The effects of floor space and nest box access on the physiology and behavior of caged laying hens

The effects of floor space and nest box access on the physiology and behavior of caged laying hens J. M. Engel,∗ T. M. Widowski,† A. J. Tilbrook,‡ K. L. Butler,§ and P. H. Hemsworth∗,1 ∗

Animal Welfare Science Centre, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC 3010, Australia; † Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1, Canada; ‡ Centre for Animal Science, University of Queensland, St Lucia, Queensland, 4072, Australia; and § Biometrics Group, Department of Economic Development, Jobs, Transport & Resources, Hamilton, VIC 3030, Australia. ABSTRACT Confinement housing appears to be at the forefront of concern about laying hen welfare. This experiment examined the effects of floor space during rearing (315 or 945 cm2 /bird) and adulthood (542 or 1648 cm2 /bird) and access to a nest box on the welfare of caged laying hens. Measurements of the normality of biological functioning, such as plasma, egg albumen and yolk and fecal corticosterone concentrations, and heterophil to lymphocyte ratios, behavioral time budgets, mortality and efficiency of productivity, and measurement of hen preferences, such as choice behavior in Y maze tests, were used to assess hen welfare. There were no effects of treatment on physiological measurements. Hens given less space during adulthood spent less time mobile, inedible pecking, drinking, and preening and spent more time resting and feed pecking and sitting (P < 0.05). Hens with access to a nest box spent more time resting (P = 0.046) and less time sham dust

bathing (P = 0.044) than hens without access to a nest box. There were no effects of space allowance on choice behavior for space or a nest box over food; however, hens with access to a nest box chose the nest box over food more than hens without access to a nest box (P = 0.0053). The present experiment provides no convincing evidence that either reducing space allowance in adulthood from 1648 to 542 cm2 /bird or eliminating access a nest box results in disruption of biological function. Less space and no access to a nest box did not increase the choice for more space or a nest box, respectively, over food in the preference tests. However, reduced floor space reduced behavioral freedom and denying access to a nest box eliminated the opportunity for the motivated behavior of laying their eggs in a discrete enclosed nest box, both of which presumably provide hens with the opportunity for positive affective experiences.

Key words: laying hen, housing, corticosterone, behavior, welfare 2018 Poultry Science 0:1–15 http://dx.doi.org/10.3382/ps/pey378

INTRODUCTION

sary minimum space requirements to perform certain behaviors, such as stretching, wing flapping, standing, and lying down. Dawkins and Hardie (1989) showed that brown laying hens used 540 to 1006 cm2 when turning, 653 to 1118 cm2 /hen when stretching wings, and 540 to 1005 cm2 /hen when ground scratching. Mench and Blatchford (2014) reported that individually housed light hybrids used, on average, 563 cm2 for standing, 1315 cm2 for turning, and 1378 cm2 for wing flapping. Using an operant method, Lagadic and Faure (1987) found that hens in groups of 4 would work to increase space above 400 cm2 /hen, but only for 25% of the time, suggesting that there may be an intermittent preference for a larger cage that is context dependent (Cooper and Albentosa, 2003). It needs to be recognized that group size/total cage size is also important as it affects the amount of free space available to the bird. For example, aggression in laying hens decreases as flock size is increased within a constant pen area (Nicol et al., 1999).

Two of the most contentious issues in relation to cage housing and hen welfare are space (Lay et al., 2011; Widowski et al., 2016) and the need for a separate nest (Cronin et al., 2012a). The literature on the effects of space allowance in conventional layer cages shows that in general, within a range of 300 to 650 cm2 per hen, as floor space decreases, mortality increases, and egg production, body weight, and efficiency of feed utilization decreases (see reviews by Barnett and Hemsworth, 2003; Widowski et al., 2016). Mench et al. (1986) reported that reducing space allowance in 2-bird cages from 1394 to 697 cm2 /bird increased plasma corticosterone concentrations. Furthermore, hens have neces-

