.1°F (29.5°C) and 66%, respectively. Broilers were transported under naturally ventilated condition using ventilated plastic transport crates arranged in stacked rows within open-sided truck compartment. The average anemometer reading of crate-level air speed was 0.6 m/s during transportation. Prior to loading, birds were allocated into assigned crates according to stocking density. The lairage area was maintained in a clean and ventilated condition.

Animal sampling

Physiological outcomes were measured at the bird level and then aggregated at the crate level. Mortality % was recorded at the crate level. Individual live body weight and body temperature were measured before and following transportation, from where body weight loss and temperature change were measured. The values were then aggregated at the crate level to examine the distance × density effect. For the hematological and biochemical parameters, carcass characteristics, and meat quality characteristics, the random selection of one bird from each crate resulted in the balanced dataset of 108 observations per trait to accommodate the results of the 2 × 3 × 2 factorial arrangement. The evaluation of the gut health parameters was done using the same distance × density dataset, maintaining the same crate and truck identification.

Haemato-biochemical stress indicator

Upon arrival, two aliquots of blood were collected for haemato-biochemical analysis from the wing vein. Blood samples for hematology were collected in blood tubes that contain an anticoagulant (EDTA), whereas blood was collected in a blood tube without an anticoagulant for biochemical analysis. Blood parameters were measured for the following metrics: total leukocyte count (WBC), with heterophils and lymphocytes, hemoglobin, red blood cells, and packed cell volume. A Complete Blood Count (CBC) was performed using a CBC analyzer (BIOELAB EC 38, E-Lab Biological Science & Technology Co., Ltd., Nanjing, China). For the biochemical analysis, corticosterone, plasma glucose, AST, triglyceride, and LDH were estimated. For detecting the corticosterone level, the competitive inhibition ELISA kit (EK248056) was used. Plasma glucose was analyzed using an automated biochemistry analyzer (BIOELAB AS-280, Germany) and reagent (Cromatest Glucose MR). AST and triglyceride level detection were conducted using E- Lab EA 200 (Nanjing, China) and the reagents are Biolin GmbH (Germany).

Relative carcass traits and meat quality assessment

Birds were humanely slaughtered by cutting the jugular vein. Carcass weight, edible carcass weight, whole intestine weight, and other carcass characteristics such as breast muscle weight and thigh muscle weight were compared with the live weight of the broiler and expressed as a percentage. The pH values of breast meat were measured at 45 minutes (pH-45 min) and at 24 hours (pH-24 hours) after slaughtering using a portable pH meter (Orion model 301; Orion, Beverly, MA). Drip loss % was measured following the methodology (Honikel, 1998); for measuring cooking loss %, the plastic bags containing the thigh and breast meat samples were immersed in a preheated water bath and cooked until the internal core temperature of the meat samples reached 75°C, and for post-cook handling treatments, they were cooled for 5 min at room temperature before reweighing (Pang et al., 2020).

Gut microbial load

A series of tenfold dilutions up to 10⁻⁵ was prepared using sterile saline solution (TAYLOR, 1962). Selective agar media were employed to culture specific bacterial species using conventional microbiological techniques. For the cultures of Escherichia coli and Lactobacillus spp., two concentrations (10⁻³ and 10⁻⁴) were selected. A 100 μL portion of the diluted samples was carefully pipetted and evenly spread onto Eosin Methylene Blue (EMB) agar for E. coli and Lactobacillus MRS agar for Lactobacillus spp., then incubated for 24 to 36 hours at 37°C in a constant-temperature incubator. On the EMB agar, a distinctive metallic green sheen was enumerated as presumptive E. coli colonies, while on the MRS agar, colonies typically appearing round, shiny, and varying in color from off-white to cream was presumed to be Lactobacilli spp.

The resulting colonies were enumerated using a colony counter, applying the following formula for colony counting:

$$CFU/gm=\frac{Numberofcolonies\times Dilutionfactor}{VolumeofsampleplatedinmL}$$

A plate count agar (manufactured by HiMedia Laboratories LLC, Mumbai, India) was used as the culture medium to determine the total aerobic plate count. This process resulted in the formation of colonies with irregular morphology. The results were expressed as the logarithm base 10 of the colony-forming units (CFUs) per gram of cecal content, with each sample being replicated three times.

