Commercial transport of spent layer breeder chickens: process and strain-specific physiological and behavioral responses

Abstract

Understanding the underlying issues related to animal transport is essential for promoting animal welfare. However, no research has focused on the transport process and its physiological effects on spent layer breeder chickens. Therefore, this study aimed to investigate a single commercial transport process in spent layer breeder chickens and evaluated its physiological and behavioral effects across three strains of focal hens: Sakura, Lohmann Julia (Julia), and Hy-Line Brown (Brown). A total of 1,611 chickens (1,547 hens and 64 roosters, aged 700 days) including various crossbreeds and lineages were transported over 32.9 km in 55 min. The total mortality rate was 0.25%. Each chicken was sequentially housed in two transport crate types and remained there until shackling. The maximum duration from initial capture to shackling was 7 hr 30 min. Temperatures inside cages and crates were higher than ambient conditions. Transported focal hens showed reduced body weight and elevated heterophil-to-lymphocyte ratios. Brown hens exhibited higher heterophil-to-lymphocyte ratios and lower core body temperatures compared to the overall mean. Additionally, transported focal hens exhibited wet plumage, shivering, and piloerection due to rain exposure, although temperatures near them remained within their thermal comfort zone. These findings reveal the complexity of the transport process and its adverse effects on spent layer breeder chickens, highlighting genetic differences among strains in physiological and behavioral responses. They also underscore the need for careful animal monitoring, preventing wetting, and adopting strain-specific transport management practices to improve animal welfare.

INTRODUCTION

Animal transport is an essential practice in the poultry industry. The transport process consists of a lot of stages including catching, crating, loading onto vehicles, transport, unloading, uncrating, and shackling [35]. In addition, the process occurs throughout various seasons and times of the year [9, 36, 50]. The number of animals dead on arrival (DoA) is considered a key indicator of animal welfare and health, as well as a source of economic loss during transport to slaughterhouses [36, 50, 56]. Major risk factors for DoA include the distance traveled [49, 50, 56], the season or month of the year [36, 50], and climatic conditions [56]. Poultry exposed to extremely high temperatures have been reported to show increased mortality, elevated core body temperature (CBT), and altered blood physiology [30, 46]. In contrast, exposure to cold temperatures has been associated with higher mortality, reduced CBT and changes in blood physiology [10, 19, 48]. Birds display various adaptive behaviors to cope with thermal stress. In high temperatures, chickens and turkeys pant to facilitate heat dissipation [31, 57], whereas in cold temperatures, they perform shiver, piloerection, and huddle together [19, 44].

In the poultry industry, two types of commercial chickens—meat-type and layers—are primarily raised. Meat-type chickens are specifically bred and raised for meat production. Once they reach the desired body weight, they are transported to slaughterhouses for meat processing [51]. Layers, also referred to as laying (hens), are raised for egg production. Depending on factors such as their egg-laying performance, the market prices of feed and eggs, and the cost of pullets, laying hens are eventually referred to as “spent,” “end-of-lay,” or “end-of-cycle” hens and are transported to slaughterhouses, often for use in processed meat products [9, 49]. Both meat-type chickens and laying hens originate from parent stock known as breeder chickens. Breeder chickens are kept for reproductive purposes and are selected based on their breed and lineage, including traits such as livability, fertility, and high meat or egg production [6]. The facilities housing breeder chickens are cleaned regularly to ensure high biosecurity and are maintained under strict hygiene conditions, with restricted human contact [12]. Once the reproductive performance of breeder chicken declines, they are classified as “spent,” “end-of-breeding,” or “end-of-cycle” chickens and are transported to slaughterhouses for meat processing, similar to that of meat-type chickens and spent laying hens.

In the poultry life cycle, transport is typically a brief and temporary event; however, the conditions during transport are often complex, and the associated welfare concerns and negative impacts cannot be underestimated. Investigating the underlying issues related to animal transport is essential for improving both animal welfare and economic outcomes. However, whereas the welfare impacts of transport have been extensively studied in meat-type broiler chickens and are gradually being investigated in spent laying hens, no research to date has focused on the transport process, physiological and behavioral effects on spent breeder chickens. Additionally, layer breeder chickens (hens) may respond differently to the transport process compared to laying hens, as most laying hens are housed in single-sex groups, whereas most layer breeder chickens (hens) are housed with, or in contact with, roosters and are managed under stricter hygiene programs with limited human contact [12].

Therefore, the present study aimed to examine the transport process of spent layer breeder chickens, including both general chickens and three strains of focal hens (Table 1), and to evaluate its physiological and behavioral effects on focal hens. The findings may offer valuable insights for stakeholders in the animal industry, contributing to enhanced animal welfare and economic value during transport.

Table 1. Timeline of key events, chicken types, and housing conditions during the transport process of spent layer breeder chickens, consisting of general chickens and focal hens.