 C 2018 Poultry Science Association Inc. Received May 5, 2018. Accepted July 23, 2018. 1 Corresponding author: [email protected]

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Hens are also highly motivated to access an enclosed nest site as shown using preference and behavioral demand tests (Cooper and Albentosa, 2003; Weeks and Nicol, 2006). Furthermore, the hen’s nesting behavior is less “settled” when a suitable nest is unavailable (Nicol 2015; Hunniford and Widowski, 2018); hens are more active and engage in locomotor behavior for a longer duration before laying their eggs. Hens may also perform stereotyped pacing, all of which have been interpreted as signs of frustration (Yue and Duncan, 2003; Appleby, 2004). This pre-laying activity could also be interpreted as searching for a nest site. However, the relationship between the lack of a nest box and stress physiology has received little scientific attention. Alm et al. (2016) and Cronin et al. (2008) found no shortor long-term differences in glucocorticoids, respectively, between hens with or without a nest box. Cronin et al. (2012b) subsequently reported that hens that exhibited a longer duration and fewer bouts of pre-lay sitting had lower plasma corticosterone concentrations regardless of whether they had a nest box. The objectives of this experiment were to determine the effects of floor space allowance and access to a nest box in caged laying hens on a broad range of wellaccepted welfare indices. Since there is growing evidence that early experience may not only affect preferences for resources such as space allocation (Faure, 1991), but also the sensitivity of the hypothalamo-pituitary adrenal (HPA) axis (Ericsson et al., 2016), this experiment also examined the effects of floor space allowance during rearing on subsequent hen welfare. Understanding animal welfare and its assessment requires the use of multiple indicators from multiple disciplines (Hemsworth et al., 2015; Tilbrook and Ralph, 2017), particularly since individuals vary in the manner in which they cope with stressors (Broom, 1986; Koolhaas et al., 1999). The conceptual framework of biological functioning is commonly used to infer compromised animal welfare on the basis that difficult or inadequate adaptation will generate welfare problems (Broom 1986; Hemsworth et al., 2015). The framework of affective state to assess animal welfare is based on the concept that the welfare of an animal derives from its capacity for affective experiences and thus a common approach is preference research on the assumption that animals make choices that are in their best interest, that is, avoid aversive stimuli and choose positive stimuli (Duncan and Fraser 1997; Fraser, 2003). The normality of biological functioning was studied using measures of the behavioral and physiological responses of hens to the housing treatments, as well as common production variables that may be adversely affected by the extent of biological activity underlying attempts to cope, such as body weight, egg production, mortality, and feather condition. The affective state was studied by measuring the preference of hens to access extra space and a nest box in a discrete choice test. Thus, risks to hen welfare in this experiment were assessed in 2 ways. First, by measuring the normality in

biological functioning (i.e., difficulty in adapting) using behavioral and physiological changes, such as increases in glucocorticoid concentrations and heterophil to lymphocyte (H:L) ratios, decreases in preening, and increases in cohort pecking, as well as changes in fitness measures, such as decreases in body weight, egg production, egg weight, extracuticular shell calcium, and feather condition. Second, by measuring the hen’s choice for either space or a nest box versus food in a discrete choice test.

MATERIALS AND METHODS All experimental procedures were conducted under approval of the University of Melbourne Animal Ethics Committee and in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council, 2013).

Treatments The treatments in the experiment were a 2 × 2 × 2 factorial, consisting of the following 3 main effects: 1. Rearing space allowance—2 levels in groups of 8 pullets per cage from 7 wk of age, 315 and 945 cm2 /bird. 2. Adult space allowance—2 levels in groups of 6 hens per cage from 16 wk of age, 542 and 1648 cm2 /bird. For convenience, this period from 16 wk of age will be labeled ‘adulthood’ or ‘adult period’ of the experiment. 3. Nest box access—2 levels, presence or absence of a nest box during adulthood. In the latter, the nest box was present but its access was blocked. The space allowances chosen met the standards of the Model Code of Practice for the Welfare of Animals in Australia (Primary Industries Standing Committee, Domestic Poultry 4th Edition). The Code of Practices requires pullets to have a space allowance of 315 cm2 /hen at 15 wk of age. This space allowance was used throughout rearing. The smaller space allowance during adulthood is higher than the requirement for cage systems in Australia purchased prior to January 2001, but slightly less than that required of cages purchased after January 2001. The larger space allowance (3-fold greater than the smaller space allowances) was chosen, as it would likely provide ample space for hens to perform their entire repertoire of behaviors (Cooper and Albentosa, 2003).

