The effects of simulated transportation conditions on the core body and extremity temperature, blood physiology, and behavior of white-strain layer pullets.

Transportation of poultry is stressful. The transportation of broilers has been well studied, while the transportation of layer pullets from rearing to laying facilities has not been thoroughly evaluated. This experiment aimed to establish the effects of temperature (T)/RH combinations and duration (D) of transport, via a 5 × 2 factorial arrangement of simulated transport conditions using 5 T/RH combinations (21°C with 30% RH [21/30], 21°C with 80% RH [21/80], 30°C with 30% RH [30/30], 30°C with 80% RH [30/80], and -15°C with uncontrolled RH [-15]), and 2 exposure D (4 or 8 h). Pullets (18-19 wk; n = 240) were obtained from 3 commercial farms (N = 3 farms). Pretreatment, birds were orally administered a miniature data logger to record core body temperature (CBT), an initial blood sample was taken (5 birds/replicate), and initial foot T was recorded. Behavior during exposure was video recorded. Following exposure, a final blood sample was taken (analyzed for heterophil to lymphocyte ratio, partial pressure of CO2, total CO2, bicarbonate, and glucose), birds were slaughtered, and data loggers were retrieved. Data were analyzed as a randomized complete block design via Proc Mixed (SAS 9.4) and significance was declared at P ≤ 0.05. There were no interactions observed for the T/RH and D combinations throughout the study. The CBT and foot T were lowest in pullets exposed to -15 compared with all other treatments. Foot T was also highest in pullets exposed to 30/80 compared with -15, 21/30, and 21/80. There was no impact of T/RH on pullet blood physiology. Activity and thermoregulatory behaviors were impacted by the T/RH combinations. Pullets exposed to 30/30 and 30/80 spent the most time panting. Pullets exposed to 30/80 also spent the least amount of time motionless. Duration had minor impacts on pullet CBT, blood physiology, and behavior. These data indicate that as a response to thermal stress, layer pullets were successful at implementing mechanisms to maintain homeostasis.

Introduction

Transportation is a fundamental component of the poultry industry; however, it can also result in animal welfare concerns. Many of the steps involved are stressors for poultry, such as catching, loading, transport, and unloading (Schwartzkopf-Genswein et al., 2012). Unlike broilers and laying hens, pullets are typically not feed-withdrawn before transportation, as they are not destined for slaughter. Rather, pullets are transported from a rearing barn to a laying barn at approximately 17 wk of age, and this transportation can be over either a short or long distance (Schwartzkopf-Genswein et al., 2012; NFACC, 2016). Stressors such as microclimate (temperature [T] and RH), vibration, noise, and the duration (D) of transport can have a large impact on the welfare of birds during transportation (Mitchell and Kettlewell, 1998, 2009).

The majority of transportation studies have focused on broiler chickens as they are most commonly transported for slaughter at the end of their production cycle. The stressor most frequently studied in relation to transport is thermal stress. Chickens are homeotherms, where the core body temperature (CBT) of inactive birds is approximately 41°C to 42°C (Mount, 1979). As a first response to thermal stress, birds will modify their behavior in order to conserve or dissipate heat (Mount, 1979). Under cold stress, birds will huddle or burrow under one another in an attempt to minimize radiation to the environment and maximize conduction between conspecifics (Mount, 1979; Strawford et al., 2011). Furthermore, pteroerection can be performed as a thermoregulatory mechanism against cold exposure, as feathers can aid in providing insulation against exposure to the cold T, trapping air between feather fibers to conserve body heat (Mount, 1979; Strawford et al., 2011). Under heat stress, birds will move away from conspecifics and lift their wings, as an attempt to maximize space usage within the crate to ensure that the maximum body surface area is exposed to the environment (Mount, 1979; Weeks et al., 1997). If maximizing body surface area alone is not enough, birds will implement evaporative heat loss mechanisms (panting) to dissipate heat (Whittow et al., 1964; Mount, 1979). However, when the RH in the environment is high, panting may become ineffective at dissipating heat from the core body (Whittow et al., 1964; Richards, 1971). It has been shown in previous research that the CBT of broilers will significantly increase as a result of exposure to T above 30°C for a 2 h D (Sandercock et al., 2001; Menten et al., 2006).