		General chickens		Focal hens
		Hens	Roosters	Sakura	Julia	Brown
Timeline of key events:
	Preparation start	8:00	8:00	10:00	10:00	10:00
	Truck departure	12:27	12:27	12:27	12:27	12:27
	Unloading start	13:22	13:22	13:22	13:22	13:22
	Shackling start	14:30	14:30	16:40	16:40	16:40
Types of chickens:		Araucana, Buff Plymouth Rock, White Leghorn lines, new White Leghorn × Araucana, Buff Plymouth Rock × Rhode Island Red, Rhode Island Red × White Plymouth Rock, White Leghorn × (Rhode Island Red × White Plymouth Rock), others	Araucana, Buff Plymouth Rock, White Leghorn lines, new White Leghorn × Araucana, White Leghorn × Araucana	White Leghorn × (Rhode Island Red × White Plymouth Rock)	White Leghorn	Rhode Island Red × White Plymouth Rock
Research center crate:
	Birds/crate	10–12	5–7	5	5	5
	Density (cm2/bird)	323–387.6	553.7–775.2	775.2	775.2	775.2
	Density (kg/m2)	58.3–69.9	–	29	24.7	29.8
Slaughterhouse crate:
	Birds/crate	10–11	5–6	10	10	10
	Density (cm2/bird)	368.7–405.6	676–811.2	405.6	405.6	405.6
	Density (kg/m2)	55.7–61.3	–	58	49.5	59.5

MATERIALS AND METHODS

This study was approved by the Animal Experiment Board of Japan Women’s University (approval number: 2023-II-1) and conducted in accordance with the university’s guidelines for animal experimentation. This study involved a single commercial transport of spent layer breeder chickens from a Chiba Prefectural Livestock Research Center (hereafter referred to as the Research Center) to a slaughterhouse in Japan. Accordingly, the transport process, and physiological and behavioral impact on the chickens were monitored within established limits, in compliance with the Research Center’s regulations and without interfering with slaughterhouse procedures.

Chicken types and housing

Layer breeder chickens (Gallus gallus domesticus) were obtained from commercial vendors and maintained as parent stock for breeding purposes at the Research Center. They were scheduled for transport as spent layer breeder chickens upon reaching 700 days of age. The spent layer breeder chickens consisted of general chickens and focal hens. They included various breeds, lineages, and crossbreeds, including Araucana and White Leghorn lines. At local livestock production sites, the use of windowless housing systems has been increasing, and laying hens are typically kept until approximately 700 days of age under these conditions. In line with this practice, the Research Center raised layer breeder chickens for a similar duration in its own windowed, open-sided breeder houses. The spent layer breeder chickens were housed either in conventional cages or in a floor system, without outdoor access. The dimensions of conventional cages for hens were 39 × 23 × 38 cm (length × width × height), with a sliding door measuring 15.5 × 16.5 cm. Conventional cages for roosters measured 60.5 × 45 × 60 cm (length × width × height), with a sliding door measuring 20.5 × 30 cm. The conventional cages were equipped with a feed trough and a drinker located at the front of each cage. The spent layer breeder chickens were maintained under comparable environmental conditions, including identical feed (crude protein: 17%, metabolizable energy: 2,850 kcal/kg) and standardized lighting cycles. Although no feeding or watering records were available, the staff confirmed that the feed troughs and drinkers were consistently kept full, indicating that feed and water were provided ad libitum. The lighting cycle included light periods from 04:00 to 07:00 hr and from 16:00 to 20:00 hr.

Among the spent layer breeder chickens housed in paired conventional cages, hens from three strains—Sakura (n=5), Lohmann Julia (Julia; n=5), and Hy-Line Brown (Brown; n=5)—were selected as focal hens to examine their physiological and behavioral responses to transport. Sakura hens are a white-feathered domestic crossbreed developed by Goto Co., Ltd., Kakamigahara, Japan. The hens lay pink-shelled eggs that resemble the color of cherry blossoms, called “Sakura” in Japanese; therefore, they are named “Sakura”. Julia hens are among the most popular commercial layers in Japan. They have white feathers and lay white-shelled eggs. Brown hens are also known locally in Japan as “Boris Brown”. These brown-feathered hens are another popular commercial layer strain and lay brown-shelled eggs. All focal hens were maintained within the same breeding facility.

Data collection during the transport process

Information on the transport process including the timeline, types of spent layer breeder chickens, housing systems, catching methods, crate sizes, vehicles used, number of spent layer breeder chickens transported, and number of dead spent layer breeder chickens was collected through direct observations and interviews with staff from the Research Center and the slaughterhouse. Mortality rate was calculated as a percentage of the number of spent layer breeder chickens that died during the period from catching in their home cages or on the floor at the Research Center to shackling at the slaughterhouse, relative to the total number of spent layer breeder chickens transported. Transport time was measured from the moment the truck’s engine was started at the Research Center until it was turned off upon arrival at the slaughterhouse. The trip distance was recorded using the truck’s trip meter.