Statistical analysis

All data was analyzed using linear mixed effects models to appropriately address the hierarchical structure of the experimental design. Transport distance, stocking density, and lairage duration were included as fixed effects. The truck was included as a random intercept to address the clustering of crates (and birds) within transport units.

For variables related to hematological, biochemical, carcass, and meat quality characteristics (2 × 3 × 2), the linear mixed model formula was included in this study by following the formula used in a previous study (Bourget, 2023).

$$Y_{ijkl}=\mu +D_i+De{n}_j+L_k+{(D\times Den)}_{ij}+{(D\times L)}_{ik}+{(Den\times L)}_{jk}+{(D\times Den\times L)}_{ijk}+T_l+{\epsilon }_{ijkl}$$

where $T_l$ denotes the random effect of the truck.

For physiological and gut health traits (2 × 3 factorial arrangement), the model contained transport distance, stocking density, and their interaction, with truck as a random effect. The models were fitted using the restricted maximum likelihood method (REML). The significance of fixed effects was assessed using chi-square tests. If significant interactions at p < 0.05 were found, treatment means were compared using Tukey’s honestly significant difference test at the appropriate level of interaction. Superscripts were allocated hierarchically on the basis of the highest-order significant interaction. Pooled standard error of the mean (SEM) was obtained from the residual mean square of the mixed model. It was obtained based on the number of experimental units per treatment combination (n = 9 for 2 × 3 × 2 datasets; n = 18 for 2 × 3 datasets). Significant differences were determined at p < 0.05. Data analysis was done using R software (version 4.4.2) (R Development Core Team, 2023), and the level of significance was declared at p < 0.05.

Results

Body physiology

Effects of transportation distance and stocking density and their interaction on body physiology of broiler are presented in Table 1. In this study, there was no statistically significant effect of transport distance, body weight loss, and stocking density on mortality rate and changes in body temperature (p > 0.05). Though the higher mortality and greater rectal temperature changes were observed at 80 km compared to 60 km, particularly at lower space allowance, the differences were not significant statistically. On the other hand, there was a significant effect of transport distance (p = 0.002) and stocking density (p < 0.001) on body weight loss. The birds that were transported over 80 km had higher weight loss than those transported over 60 km.

Table 1.

Effects and Interaction of transportation distance and stocking density on body physiology of broiler.

Parameters	Effects							Interaction
	60 Km			80 Km			SEM	Distance (D)	Density (Den)	D×Den
	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2
Mortality %	9.259	5.555	3.703	12.963	7.407	5.555	3.931	0.511	0.595	0.963
Body Weight Loss %	1.978b	2.504a	2.911a	2.805a	2.960a	3.078a	0.143	0.002	<0.001	0.068
Body Temperature Change	0.755	0.955	0.572	0.950	1.083	0.666	0.220	0.720	0.468	0.973

Presented values are least squares means.

SEM (pooled) is derived from residual variance of the mixed model.

(^a-b) denotes significant difference among treatment combination.

Significant interaction was detected at (p < 0.05).

Gut health

Effects of transportation distance and stocking density and their interaction on gut health of broiler are presented in Table 2. In this study, the transport distance significantly affected the counts of Escherichia coli (p < 0.001), and for stocking density, it was (p < 0.001). A significant interaction between the two factors, distance and density, was found (p < 0.001). This indicates that the increase in the counts of Escherichia coli as the distance of transport increases was dependent on the available space. The counts of Escherichia coli were generally higher in the 80 km trial compared to the 60 km trial.

Table 2.

Effects and Interaction of transportation distance and stocking density on cecal bacterial count of broiler.