Animals, Housing, Husbandry The experiment was conducted over 30 mo. Sixtyfour female Hy-line Brown pullets in each of 4 time replicates (256 pullets in total) were beak-trimmed at 1 d of age. At 7 wk of age, they were transported

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BEHAVIOR AND WELFARE OF CAGED LAYING HENS

approximately 1.5 h from a commercial poultry farm in Kinglake West (Victoria, Australia; 37◦ 28’S, 145◦ 14’E) to the research facility in Werribee (Victoria, Australia; 37◦ 55 S,144◦ 40 E) and 8 pullets were ad hoc allocated to each of 8 2.5 m2 cages and 8 7.5 m2 cages within one room of an environmentally controlled layer facility containing 66 Victorsson Trivselburen furnished cages (AB Broderna, Victorsson, Sweden). The cages were 455 mm high at the rear and were modified to meet the experimental requirements in that the perches were removed and the dust baths blocked in the experiment. Each cage provided 2 water nipples, shared between the back-to-back cages, and water was available for ad libitum consumption. Pullets were vaccinated per industry standard and ad libitum fed a commercially available grower pellet (15.5% CP). The hens remained in these rearing cages until 16 wk of age. A second room was set up to house the hens from 16 to 29 wk of age. This room had 2 parallel banks of cages (left and right banks) that ran from the front to the back of the room, in a manner that allowed the size of the cages to be manipulated by adjusting the placement of the sides of the cages along the rows. The banks were placed at the height of a top tier in a 3-tier shed, so that video cameras could be placed in the area normally under the cages. The 2 banks backed onto each other, but were separated by a solid wall, and the front of the cages in the 2 different banks faced opposite directions. Within each time replicate, the portion of the banks of cages toward the front and back halves of the room were randomly allocated to contain only hens from small or large rearing cages (i.e., the front half only contained hens from small rearing cages and the back half only contained hens from large rearing cages or vice versa). Within each half at each time replicate, from the front to the back of the 2 banks, a random choice (from front to back) of a small and then a large adult pen or a large and then a small adult pen was made. Within a time replicate, the 2 pens backing onto each other, but in different banks, were restricted to be the same size. In our descriptions, we will designate the 2 same sized pens backing onto each other as a row. Within each half of the bank, nest box access was then allocated to row-bank combination using a 2 treatment latinsquare randomization. The sources of variation for this design (dummy analysis of variance) are summarized in Table 1. At 16 wk of age, 48 hens were randomly allocated (within their rearing treatment) to the adult cages, and thus, new groups were formed. Once in the adult cages, hens were fed a commercially available pre-lay pellet (15.5% CP) for 2 wk, followed by a formulated layer diet based on 95 g feed consumed/hen/day ad libitum (15% CP). The nest boxes (24 cm × 40 cm × 20 cm; w × d × h) consisted of solid, steel sides and topped with a dust bath to which access was blocked. The floor of each nest box was lined with a piece of Astroturf measuring the area of the nest box.