Transport process at the Research Center

All spent layer breeder chickens scheduled for transport, including the focal hens, had ad libitum access to feed and water until the time of catching for transport. Catching was performed manually from the initiation of transport preparation at the Research Center through to shackling at the slaughterhouse. Research Center staff captured the chickens using a single-wing catching technique. During preparation stage, spent layer breeder chickens were captured individually and sequentially placed into two types of transport crates positioned nearby. Initially, the chickens were removed from their home cages or from the floor of the breeding facilities by ten Research Center staff members and placed into the Research Center’s plastic crates (length × width × height: 51 × 76 × 24 cm), which had a top opening measuring 20 × 27 cm. Each crate contained 5–12 spent layer breeder chickens. For the focal hens, chickens housed in pairs in cages were first grouped by strain, and five focal hens from each strain were placed into a Research Center crate. The stocking densities in the Research Center crates ranged from 323.0 to 775.2 cm²/bird (Table 1). The crates were then loaded onto a small specialized vehicle, stacked in two rows with two to five crates per row (Fig. 1A), and transported by staff from the breeding facilities to a roofed open holding area near the entrance (Fig. 2). Upon arrival, the crates were unloaded and placed either on the loading platform or around the truck.

Fig. 1.

(A) A photo showing a specialized transport vehicle carrying crates from the breeding house to the entrance holding area. (B) A photo taken inside the truck’s loading platform, showing stacked crates. Spent layer breeder chickens are being transferred into slaughterhouse crates for transport, beside the mesh curtains covering the wall.

Fig. 2.

Schematic diagram of the spent layer breeder chicken transport process from the Research Center’s breeding house to the slaughterhouse holding area. The chickens are sequentially placed in two different types of transport crates—those from the Research Center and the slaughterhouse—and transported using two types of vehicles: a specialized vehicle and a truck.

From this point onward, subsequent procedures were carried out by three slaughterhouse staff members. They used either a single-wing or a both-wing catching technique. The spent layer breeder chickens were removed from the Research Center’s crates and transferred to the slaughterhouse’s plastic crates (length × width × height: 52 × 78 × 24 cm), which had a top opening measuring 20 × 27 cm (Fig. 1B). Each crate contained 5–11 spent layer breeder chickens. The focal hens were then combined with five additional general chickens (hens), resulting in groups of 10 hens per slaughterhouse crate. The stocking densities in the slaughterhouse crates ranged from 368.7 to 811.2 cm²/bird. These crates positioned near the truck were loaded onto the truck’s loading platform. They were stacked starting from the front of the platform and arranged into a block consisting of four rows, each containing six to seven crates. The crates containing the focal hens were placed side by side on the floor at the rear of the loading platform, after which the rear door was closed. The vehicle was a passively ventilated canopy truck, with the loading platform enclosed by mesh curtains along both side walls; the rear section was similarly covered once the door was closed. The truck then departed, and the spent layer breeder chickens inside the crates were transported from the Research Center to the slaughterhouse.

Transport process at the slaughterhouse

A total of 1,611 spent layer breeder chickens (1,547 hens and 64 roosters), were transported. The transit lasted 55 min and covered a distance of 32.9 km. Upon arrival, the crates were unloaded from the truck and stacked in the slaughterhouse’s holding area by five staff members. The crates were stacked starting from the rear of the holding area, with six to seven crates arranged per stack. The crates containing the focal hens were placed side by side on the floor. The holding area was an open, roofed space where the spent layer breeder chickens remained in their crates until preparation for shackling prior to slaughter. The total duration of the transport process—from initial catching to shackling—ranged from approximately 6 hr 30 min to 7 hr 30 min. The day of the transport was a rainy spring day. The spent layer breeder chickens were briefly exposed to rainfall in an unroofed area during the transfer from the breeding houses to the entrance holding area, as well as during both the loading and unloading stages. Additionally, rain was observed entering the loading platform through the mesh curtains during transport.

Blood sampling

Focal hens (n=5 per strain) were randomly captured from their home cages and transport crates, and blood samples were collected via the brachial vein to analyze the heterophil-to-lymphocyte (H/L) ratio. Two blood smears were prepared immediately for each hen and subsequently stained using a Diff-Quick stain kit (Sysmex, Kobe, Japan). The numbers of leukocyte granulocytes (heterophils, eosinophils, basophils) and non-granulocytes (lymphocytes, monocytes) were counted in the monolayer area of the smears under the microscope field of view until a total of 100 cells were observed [18, 26], using digitized images captured with a 100× oil immersion microscope (BX63; Olympus, Tokyo, Japan) equipped with a digital camera (DP23 camera system; Olympus). The ratios counted from duplicate smears were averaged to obtain a single value. Blood sampling was conducted at two time points: first, at the Research Center breeding house in the afternoon, two days before transport (before transport); and second, at the slaughterhouse holding area after transport (after transport).

Body weight and feather scores

Focal hens were weighed, and their plumage condition was assessed and scored for damage and dirt across seven body regions: head, neck, back, belly, wings, legs, and tail. Feather damage and dirt were evaluated using a 3-point scale: score 1 (<25% of the plumage missing or stained), score 2 (≥25% but <50% missing or stained), and score 3 (≥50% missing or stained). The scores were averaged to generate whole-body scores for feather damage and dirt. Body weight measurements and feather scoring were also conducted at two time points, as described in the blood sampling. Identification number tags were attached to the wings and legs, after which the birds were returned to their home cages or transport crates.