Parameters	Effects							Interaction
	60 Km			80 Km			SEM	Distance (D)	Density (Den)	D×Den
	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2
E. coli (log10 CFU/g)	5.492c	5.490c	6.112b	6.748a	5.708b	6.873a	0.064	<0.001	<0.001	<0.001
Lactobacillus spp. (log10 CFU/g)	5.018b	4.942b	5.449a	5.077b	4.816b	4.902b	0.049	0.627	<0.001	<0.001

Presented values are least squares means.

SEM (pooled) is derived from residual variance of the mixed model.

(^a-b) denotes significant difference among treatment combination.

Significant interaction was detected at (p < 0.05).

On the other hand, the Lactobacillus spp. count did not show a response to transport distance alone, since the effect was not significant (p = 0.627). However, the Lactobacillus spp. populations showed a highly significant response to stocking density (p < 0.001), and the interaction between transport distance and stocking density was also highly significant (p < 0.001). These findings suggest that the short transportation distance variation alone was insufficient to significantly alter beneficial microflora, while combined with higher stocking density, the cumulative transportation stress may have contributed to the change in Lactobacillus population during transportation.

Haemato-biochemical stress indicator

Effect of transportation distance, stocking density, and lairage and their interaction on haemato-biochemical stress indicator of broiler are presented in Table 3, Table 4. The hematological and biochemical parameters showed a distinct trend in response to transport distance, stocking density, and lairage duration. Most stress-related parameters, such as white blood cell count (WBC), H: L, corticosterone, AST, LDH, and plasma glucose, were higher in birds transported to 80 km compared to 60 km. An increase in space allowance from 0.65 to 1.30 square feet per bird resulted in lower values of WBC, H:L ratio, Hb, corticosterone, triglycerides, and plasma glucose. Birds subjected to 3 hours of lairage after transport also had lower values of WBC, H:L ratio, corticosterone, LDH, AST, and triglycerides compared to birds without lairage.

Table 3.

Effect of transportation distance, stocking density, and lairage on haemato-biochemical stress indicator of broiler.

Parameters	Distance 80 km						Distance 60 km						SEM
	Lairage 0 Hour			Lairage 3 Hour			Lairage 0 Hour			Lairage 3 Hour
	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2
WBC 109/L	29.294a	27.629a	17.475b	22.237a	21.854a	16.013b	25.526a	19.463b	19.424b	14.591b	12.845b	12.158b	1.126
H: L	0.821a	0.851a	0.771b	0.719a	0.705a	0.681b	0.708a	0.758a	0.603b	0.589a	0.604a	0.447b	0.025
RBC 1012/L	3.603b	3.076b	3.560b	3.533b	3.380b	3.085b	4.399a	2.847c	3.003b	3.530b	2.870c	2.910c	0.131
HB (g/dL)	13.478a	11.400b	10.030b	12.063a	10.721b	9.869b	10.961b	10.489b	10.438b	10.255b	10.380b	9.402b	0.410
PCV (%)	39.781b	34.248b	47.193a	34.839b	38.793b	39.606a	44.700a	34.611b	34.494b	38.621a	32.724b	30.496b	1.775
Corticosterone (ng/ml)	10.397a	10.031a	9.008a	8.960a	7.550b	9.172a	9.429a	9.787a	6.963b	7.738b	5.515c	5.192c	0.364
LDH (U/L)	1375a	1155a	1436a	1119a	1007a	1143a	1032b	949b	965b	611b	742b	604b	70.567
Triglyceride (mg/dL)	56b	62a	39c	45b	40b	35c	74a	61a	48b	50b	44b	43b	3.220
Plasma Glucose (mmol/L)	14.505a	12.593b	12.231b	12.783a	12.534b	12.808b	13.556a	11.416b	11.491b	12.414a	11.350b	11.247b	0.316
AST (U/L)	494a	536a	563a	302b	339b	404b	453a	459a	334b	443a	301b	380b	28.791

Presented values are least squares means.

SEM (pooled) is derived from residual variance of the mixed model.

(^a-c) denotes significant difference among treatment combination.

Table 4.

Interaction among transportation distance, stocking density, and lairage on haemato-biochemical stress indicator of broiler.