Table 1. Sources of variation (analysis of variance) in the experiment. Source of variation Time replicate stratum Residual Half within time replicate stratum Rearing cage size (R) Residual Rows within halves stratum Adult cage size (A) A.R Residual Banks within halves stratum A.Nestbox A.R.Nestbox Residual Pen stratum Nestbox R.Nestbox Residual Total

Degrees of freedom 3 1 3 1 1 6 1 1 6 1 1 6 31

In both rooms used in the study, the thermostats were set at 21◦ C with the average temperature maintained at approximately 17◦ C during the dark period and 23◦ C during the light period; however, humidity within the research facility was difficult to maintain. Lighting was provided by incandescent bulbs and controlled by a computer. At placement, the birds were on an initial light:dark cycle of 10L:14D. Day length was increased until the hens were exposed to an ongoing lighting regimen of 14L: 10D at 29 wk of age. The birds were housed at approximately 20 lx (1.86 foot candles) when lights were on.

Measurements Physiology. Two laboratories for convenience were used to analyze plasma corticosterone and corticosterone metabolites in eggs and excreta samples. One laboratory analyzed the samples from replicates 1 and 2, and the other analyzed the samples from replicates 3 and 4. The extraction methods used by each laboratory are described below Baseline Plasma Corticosterone Collection and Extraction. Blood was collected from each hen 1 day per week at 26 and 27 wk of age from 1:300 to 1:400 h. All samples analyzed were taken within 2 min of the hen being removed from her home cage in order to avoid an acute stress response to capture and handling influencing baseline plasma cortisol concentrations (i.e., to avoid handling confounding baseline measures; Broom and Johnson, 1993). Whole blood was collected via the wing vein using a 4.5 mL heparin-coated syringe with a 23-G needle. Samples were kept on ice (<1 h) until they were centrifuged and the plasma poured into 1 mL tubes. The samples were then pooled within cage and over the 2 collection days to establish a sample for baseline corticosterone concentrations. Samples were frozen at –20◦ C until analyzed for corticosterone concentration.

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Laboratory 1. Plasma samples were not extracted. Laboratory 2. Plasma samples were extracted following the methods described by Downing and Bryden (2008). Briefly, plasma samples (0.1 mL) were dispensed into glass culture tubes and 3 mL of diethyl ether were added. The contents were shaken for 10 min. The tube and contents were placed in a −80◦ C freezer and when the aqueous phase was frozen, the solvent fraction was poured into a 12 × 75 mm culture tube. The diethyl ether was removed by heating it under a constant flow of nitrogen gas. The precipitate was the dissolved in 0.1 mL PBS and analyzed. Adrenocorticotropic Hormone (ACTH) Challenge Collection and Extraction. Hens were challenged with adrenocorticotropic hormone (ACTH) at 28 wk of age to investigate the maximum corticosterone response to ACTH. Hens were injected into the thigh muscle with 0.5 mL (12.5 IU) ACTH (Synacthen, Ciba Geigy, AllHank Trading, South Melbourne, Victoria, Australia) using a 1 mL syringe with a 25-G needle (Barnett et al., 2009). After 1 h, blood was collected as described previously. Plasma was pooled for each cage and samples were frozen at –20◦ C until analyzed for corticosterone concentration. Samples were extracted following the protocol for the plasma. White Blood Cell (WBC) Counts (Haematology). Blood was sampled from all hens at 29 wk of age for white blood cell (WBC) counts which were used to obtain H:L ratios. Whole blood was collected via the wing vein using a 4.5-mL heparin-coated syringe with a 23-G needle. These blood samples were transported on ice to IDEXX Laboratories (Brisbane, Australia), and the absolute numbers of heterophils and lymphocytes were measured on individual hen samples in an autoanalyzer CellDyn 3700 (Abbott Diagnostic Division, Abbott Park, IL). Automated counting of blood leukocytes has been validated for chickens using an earlier model of this machine (Post et al., 2003a). Whole Egg Collection, Analysis, Separation, and Extraction. Eggs were collected over the 2 d prior to blood collection at 26 and 27 wk of age. On collection days, all eggs were recorded as to the time at and cage from which they were collected. Eggs were labeled with a corresponding number and collected. Eggs were then analyzed for extracuticular calcium following the methods of Reynard and Savory (1999), using a colorimeter (Hunter Lab Miniscan XE, Reston, VA). The device was placed over the broad end of the egg where calcium dusting is most likely to occur (Mills et al., 1987). For each egg, 3 dry readings were taken before the shell was wiped with a damp cloth and 3 wet readings were taken. Using the L-score (a ranking of white (100) to black (0)), the dry and wet readings were averaged and the wet reading was subtracted from the dry reading to yield a dusting score. Following extracuticular calcium analysis, eggs were weighed and separated into the albumen and yolk. Each component was weighed and a sample of each was retained (yolk, 4 to 6 g; albumen, 10 to 12 g). Samples