CBT

Focal hens were orally administered a miniature data logger (iButton Thermochron DS1921G-F5#, KN Laboratories, Osaka, Japan) to record their CBT at 5-min intervals. These data loggers were later retrieved from the crop or gizzard by slaughterhouse staff, and CBT data were finally collected from four Sakura, three Julia, and three Brown hens. These data collection was organized into five distinct stages of the transport process: 2 hr prior to the start of transport preparation at the Research Center breeding houses (before preparation), during transport preparation at the Research Center (preparation), while in transit by truck from the Research Center to the slaughterhouse (transport), the removal of crates from the truck and their transfer and placement at the slaughterhouse holding area (unloading), and during the stay at the slaughterhouse holding area after transport (holding area).

Temperature and relative humidity (RH)

Three data loggers (iButton Hygrochron #1923; KN Laboratories) per strain were used to record the temperature and RH near the focal hens inside the cages and crates at 5-min intervals. An additional five data loggers were placed to record the ambient temperature and RH in the area approximately 1.5 m surrounding the cages and crates, also at 5-min intervals. These data collection was also organized into five distinct stages of the transport process, as described in CBT. These loggers were positioned to capture environmental conditions across all previously defined transport stages where the focal hens were present: from the breeding house, through transfer to the entrance holding area, during loading, inside the transport truck, during unloading, and at the slaughterhouse holding area after transport.

Behavioral observations

The behaviors of focal hens were recorded using video cameras (Handycam HDR-XR550, Sony, Tokyo, Japan) from approximately 1 m away for approximately 1 hr during transport and for 2 hr 10 min at the slaughterhouse holding area after transport. Behavioral data were collected using instantaneous sampling at 1-min intervals. Behavioral categories were as follows: resting (sit-resting, stand-resting), feather pecking (gentle feather pecking, severe feather pecking), foraging (pecking at objects, scratching the floor, exploring), preening (including body and feather shaking, wing stretching), agonistic behavior (aggressive pecking, threats, escaping), huddling, moving (walking), shivering, piloerection, panting, vocalization, and others. Behavioral observations were conducted at two time points: during transport and at slaughterhouse holding area after transport (after transport).

Statistics

All statistical procedures were analyzed in JMP Student Edition 19.1.0 (SAS Institute, Inc., Cary, NC, USA). A generalized liner mixed model (GLMM) was used for body weight, H/L ratio, feather scores, CBT, and behaviors (resting, sit-resting, stand-resting, moving, preening, and others). Chicken type (focal hens of Sakura, Julia, and Brown), stage, and their interaction were included as fixed effects, whereas chicken ID of the focal hens was included as a random effect in the model. The model was fitted using a Gaussian distribution for body weight, H/L ratio, feather scores, and CBT. The model for resting and moving assumed a Poisson distribution, whereas sit-resting, stand-resting, preening, and others assumed a negative binomial distribution. A generalized chi-square to degrees of freedom ratio close to 1 was used to assess model fit. For body weight, H/L ratio, and feather scores, the stages were defined as before and after transport. For CBT, the stages were defined as before preparation, preparation, transport, unloading, and holding area. For behaviors, the stages were defined as during and after transport. The results of fixed effect testing were treated as the main effects. If a main effect showed a significant effect (including trends), the results of effect coding that demonstrated this significance were also reported. Feather pecking, gentle feather pecking and foraging during and after transport were analyzed by Wilcoxon signed-rank test. Data are presented as mean ± standard error (SE) in Table 2 for body weight, H/L ratio, feather scores; in Table 3 for behaviors; and in Fig. 3 for CBT. Severe feather pecking, vocalization, shivering, and piloerection occurred infrequently and therefore were not statistically analyzed, though the data are presented in Table 3. Statistical significance was set at P<0.05, and trends were noted for 0.05 ≤ P<0.1.

Table 2. Physiological changes before and after transport among focal hens.

Parameter	Stage	Sakura	Julia	Brown
Body weight (kg)	Before transport	2.38 ± 0.15	2.03 ± 0.18	2.47 ± 0.07
	After transport	2.33 ± 0.15	1.98 ± 0.17	2.36 ± 0.06
H/L ratio	Before transport	0.46 ± 0.08	0.33 ± 0.05	0.79 ± 0.14
	After transport	1.25 ± 0.26	1.27 ± 0.29	2.11 ± 0.26
Feather damage score	Before transport	1.88 ± 0.17	1.83 ± 0.21	1.75 ± 0.19
	After transport	1.90 ± 0.12	1.88 ± 0.20	1.75 ± 0.20
Feather dirt score	Before transport	1.15 ± 0.07	1.25 ± 0.07	1.08 ± 0.05
	After transport	1.15 ± 0.05	1.13 ± 0.10	1.10 ± 0.03

Data are presented as mean ± SE.