Parameter	Distance (D)	Density (Den)	Lairage (L)	DxDen	DxL	DenxL	DxDenxL
WBC	0.023	<0.001	<0.001	<0.001	0.099	0.118	0.291
H: L	0.002	<0.001	0.001	0.322	0.747	0.731	0.691
RBC	<0.001	<0.001	<0.001	<0.001	0.002	0.001	0.006
HB	<0.001	0.611	0.225	0.001	0.387	0.519	0.319
PCV	0.050	<0.001	0.015	<0.001	0.748	0.498	0.136
Corticosterone	0.060	<0.001	0.001	0.045	0.728	<0.001	0.195
LDH	<0.001	0.677	<0.001	0.170	0.242	0.295	0.838
Triglyceride	<0.001	<0.001	<0.001	0.012	0.029	0.006	0.111
Plasma Glucose	0.039	<0.001	0.012	0.787	0.372	0.190	0.292
AST	0.415	0.002	0.855	0.002	0.011	0.001	0.087

Significant interaction was detected at (p < 0.05).

Statistical analysis revealed significant main effects of distance, density, and lairage (p < 0.05), on most parameters. Significant interaction effects (p < 0.05) of Distance × Density were found for WBC, RBC, Hb, PCV, corticosterone, triglycerides, and AST. Interactions involving Distance × Lairage were significant for RBC (p = 0.002), triglycerides (p = 0.029), and AST (p < 0.011). A significant three-way interaction of distance × density × lairage was observed only for RBC count (p = 0.006), while other variables failed to show significant three-way interactions (p > 0.05), indicating little synergistic effect of the three variables.

Carcass traits

Effect of transportation distance, stocking density, and lairage and their interaction on carcass traits of broiler are presented in Table 5, Table 6. Carcass characteristics showed moderate differences among the various treatments. The carcass weight was significantly affected by the distance the birds were transported (p < 0.001). On average, the carcass weights at 80 km distance were higher, and there was a minor reduction in weight after 3 hours of lairage. Stocking density, however, did not show any significant difference (p = 0.152) for carcass weight. In terms of the edible carcass and spleen, weight was found to be unaffected by any of the variables and their interactions (p > 0.05). The breast yield was found to have a significant response to stocking density (p = 0.002) and also to have a significant distance × density interaction (p < 0.001). For the thigh yield, there was a significant influence from stocking density (p = 0.002). The percentage of whole intestine was significantly influenced by density, lairage, and also by the distance × density interaction (p < 0.05). For all the carcass variables, there were no significant three-way interactions (distance × density × lairage), except carcass weight.

Table 5.

Effect of transportation distance, stocking density, and lairage on carcass traits of broiler.

Parameters	Distance 80 km						Distance 60 km						SEM
	Lairage 0 Hour			Lairage 3 Hour			Lairage 0 Hour			Lairage 3 Hour
	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2
Carcass weight%	76.847a	76.756a	76.504a	75.592a	76.122a	75.668a	75.328b	75.963a	76.035a	75.923a	75.294b	75.347b	0.283
Edible carcass weight%	66.154	66.823	66.081	65.110	66.527	65.328	66.198	65.782	65.955	66.283	65.428	64.902	0.402
Breast weight%	24.957a	26.345a	25.500a	25.845a	24.898a	24.860a	26.324a	24.742b	24.362b	25.906a	24.340b	24.423b	0.417
Thigh weight%	8.467b	8.814a	9.010a	8.331b	8.433a	8.406a	8.227b	8.847a	9.135a	8.262b	8.325a	8.260a	0.191
Whole intestine weight%	8.688a	7.675a	7.283b	7.283a	6.837a	6.441b	7.493b	8.153a	7.142b	6.785b	7.773a	6.958b	0.241
Spleen weight%	0.069	0.082	0.081	0.070	0.157	0.081	0.080	0.094	0.095	0.090	0.110	0.106	0.024

Presented values are least squares means.

SEM (pooled) is derived from residual variance of the mixed model.

(^a-b) denotes significant difference among treatment combination.

Table 6.

Interaction among transportation distance, stocking density, and lairage on carcass traits of broiler.