If the bird is still experiencing thermal stress after implementing behavioral changes, then physiological changes are likely to occur. Under cold stress, to further conserve heat for the core body, blood vessels to the extremities (feet and comb) will vasoconstrict to restrict blood flow (Midtgard, 1989; Wellehan, 2014). However, if the ambient T becomes too cold, the extremities can be susceptible to frostbite, and hypothermia may occur (Mount, 1979; Wellehan, 2014). Previous studies have consistently found that the CBT of broilers will decrease when exposed to T below −5°C for 3 h (Dadgar et al., 2011, 2012a). If thermoregulatory mechanisms against cold stress are unable to maintain the bird's CBT, their welfare can be negatively impacted, and mortality may occur. When under heat stress, blood vessels to the extremities (feet and comb) will vasodilate, causing increased blood flow, which can aid in dissipating heat through convection and radiation through the bare skin (Whittow et al., 1964; Richards, 1971; Midtgard, 1989).

Thermal stress can also alter the blood physiology of birds. Measuring various blood physiology parameters such as blood glucose, acid–base balance parameters, blood gas concentration, dehydration markers, and the heterophil to lymphocyte (H/L) ratio may give an indication of the degree of stress experienced by birds during transportation. Due to dehydration, the amount of fluid in the blood will decrease, increasing both the hematocrit and hemoglobin values (Thomas et al., 2008; Bergoug et al., 2013). However, it has also been hypothesized by previous research that the hematocrit and hemoglobin values may decrease through hemodilution, which is an adaptive response to evaporative heat loss where water is lost through the extracellular fluid rather than through the plasma (Borges et al., 2004; Scanes, 2016). In addition, the body will lose water (via dehydration and defecation) and electrolytes (through mainly defecation), resulting in low blood sodium levels and hyponatremia (Thomas et al., 2008). Furthermore, as a result of stress during transportation, corticosterone in the body will increase, causing glucose to be mobilized from the fat stores in the body through glycogenolysis, and from protein stores through gluconeogenesis (Zhang et al., 2009; Sherwood et al., 2013). This glucose will then be used as energy to respond to stressors and thermoregulate against heat or cold stress. As a result of evaporative heat loss during heat exposure, blood gases and the acid–base balance in the blood will change, causing a shift toward a respiratory alkalosis (Koelkebeck and Odom, 1995; Lumb, 2000). Hyperventilation during heat exposure will increase the elimination of CO2, resulting in a decrease in the partial pressure of carbon dioxide (pCO) and total carbon dioxide (tCO) in the blood, causing an increase in the blood pH (Ahmad and Sarwar, 2006; Sherwood et al., 2013). Finally, the H/L ratio has been shown to be a reliable indicator of chronic stress in poultry (Felver-Gant et al., 2012). Stress will cause corticosterone to increase which will, in turn, result in either an increase or decrease in the number of heterophils and/or lymphocytes, ultimately causing an increase in the H/L ratio (Blecha, 2000; Elsayed, 2014). Therefore, changes in the blood physiology of pullets during transport may indicate that they were subjected to thermal stressors during transport; thus this may indicate poor welfare.

Since most studies regarding poultry transportation have focused on broilers, very little is known regarding the transportation of pullets. The objective of this study was to quantify how exposure-simulated transport conditions (hot and neutral T, at a high and low RH, as well as cold T) over a D of 4 or 8 h affects pullet behavior, physiology, and welfare. This was achieved by evaluating CBT, extremity T, behavior, and blood physiology.