Table 3. Behaviors exhibited in focal hens during and after transport.

Behaviors exhibited (%)		Stage	Sakura	Julia	Brown
Resting		During transport	97.00 ± 1.53	97.67 ± 0.41	97.68 ± 0.67
		After transport	81.37 ± 2.19	79.39 ± 3.02	81.98 ± 3.37
	Sit-resting	During transport	90.67 ± 6.88	96.00 ± 0.41	97.33 ± 0.85
		After transport	65.34 ± 6.71	60.15 ± 4.23	56.18 ± 10.01
	Stand-resting	During transport	6.33 ± 5.51	1.67 ± 0.53	0.33 ± 0.33
		After transport	16.03 ± 5.54	19.24 ± 2.44	25.80 ± 9.90
Feather pecking		During transport	0	0	1.33 ± 0.33
		After transport	2.29 ± 0.76	0.31 ± 0.19	6.41 ± 2.31
	Gentle feather pecking	During transport	0	0	1.33 ± 0.33
		After transport	2.29 ± 0.76	0.31 ± 0.19	3.66 ± 1.19
	Severe feather pecking	During transport	0	0	0
		After transport	0	0	2.75 ± 1.42
Foraging		During transport	0	0	0
		After transport	7.18 ± 1.67	11.76 ± 2.78	5.04 ± 1.83
Moving		During transport	1.67 ± 0.75	1.00 ± 0.67	0.67 ± 0.41
		After transport	6.72 ± 1.12	2.44 ± 0.88	2.29 ± 0.54
Preening		During transport	1.00 ± 0.67	0.33 ± 0.33	0.33 ± 0.33
		After transport	0.76 ± 0.34	3.97 ± 1.06	1.53 ± 0.76
Vocalization		During transport	0	0	0
		After transport	0	0	2.14 ± 1.12
Shivering		During transport	0	0	0
		After transport	0.15 ± 0.15	1.37 ± 0.37	0.15 ± 0.15
Piloerection		During transport	0	0	0
		After transport	0.46 ± 0.31	0.46 ± 0.19	0.15 ± 0.15
Others		During transport	0.33 ± 0.33	1.00 ± 0.67	0
		After transport	0.92 ± 0.29	0.31 ± 0.19	0.31 ± 0.19

Data are presented as mean ± SE.

Fig. 3.

Changes in the core body temperature (CBT) of focal hens inside the cages and crates during each stage of the transport process. Data are presented as mean ± SE.

RESULTS

Mortality rate

The overall mortality rate was 0.25% (4 hen deaths out of 1,611 spent layer breeder chickens consisted of general chickens and focal hens) and no mortality occurred among the focal hens.

Body weight

Stage (F_1,12=44.58, P<0.001) had a significant effect on the response variable, and effect coding showed that body weight significantly decreased after transport (estimate= −0.03, P<0.001, 95% confidence interval (CI) [−0.04, −0.02]) compared to the overall mean. The interaction between chicken type and stage (F_2,12=2.99, P=0.088) showed a marginal trend. Effect coding further indicated that the body weight changes in Brown hens after transport (estimate= −0.01, P<0.05, 95% CI [−0.03, −0.002]) were significantly different from the expected overall mean, suggesting that body weight changes across stages may differ among strains. Animal type (F_2,12=2.52, P=0.121) did not show a significant difference in body weight.

H/L ratio

Chicken type (F_2,12=4.40, P<0.05) and stage (F_1,12=62.50, P<0.001) had significant effects on the response variable. Effect coding also showed that Brown hens (estimate=0.41, P<0.05, 95% CI [0.10, 0.71]) had higher H/L ratios compared to the overall mean, and H/L ratios significantly increased after transport (estimate=0.50, P<0.001, 95% CI [0.36, 0.64]). The interaction between chicken type and stage did not show a significant difference (F_2,12=1.52, P=0.257).

Feather score

Feather scores for damage and dirt remained comparable before and after transport. Feather damage scores were not affected by chicken type (F_2,12=0.15, P=0.859), stage (F_1,12=0.45, P=0.515), or their interaction (F_2,12=0.15, P=0.862). Feather dirt scores were not affected by chicken type (F_2,12=0.88, P=0.438), stage (F_1,12=0.68, P=0.425), or their interaction (F_2,12=1.31, P=0.303). Although all focal hens showed moist plumage following transport, this did not appear to affect the feather scores.

CBT

Chicken type (F_2,7=4.39, P=0.057) had a marginal effect on CBT, and effect coding showed that Brown hens (estimate= −0.40, P<0.05, 95% CI [−0.73, −0.07]) had a significantly lower CBT than the overall mean. Stage (F_4,28=4.31, P<0.01) had a significant effect on CBT, and effect coding showed that unloading (estimate= −0.14, P<0.01, 95% CI [−0.22, −0.05]) was significantly associated with a lower CBT than the overall mean. The interaction between chicken type and stage (F_8,28=3.00, P<0.05) had a significant effect on CBT. Effect coding showed that the change in CBT for Brown hens differed from the expected overall mean during unloading (estimate=0.11, P=0.070, 95% CI [−0.01, 0.23]) and holding area (estimate= −0.20, P<0.01, 95% CI [−0.32, −0.08]), indicating that the change in CBT across stages varied depending on strains.