Parameter	Distance (D)	Density (Den)	Lairage (L)	DxDen	DxL	DenxL	DxDenxL
Carcass weight	<0.001	0.152	0.150	0.165	0.001	0.034	0.034
Edible carcass weight	0.937	0.762	0.882	0.349	0.160	0.361	0.405
Breast	0.028	0.002	0.502	<0.001	0.138	0.809	0.099
Thigh	0.375	0.002	0.898	0.610	0.654	0.056	0.701
Whole intestine	0.002	0.010	0.075	0.002	0.214	0.548	0.931
Spleen	0.751	0.888	0.768	0.998	0.856	0.989	0.509

Significant interaction was detected at (p < 0.05).

Meat quality

Effect of transportation distance, stocking density, and lairage and their interaction on meat quality of broiler are presented in Table 7, Table 8. Meat quality attributes were variably affected by transport distance, stocking density, and lairage time. Early post-mortem pH (45 min) of breast muscle was significantly affected by transport distance (p < 0.001) and the interaction between distance and density (p = 0.002). In contrast, pH at 24 h was unaffected by almost all factors except distance (p = 0.026). Cooking loss for breast meat was significantly affected by lairage (p = 0.035) but without a significant three-way interaction. For thigh meat, cooking loss was affected by all possible interactions, except lairage (p > 0.05). For drip loss of breast meat, significant effects were found for lairage (p < 0.001) only. The drip loss of thigh meat was affected by density and three-way interactions (p < 0.001).

Table 7.

Effects of transportation distance, stocking density, and lairage on meat quality of broiler.

Parameters	Distance 80 km						Distance 60 km						SEM
	Lairage 0 Hour			Lairage 3 Hour			Lairage 0 Hour			Lairage 3 Hour
	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2	0.65 ft2	0.86 ft2	1.30 ft2
pH 45 min	5.987a	5.913b	5.887b	5.943a	5.909b	5.869b	5.895b	5.869c	5.896b	5.881b	5.845c	5.887b	0.014
pH 24 hour	5.816a	5.747a	5.815a	5.686a	5.674a	5.721a	5.662b	5.693b	5.669b	5.614a	5.729a	5.741a	0.049
Cooking loss of breast muscle	29.526a	29.561a	29.525a	27.302b	25.438b	25.393c	30.318a	29.942a	29.521a	29.113a	27.273b	26.237b	0.394
Cooking loss of thigh muscle	30.755a	28.271a	29.472a	26.721b	25.475b	26.311b	27.657b	29.900a	28.722a	27.472b	26.858b	25.758b	0.522
Drip loss of breast muscle	5.363a	5.116a	4.710b	3.614a	3.548a	3.323b	5.305a	4.657a	4.642b	3.945a	3.451a	2.988b	0.248
Drip loss of thigh muscle	5.204a	3.464b	2.972b	3.280b	3.078b	3.375b	4.697a	4.415a	3.567b	4.473a	3.264b	2.943b	0.236

Presented values are least squares means.

SEM (pooled) is derived from residual variance of the mixed model.

(^a-c) denotes significant difference among treatment combination.

Table 8.

Interaction among transportation distance, stocking density, and lairage on meat quality of broiler.

Parameter	Distance (D)	Density (Den)	Lairage (L)	DxDen	DxL	DnxL	DxDenxL
pH 45 min	<0.001	0.330	0.476	0.002	0.312	0.868	0.485
pH 24 hour	0.026	0.900	0.489	0.532	0.402	0.457	0.829
Cooking loss of breast muscle	0.166	0.359	0.035	0.600	0.208	0.025	0.855
Cooking loss of thigh muscle	0.004	0.009	0.863	<0.001	0.011	0.007	0.009
Drip loss of breast muscle	0.895	0.097	0.002	0.653	0.533	0.657	0.572
Drip loss of thigh muscle	0.130	0.002	0.502	0.005	<0.001	0.145	<0.001

Significant interaction was detected at (p < 0.05).