Materials and methods

The experimental protocol for this research was approved by the University of Saskatchewan Animal Care Committee and was performed under the guidelines of the Canadian Council of Animal Care (1993, 2009).

Experimental Design

This study was a 5 × 2 factorial arrangement with 5 T/RH combinations and 2 D of simulated transportation (4 or 8 h). A hot (+30°C), and neutral (+21°C) T were chosen with a high (80%) and low (30%) RH, as well as a cold T (−15°C) with uncontrolled RH, which resulted in the following 5 combinations: −15°C uncontrolled RH (−15), +21°C 30% RH (21/30), +21°C 80% RH (21/80), +30°C 30% RH (30/30), and +30°C 80% RH (30/80).

Birds and Housing

A total of 240 white-feathered commercial layer pullets were obtained from 3 commercial farms (2 Lohmann LSL-Lite and 1 Dekalb White) within a 250-km radius of Saskatoon, Saskatchewan. Pullets were obtained at 18 to 19 wk of age, and were given a 3 to 5 d acclimatization period. Pullets were housed in 1 floor pen (3.9 m × 3.0 m) with straw litter, with feed and water provided ad libitum. Feed was acquired from the farm of origin and was provided via 3 aluminum tube feeders (38 cm pan diameter). Water was provided with 2 bell drinkers (36 cm diameter). The lighting program from the farm of origin was maintained via incandescent bulbs.

Prior to Chamber Exposure

Prior to the simulated transport exposure, 8 pullets were randomly assigned to one of the T/RH and D combinations (N = 40). Pullets were not feed-withdrawn prior to chamber exposure. Each pullet was wing banded for identification purposes, and was orally administered a miniature data logger (iButton Thermochron DS1922L; Maxim Integrated, San Jose, CA), which travelled to the crop/gizzard and recorded CBT every minute. An initial blood sample was taken from the brachial vein into a dipotassium EDTA tube (5 birds/replicate). All 5 samples were used for H/L analysis which was performed by preparing a blood smear. The slides were stained (PROTOCOL Hema 3; Fisher Scientific, Ottawa, Canada) and read under 100× oil magnification (microscope B-290TB; Optika, Bergamo, Italy) by counting the number of heterophils and lymphocytes within a field of view until a total of 100 cells was reached. Immediately after collection, 3 blood samples were analyzed using a CG8+ cartridge in the i-STAT1 handheld analyzer (Abbott Point of Care Inc., Princeton, NJ). Blood physiology parameters analyzed (3 birds/replicate) included blood glucose (mmol/L), sodium (mmol/L), hematocrit (% packed cell volume), hemoglobin (mmol/L), pH, base excess (BE), pCO2 (mmHg) and oxygen (pO; mmHg), tCO2 (mmol/L), oxygen saturation (sO; %), and bicarbonate (HCO; mmol/L). Afterward, pullets were placed into 1 of 2 crates for a 15-min lairage period to obtain baseline readings for CBT, and then transported to the environmental chambers (College of Engineering, University of Saskatchewan, Saskatoon, Canada). After transportation, an initial foot T reading was taken individually from all pullets using a thermocouple attached to a multimeter (Omega HH509; Omega Engineering Inc., Laval, Canada), and a plastic clip to ensure the thermocouple remained in place on the middle toe of the right foot. Pullets were then transferred into the treatment crates (stocking density 45.5 kg/m2) and placed in the environmental chamber with 1 crate per treatment D.