Temperature

Temperatures differed among the inside and outside of the cages and crates. The temperatures measured near the chickens were higher than the ambient temperatures (Near hens: Range: 20.68–26.37°C; Ambient: Range: 19.97–22.34°C). The temperatures measured near each strain ranged as follows: near Sakura, 20.94–25.13°C; near Julia, 21.16–23.83°C; near Brown, 20.68–26.37°C. The temperatures measured near the chickens between the preparation and holding area stages ranged 22.85–26.37°C.

RH

The RH measured in the ambient and near the chickens ranged as follows: ambient, 90.74–95.51%; near Sakura, 84.22–94.88%; near Julia, 88.28–96.67%; near Brown, 83.4–86.87%.

Behaviors

Resting was the most frequently observed behavior. Stage (F_1,24=22.92, P<0.001) had a significant effect on resting, and effect coding showed that resting significantly decreased after transport (estimate=exp (−0.09), P<0.001, 95% CI [−0.13, −0.05]). Sit-resting was significantly affected by stage (F_1,10.8=49.97, P<0.001), and effect coding indicated a significant decrease after transport (estimate=exp (−0.22), P<0.001, 95% CI [−0.29, −0.15]). Stand-resting was significantly affected by stage (F_1,24=51.94, P<0.001), and effect coding showed a significant increase after transport (estimate=exp (1.35), P<0.001, 95% CI [0.96, 1.74]). The interaction between chicken type and stage (F_2,23.9=4.18, P<0.05) also had a significant effect on stand-resting, and effect coding showed that the increase in Brown hens after transport (estimate=exp (0.77), P<0.05, 95% CI [0.12,1.41]) was significantly greater than the expected overall mean, indicating a strain-specific response of stand-resting across stages. Feather pecking tended to increase after transport in Brown hens (Wilcoxon: P=0.062). Gentle feather pecking did not differ between stages (Wilcoxon: Sakura, P=0.125; Julia, P=0.5; Brown, P=0.312). Foraging tended to increase after transport in all strains of focal hens (Wilcoxon: all P=0.062). Moving was significantly affected by stage (F_1,24=15.12, P<0.001), and effect coding showed a significant increase after transport (estimate=exp (0.58), P<0.001, 95% CI [0.27, 0.89]). Chicken type (F_2,16.2=2.99, P=0.077) had a marginal effect on moving. Preening was significantly affected by stage (F_1,23.2=7.04, P<0.05), and effect coding showed a significant increase after transport (estimate=exp (0.61), P<0.05, 95% CI [0.13, 1.10]). The interaction between chicken type and stage (F_2,22.3=3.28, P=0.056) had a marginal effect on preening, and effect coding showed that the increase in preening after transport tended to be greater in Julia hens (estimate=exp (0.61), P=0.078, 95% CI [−0.07, 1.30]) than the expected overall mean, indicating that the change in preening between stages may differ among strains. Severe feather pecking and vocalization were observed among Brown hens after transport. Shivering and piloerection were also observed after transport. Others were not affected by chicken type (F_2,20.1=1.68 × 10⁻⁴, P=0.999), stage (F_1,24=1.55 × 10⁻⁴, P=0.990), or their interaction (F_2,24=1.24, P=0.305).

DISCUSSION

Spent laying hens are often transported over longer distances for processing due to the limited number of poultry processing facilities available for them, compared to those for broiler chickens and turkeys raised for meat production [33]. The mortality of spent laying hens increases with longer transport distances, with rates of 0.338% for hens transported up to 50 km and 0.801% for those transported between 201 and 300 km [49]. In the present study, the transport distance of 32.9 km was below the 50 km threshold, and the maximum duration from initial catching to shackling—7 hr 30 min—was also shorter than the suggested limit of 10 hr [14]. Although the total mortality rate of spent layer breeder chickens (0.25%) in the present study appeared to remain within the range of previously reported transport-related mortality rates for spent laying hens (0.18–1.397%) [9, 40, 49], it exceeded the 0.1% threshold that is generally considered indicative of compromised animal welfare during transport [14]. Additionally, crate densities for focal hens (49.5–59.5 kg/m²) exceeded recommended threshold for spent breeders (49 kg/m²) [14].

During preparation for transport in the present study, all spent layer breeder chickens were sequentially housed in two types of transport crates and transported using two types of vehicles. These processes appeared to be complex for both the birds and the personnel involved. However, breeder chickens are typically kept under strict hygiene conditions to ensure high biosecurity, with minimal human contact [12]. Consequently, restricting human access, including slaughterhouse staff, to the breeding facilities and their surrounding areas, along with the exclusive use of transport crates specifically designated for the Research Center and their transfer to the entrance holding area by Research Center staff, is a critical measure for safeguarding the biosecurity for the remaining breeder flocks.