Discussion

Body physiology

In the present experiment, the transport distance and stocking density were found to have no statistically significant impact on the mortality rates and body temperature changes. Notably, the mortality rate in this study was comparatively higher compared to previous findings; however, birds were clinically examined prior to transportation with no obvious signs of infectious diseases or outbreaks. The combined effect of summer daytime transportation with higher ambient temperature and humidity may influence the mortality rate, though subclinical factors cannot be completely excluded. This is in contrast to the previous experiments conducted under hot-humid summer conditions, where the mortality rates and body temperature changes were found to increase significantly with the increase in transport distances and stocking densities, especially when both factors were simultaneously increased (Hussnain et al., 2020a; Hussnain et al., 2020b). The absence of significant interactions in the present experiment could have been due to the moderate transport distances and environmental conditions, which were within the critical thermal limits.

In contrast, body weight loss was significantly influenced by both transport distance and stocking density, although the interactive effect was not significant. This result partially agrees with previous findings that transport duration contributes to weight loss caused by fasting, dehydration, and increased metabolic rate (Bowen et al., 2007). Unlike previous findings of the impact of stocking density on the degree of weight loss due to extreme heat or cold stress (Yu, Oketch, et al., 2024), the influence of transport distance on weight loss in the present experiment was consistent across all stocking densities. This implies that in the experiment, the distance of transport was the major stress on the broiler, and the space available did not increase the stress sufficiently to affect mortality rate and temperature regulation.

Gut health

The results of the present study suggest that transport distance and stocking density have a joint effect on cecal microbial balance, as shown by the increased counts of E. coli at longer transport distances and the effect of stocking density on Lactobacillus populations. In this study, broilers underwent 6 h of feed withdrawal prior to transportation, therefore, the observed changes in culturable E. coli and Lactobacillus population may not solely reflect extensive bacterial proliferation during the relatively short transportation period. Instead, short-term pre-slaughter stressors such as feed withdrawal, stocking density, and transportation may alter recoverable bacterial load and gut microbial balance through intestinal emptying, fecal contamination, bacterial shedding, and microbial redistribution (Abidin et al., 2023); (Thompson et al., 2008). The significance of the interaction effects on the two microbial groups suggests that the effect of transport distance on gut microbiota is modified by space allowance. The findings are consistent with the previous observations that high stocking density alters gut microbiota, increasing the counts of E. coli and reducing Lactobacillus, as a result of stress-induced dysbiosis (Angel et al., 2015; Dong et al., 2025).

The observed density-dependent response may be explained by the effects of transport stress on the endocrine and immune systems. Studies have shown that corticosterone, which rises after stress from transport, is also strongly correlated with the intestinal barrier dysfunction (leaky gut) that occurs as a function of the stress of these conditions impairing intestinal integrity and increasing intestinal permeability among the gastrointestinal tracts of broiler chickens (Ruff et al., 2020). The sudden changes in microbes in response to acute heat stress further emphasize the susceptibility of the cecal microbial ecosystem to acute stressors (Campos et al., 2025). So, the findings of the present study suggest that stocking density affects the susceptibility of broilers to transport-induced dysbiosis, while increased transport distance acts as a stressor that exacerbates microbial imbalance in broilers under limited space.

Haemato-biochemical stress indicator

The current study showed that the transport distance, stocking density, and lairage time affected hematological and biochemical stress parameters, with increased transport distance and stocking density increasing WBC, H:L ratio, corticosterone, glucose, LDH, and AST in broilers. The 3-h lairage reduced the stress response to a certain extent. All these features are consistent with the effects of pre-slaughter stress, which may be predominantly a rapid impact via the HPA axis, triggering a cascade of endocrine, immune, and metabolic effects (Rodrigues et al., 2017; Gou et al., 2021a). The increased H:L ratio observed with longer trips and higher stocking density is consistent with the idea that the H:L ratio is a sensitive integrative indicator of cumulative stress, especially under conditions of high handling pressure and confinement (Rodrigues et al., 2017; Miah et al., 2025).