During Chamber Exposure

Simulated transportation conditions were achieved using 2 environmental simulation chambers (2.1 m × 3.4 m; College of Engineering, University of Saskatchewan) (Figure 1). Chamber conditions were monitored and adjusted if needed in real time via a thermocouple and multimeter (Omega HH509) and RH sensor (HM1500LF; Measurement Specialties, Inc., Toulouse, France). In addition, there were 3 T and RH data loggers (iButton Hygrochron DS1923-F5#; Maxim Integrated) in each chamber with one located on the wall to record chamber conditions and one in each crate at bird level that were set to record T and RH every minute. Two infrared camera systems (Panasonic WV-CF224FX; Panasonic Corporation of North America, Newark, NJ) were placed on the wooden table inside each chamber, with 1 camera observing each crate. Behavior was evaluated from the recording at 5-min intervals via scan sampling over the entire D of exposure (4 or 8 h). The behaviors evaluated included activity (motionless, active, shuffle), thermoregulatory (burrow, shiver, pteroerection, panting, survey), pecking (object peck, bird peck), and other actions (preen, head shake, gulp, scratch, unidentifiable) which are defined in the ethogram (Table 1).

Figure 1

Image of the interior of one of the environmental simulation chambers used in this study.

Table 1

Ethogram of behavioral activities monitored during the study and the criteria for each behavior; all behaviors are mutually exclusive, except for panting (Webster and Hurnik, 1990; Hurnik et al., 1995; Webster, 2000; Henrikson et al., 2018).

Category	Behavior	Description
Activity	Motionless	Bird may be standing or in a crouched position where its body is in contact with the floor of the crate (difficult to determine via videos). Bird is motionless and may be in a collected posture with its head retracted, and eyes may be open or closed
Activity	Active	Bird is moving its feet and/or wings and is changing position/location within the crate
Activity	Shuffle	Bird is moving its feet while moving its body side to side. Minimum of 1 series of side-to-side movements to be considered. Bird resets itself near the same position as when behavior began
Thermoregulatory	Burrow	Birds are actively attempting to burrow underneath other birds
Thermoregulatory	Shiver	Repeated quivering of the wings and/or body in order to produce body heat
Thermoregulatory	Pteroerection	Feathers are erect or being ruffled in an organized manner
Thermoregulatory	Panting	Increase in breathing of the bird characterized by an open mouth, polypnea, or increased thoracic movements
Thermoregulatory	Survey	Movements of the bird's head suggesting surveillance of the bird's environment
Pecking	Bird peck	Bird is using its beak, in short and quick forward motions of the head, to make contact with other birds
Pecking	Object peck	Bird is using the beak, in short and quick forward motions of the head, to make contact with either the crate or the sensors on the crate
Other	Preen	Beak is used to manipulate the feathers on the bird's own body
Other	Head shake	Rapid side-to-side movements of the head. Bird is immobile. Minimum of 2 series of side-to-side movements to be considered
Other	Gulp	Head of bird is pointed vertically upward, beak is opened, and bird takes a large gasp of air
Other	Scratch	Bird raises its leg over its wing and repeatedly rubs its head with its talons
Other	Unidentifiable	Bird cannot be seen, or behavior cannot be characterized

Post Chamber Exposure

At the end of the exposure D, the crate was removed from the chamber. A final foot T was taken for each pullet and a second blood sample was collected from the same 5 birds per crate. Blood samples were again analyzed for H/L ratio (5 birds/replicate) and blood physiology (3 birds/replicate). The pullets were then euthanized via electric stunning and exsanguination. The data logger was retrieved from the crop/gizzard and the data were downloaded (OneWire Viewer; Maxim Integrated) for further analyses. Delta (Δ) CBT was calculated for every hour by considering the average hourly CBT during exposure and subtracting the average baseline reading prior to exposure.

Statistical Analyses

Data were analyzed as a randomized complete block design with the farm of origin as the block, and crate was used as the experimental unit. A block design was chosen to control variability arising from the farm or genetic line. Data analyses were performed using SAS 9.4 (SAS Institute, Cary, NC). Data were verified for normality prior to analyses using Proc Univariate and were log transformed when necessary. They were analyzed via one-way ANOVA using the Proc Mixed procedure with means separation conducted using the Tukey test. Treatment means and pooled SEM were determined via Proc Means. Differences were considered significant at P ≤ 0.05 and trends were noted when 0.05