In response to physiological stressors, the number of lymphocytes decreases, whereas the number of heterophils increases in fowl [18]. An H/L ratio of 0.5 is considered optimal [18]. An increase in the H/L ratio has been reported following catching and placement, as well as during heat stress associated with transport and lairage [31, 42]. Humans are the main source of handling stress since they are perceived by the birds as predators [16]. In the present study, the H/L ratio of focal hens increased after transport, indicating a physiological response to transport-related stress. When birds are stressed, the hypothalamus–pituitary–adrenal (HPA) axis is activated, leading to excessive secretion of corticosterone into the bloodstream [3, 18]. This hormonal response results in hypotrophy of the lymphoid organs, which in turn partially leads to an increased H/L ratio [18]. Plasma concentration of corticosterone can be changed based on a circadian rhythm [59] or the ovulatory cycle [58], which may affect the response to a stressor. High-fear chickens show an increased H/L ratio compared to low-fear chickens, whereas no differences are found in the adrenal corticosterone response to adrenocorticotropic hormone (ACTH) [3], indicating that H/L ratio does not directly reflect circulating corticosterone levels. Therefore, while the H/L ratio is a useful indicator of stress, it is recommended that responses to stressors be interpreted in a comprehensive and multidimensional manner, using multiple measures such as corticosterone levels.

Brown hens in the present study exhibited a higher H/L ratio, which may reflect a genetically distinct response to the transport process [7, 26], consistent with previous findings that brown egg layers show greater stress indicators than white egg layers [4]. In addition, Brown hens showed a different pattern of change in body weight. On the other hand, the three layer-breeder strains in the present study exhibited similar body weights, even though the average body weight of brown-feathered layers has previously been reported to be higher than that of white-feathered layers [9]. Therefore, these findings suggest that potential physiological differences and similarities between commercial layers and layer breeders may be influenced by housing conditions [12]. However, findings from layer studies may not fully apply to breeder populations; therefore, further studies are needed to clarify the physiological differences and similarities between commercial layers and layer breeders. The weight loss observed after transport was likely due to deprivation of feed and water for over 6 hr following capture and crating [9, 52], as well as water loss during transport [53].

When chickens are deprived of food in an unfamiliar environment, they tend to limit their movement to conserve energy [47]. Similarly, the focal hens in the present study, which most frequently exhibited immobile resting behavior during and after transport, likely did so as a means of conserving energy. After transport, all three breeders of focal hens showed a reduction in stationary resting behavior, indicating an increase in activities such as feather pecking, foraging, moving, and preening. Feather pecking is classified into two types: gentle feather pecking and severe feather pecking. Gentle feather pecking is a behavior in which the pecker lightly pecks at the tips of the recipient’s feathers, and the recipient generally does not react [41]. Severe feather pecking involves the pulling and removal of the feather, and sometime ingesting them, which causes feather loss and pain in the recipient [41]. The recipient may react with pain vocalization (squawk) [41], as observed in Brown hens. Gentle feather pecking is considered a form of social exploration and is frequently directed toward unfamiliar individuals [38]. Foraging is a highly valued behavioral need and an instinctive activity in domestic fowl [32, 54]. However, pecking behaviors in poultry, including feather pecking and cage pecking, have been reported to be associated with frustration arising from an inability to perform certain natural behaviors [11, 24]. Additionally, severe pecking is considered a form of redirected pecking that occurs when birds are frustrated in their normal activities, such as foraging, comfort and exploratory behaviors [54]. The day of transport in the present study was rainy, and all focal hens were observed to be wet during the holding area stage after transport. It is unlikely that the focal hens were accustomed to wet plumage or exposure to rainfall, as they had previously been housed in cages without outdoor access. Therefore, in the present study, it is possible that the exhibition of feather pecking by hens may have resulted from their perception of wet plumage as unfamiliar, prompting pecking behavior, while they were also motivated to explore the unfamiliar environment and conserve energy by resting immobile [5, 27]. Simultaneously, the focal hens may have experienced stress and frustration caused by the novel environment, unfamiliar conspecific, and high crate density, which could have elicited behaviors such as foraging and feather pecking. Regarding strain differences, the incidence of severe feather pecking observed among Brown hens may reflect the darker feather pigmentation of Brown hens [22]. Preening, including feather and body shaking behavior, is considered a comfort behavior and maintain the function and integrity of the integument [34]. Conversely, it has also been suggested that displacement preening, which is thought to be associated with frustration, increases when feeding is thwarted in birds [13, 62]. In the present study, preening behavior including feather and body shaking was observed to increase after transport. Although the focal hens appeared to be drying their wet plumage to maintain its function, they may also have performed redirected preening behavior as an expression of frustration and stress after more than 6 hr of feed and water deprivation following catching and crating.