The increase in corticosterone and plasma glucose with increased transport distance supports the view that the HPA axis is activated and that energy reserves are being called upon to meet the demands of transport stress. Also, daytime transportation with significantly increased corticosterone is aligned with the findings of (Lauber et al., 1987), who found the corticosterone secretion in broilers followed diurnal variation with higher daytime concentrations. Previous research has shown that corticosterone tends to increase with short to moderate-distance transport, while the response in plasma glucose is dependent on the duration of fasting and the extent to which glycogen reserves are depleted (Gou et al., 2021a). In the present study, the 3-hour resting time in the lairage resulted in the reduction of the concentration of certain stress biomarkers, in line with other studies that have shown that the resting time partly normalizes the concentration of corticosterone and reduces the sudden endocrine responses to stress (Rodrigues et al., 2017; Zhang et al., 2009).

However, the results also indicate that there are interactions between the distance × density, and the density × lairage on the traits, suggesting that the space allowance that the birds receive does not act alone, since the appearance of the physiological stress signals depends on the density and that density heightens the stress sensitivity of the birds under transport stress (Miah et al., 2025). The increase in LDH and AST levels with an increase in distance is consistent with research indicating that stress prior to slaughter and hot temperatures can increase muscle-damaging enzymes (Xing et al., 2015). The literature, however, indicates that these enzymes' responses are dependent on certain thresholds and may not be elevated during moderate transport conditions (Zheng et al., 2020b; Gou et al., 2021a). The study finds evidence of a limited three-way interaction, largely involving red blood cells, consistent with past research that found that substantial joint effects of the duration of transport, density, and lairage time are uncommon. It also indicates that enzyme measures generally indicate more severe or protracted stress, rather than the usual pre-slaughter stress (Nasr et al., 2021; Dong et al., 2025).

Despite the present study’s distance difference of only 20 km, we have observed significant alterations in stress physiology parameters. These differences may be attributed to the cumulative efforts of transportation-associated stressors under summer conditions. Physiological stress indicators are highly sensitive to transportation duration, even during relatively short-distance transport. Findings of (Gou et al., 2021b) suggest a 0.5 h transportation significantly affected different plasma biochemical variables. Overall, the findings suggest transportation distance and density affected the intensity of physiological disruption, and lairage provided a partial recovery.

Carcass traits

The results indicate that the distance of transportation and the lairage time have significant effects on carcass weight. On the other hand, stocking density mainly influences cut yields (breast and thigh) and intestine percentage, while no three-way interactions were noted. The increase in carcass weight at the longer distance contrasted with previous studies that have found decreases in live weight and carcass weight following extended transport distances, which have been attributed to dehydration, fasting, and catabolic effects of transport stress (Utomo et al., 2019; Hussnain et al., 2020b). The difference in results may be due to the range of transport times used in this experiment and/or the relatively moderate stress levels to which the carcasses were subjected, as the loss in carcass weight was not sufficient to cause a loss in overall yield. The substantial impact of stocking density on breast and thigh yields is consistent with the evidence that density affects carcass composition, especially on important cuts (Nasr et al., 2021). Previous studies have demonstrated that density can decrease carcass and cut yields, especially in thermal stress conditions (Thema et al., 2022). The distance x density interactions for edible carcass yield, breast yield, and whole intestine percentage indicate that the effect of space allowance was influenced by transport challenge, consistent with the previous evidence that density affects transport stress (Hussnain et al., 2020a).

Lairage had a significant impact on carcass weight, edible carcass yield, thigh yield, and intestine percentage, including a decrease in intestine proportion after 3 h, which was likely the result of continued fasting. Although lairage is thought to be a period of partial physiological recovery, the effect of fasting over this period may contribute to carcass shrinkage (Utomo et al., 2019). Most importantly, the lack of significant three-way interactions between transport distance, stocking density, and lairage time indicate a cumulative rather than a synergistic effect of pre-slaughter stressors on carcass and organ characteristics. Overall, the results indicate that, although individual factors had a significant impact on individual yield components, combined effects did not result in increased or synergistic effects under the present conditions.

Meat quality

The results of the present study showed that early post-mortem pH (45 min) was affected by transport distance and stocking density, and there was a significant interaction between the two factors, while the ultimate pH (24 h) remained unaffected. This indicates that the stress experienced by the animal prior to slaughter affected the rate of early post-mortem glycolysis rather than the final rate of acidification. Other studies have shown that stress caused during transport accelerates the rate of early post-mortem glycolysis due to the activation of the sympathetic system and increased breakdown of glycogen (Hussnain et al., 2020b; Zheng et al., 2020b).

On the other hand, lack of change in pH at 24 hours in this study suggests that glycogen depletion was not sufficient to alter ultimate pH, unlike what is described in DFD-like conditions resulting from prolonged or intense stress (Rodrigues et al., 2017). The significant interaction between density and distance on pH 45 min also supports the evidence that density affected the metabolic response to transport stress rather than independently affecting it. The water-holding capacity-related traits were more consistently influenced by lairage and density than by transport distance. Cooking loss of breast was affected by all three factors, whereas cooking loss of thigh and drip loss were mainly influenced by lairage and density, with few higher-order interactions. This is partly consistent with the suggestion that increased transport time and crating density under thermal stress cause increased drip and cooking losses because of denaturation of proteins and rapid pH decline in the early period (Hussnain et al., 2020b).

However, in the present study, reductions in cooking and drip losses after 3 h of lairage indicate some degree of physiological stabilization prior to slaughter. Such improvements in water-holding capacity have been demonstrated for short recovery periods with optimum conditions of holding (Xing et al., 2016). Significant three-way interactions were found for some of the variables (thigh cooking loss and drip loss); however, these were not extensive. This is consistent with previous findings that strong synergistic effects of transport distance, stocking density, and lairage time are uncommon (Hussnain et al., 2020c). In summary, the present study has demonstrated the effects of transport distance on early metabolic response, stocking density on stress intensity, and lairage on water-holding capacity with pH remaining stable under the experimental conditions.

Conclusion

In the present study, higher transportation distance and lower stocking density resulted in higher body weight loss during transportation. The increased transport distance showed a positive correlation with the stress response and alteration of metabolic markers, while the increased space allowance reduced the negative effects of transportation on the physiological and biochemical parameters. Lairage for 3 h tended to support partial recovery, as evidenced by the reduction in key stress-related variables and improvements in meat quality characteristics. Although significant two-way interactions were found for several variables, three-way interactions were limited, suggesting that the joint effects of transport distance, stocking density, and lairage were mostly additive rather than strongly synergistic. The results of the study suggest that optimizing stocking density and lairage can help mitigate transport-related stress and improve welfare and meat quality.

Limitations

Though the present study presented a detailed evaluation of transport distance, stocking density, and lairage duration on broiler welfare and meat quality, some limitations of the present study should be pointed out. Transport distances were chosen based on the most common transport routes in the study area, which included the northeast region of Bangladesh. Very short or long-distance commercial transport was not included in the present study. The present study was conducted under a single season with daytime ambient conditions. The assessment of physiological stress was primarily based on hematobiochemical parameters, without measuring detailed immune and oxidative stress markers, inflammatory cytokines, injuries, or behavioral parameters. Additionally, the study was conducted on a single broiler strain and a single slaughter age. This limits the generalizability of the results to other broiler strains or different market weights. Future studies should consider different transport conditions and more sophisticated parameters to gain a better understanding of the effects of transport on broilers.

Author contribution

Md. Fauzul Anam Fahim: Writing-Original Draft, Investigation, Formal Analysis. Bijoy Banik: Formal Analysis, Investigation. Proshanto Singha Pranto: Investigation, Validation. Md Asibul Hasan: Formal Analysis, Investigation. Prosenjit Choudhury: Investigation, Validation. Md Mahfuzur Rahman: Investigation, Validation. Md Sodor Uddin: Formal Analysis, Investigation, Validation. Md. Nazim Uddin: Conceptualization, Supervision. Shah Ahmed Belal: Writing-Original Draft, Conceptualization, Validation, Supervision.

Disclosures

The authors declare no conflict of interest.