All three breeder strains of focal hens exhibited shivering and piloerection after transport, although these behaviors were not frequently observed. In cold conditions, birds typically exhibit behaviors such as shivering, piloerection, and huddling [19, 44]. Shivering is an acute mechanism of heat production, involving repetitive contraction and relaxation of muscle that generates heat through multiple processes [39]. Plumage plays an important role in reducing thermogenic requirements by providing insulation and trapping warm pockets of air around the body [43]. The depth of feather layers can be increased through piloerection [20]. The insulation properties of plumage are influenced by both the thermal conductivity of each feather and the collective abilities of the feathers to trap a layer of air [28]. To maintain CBT, body must produce heat by increasing thermogenic activity, such as through shivering, when environmental temperatures fall below the lower critical temperatures [14]. Spent laying hens often have poor feather coverage and, due to limited energy, protein and calcium reserves [17, 37], may have difficulty thermoregulating and responding effectively to thermal stress during transport [25]. Temperatures inside the cages and crates in the present study appeared to differ from those outside [2, 23], likely due to temperature gradients resulted from limited airflow and heat generated by hens in close proximity within the enclosures [2, 23]. In broiler chicken transport, ambient temperatures of 8°C can induce hypothermia when feathers are wet, whereas dry-feathered birds can tolerate ambient temperatures as low as −4°C in well-ventilated vehicles [21]. In contrast, the transport of spent layer breeder chickens in the present study occurred during the spring season rather than the colder winter season, and temperatures near the focal hens remained within their thermal comfort zone (22–28°C) [14, 55]. Although Brown hens (40.9°C) showed significantly lower CBT and a different pattern of CBT changes across stages compared to the overall mean, indicating a strain-specific response, the CBT of all three strains of focal hens remained within the normal range (41–42°C, with fluctuations of ± 1°C) [10, 14]. Therefore, in the present study, shivering may have resulted from the combined effects of disrupted feather insulation due to wetting, increased heat loss through air movement and evaporation from wetted surfaces, inadequate coverage, and low reserves of energy, protein, and calcium reserve [15, 25]. Additionally, one Julia hen did not seem able to maintain her normal wing position during shivering (Fig. 4). She stood up after being pushed by the movement of another bird on her right and then began shivering. She did not appear able to maintain her original, elevated wing posture, causing her wings gradually shifted into a drooped position and the wing position repeatedly shifted between an upper position and a lower position. In response to cold condition, birds retract their legs into their feathers in a sitting position (leg-tuck posture) [8, 61] to conserve body heat by using warm air trapped in feathers to reduce thermogenic demand [8, 60]. This posture reduces in exposed surface area and minimizes heat loss from the legs and feet [29]. The Julia hen may have been sitting to conserve body heat; however, she was accidentally pushed by another hen, which may have forced her to stand up suddenly. This sudden movement would have exposed more of her legs, causing increased heat loss and expression of shivering due to the legs being exposed to the air. The pectoralis flight muscle of birds is the primary site of shivering thermogenesis [45], and shivering is known to cause muscle injury and fatigue [1]. Therefore, damage to and fatigue of the pectoralis flight muscle caused by shivering under cold stress during the transport process may have contributed to the bird’s drooped wings. It may not be a universal feature for chickens to be exposed to rain during transport. However, water (e.g., mist) exposure is commonly observed while chickens are kept in the slaughterhouse holding area. Such exposure can occur when birds are placed near areas where vehicles used for transport, empty used crates, or floors are being cleaned with high-pressure washers. To mitigate such issues, transport procedures should take place in a roofed area, using solid curtains or protective covers to prevent rain or water exposure and to keep the chickens dry [14].

Fig. 4.

A Julia hen showed a change in wing position during shivering at the slaughterhouse holding area. This behavior was observed in only one hen, and whether it can be reproduced remains unclear. (A) The wings (left-side hen) were held in their normal, elevated position. (B) She shifted her wings to a drooped position during shivering, compared to the original posture.

The present study focused on and demonstrated, for the first time to our knowledge, the transport processes and the physiological, behavioral, and welfare impacts on spent layer breeder chickens, an important and novel area that helps bridge a research gap, as previous transport studies have largely focused on meat-type chickens and spent laying hens. These findings revealed the complexity of the transport process and its adverse effects, highlighting genetic differences in physiological and behavioral responses among spent layer breeder chickens. A distinct feature of this process was the transport of multiple breeds, lineages, and crossbreeds within a single shipment, unlike meat-type chickens and spent laying hens, which are typically transported as a single strain or a few strains only. This makes careful animal monitoring and strain-specific transport management essential to improve welfare. However, the present study has some limitations, including the use of only a single transport event and a small number of chickens and strains. The limited sample size likely reduces statistical power, which may contribute to certain results being reported as trends or to an inadequate interpretation of the findings. Additionally, only one bird exhibited drooped wings, and it remains unclear whether this phenomenon occurs in other flocks under cold conditions. The evaluation of wing position and feather wetness also requires standardization. Further research on additional breeder strains is required to enhance animal welfare during transport.

CONFLICT OF INTEREST

Authors declare no conflict of interests for this article.

Funding Statement

This work was supported by JSPS KAKENHI Grant Number JP23K05472 to YM and research funding from JWU to SF.