were pooled within cage and then pooled again for the 4 d of collection. They were then frozen at –20◦ C until analysis. Laboratory 1. Egg yolk was extracted following the methodologies described by Cook et al. (2009). Briefly, egg yolk (0.5 g) was added to 1 mL of distilled water and vortexed until mixed. The mixture was extracted with 3 mL hexane:diether (30:70 v/v), vortexed, and left to settle before being snap-frozen in an ethanol/dry ice bath. The supernatant was collected and dried. One milliliter of ethanol was added to the samples which were then frozen at –20◦ C overnight. The samples were centrifuged the next day and the supernatant removed and dried once more before being suspended in PBS and analyzed. Egg albumen was extracted following the methodologies of Downing and Bryden (2008). Briefly, egg albumen (5 g) was added to 5 mL of distilled water. These were mixed, and 0.5 g of the mixture was extracted with 4 mL of diethyl ether, shaken for 10 min, and then frozen at –80◦ C. The supernatant was then collected and dried. The samples were suspended in PBS and analyzed. Laboratory 2. Egg yolk was extracted using the protocol described for laboratory one. The only exceptions being that 0.1 g of yolk was added to 0.5 mL of distilled water and the ethanol mixture was frozen at – 80◦ C overnight. Egg albumen samples were extracted following the same procedures as laboratory 1, except 0.5 g of albumen was mixed with 1 mL of distilled water. Excreta (Fecal) Collection and Extraction. Excreta samples were also collected over the 2 d prior to baseline blood sampling at 26 and 27 wk of age. Collection occurred over 3 1-h collection periods on each day. At this time, manure belts were cleared of excreta and greaseproof paper was placed under each cage. At the end of each hour, excreta (feces and uric acid) were collected into an aluminum container. At the end of the day, samples were weighed and placed in a drying oven at 60◦ C for 48 h. Once dry, samples were weighed and ground. Samples from each cage were pooled over the 4 sampling days and frozen at –20◦ C until extraction and analysis. Laboratory 1. Ground, dried excreta were extracted using the methodologies described by Wasser et al. (1994). Briefly, ground, dried excreta (0.1 g) were extracted with 1 mL of 80% methanol. The samples were then vortexed for 30 min and centrifuged. The supernatant was dried and then suspended in PBS for analysis. Laboratory 2. Ground, dried excreta were extracted following the methodologies described by Brown et al. (1994). Briefly, ground, dried excreta (0.1 g) were boiled in 3 mL of 90% ethanol for 20 min and centrifuged for 10 min at 500 × g. The supernatant was removed and the process repeated. The supernatants were pooled and dried under a stream of nitrogen. The extracts were then suspended in PBS and diluted 1:20 before being analyzed.

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Table 2. Postures and behaviors measured in the behavioral time budgets. Postures Sitting Standing Squatting (Penguin Posture) Behaviors Mobile Immobile Resting Preening Eating Drinking Inedible pecking Cohort pecking Stretch Oviposition Sham dust bathing

Definition Hen’s legs are approximately parallel to the cage floor with plumage of chest and/or belly in contact with the cage floor The hen’s body is not in contact with the cage floor, legs are extended Keel is up and the cloaca pointing downward while the hen is in oviposition

Walking or running Staying in place Hen immobile and performing no other listed behaviors Hen cleaning feathers or scratching at body Hen visibly pecking into the feeder Hen visibly pecking at the nipple drinker Hen pecking at inedible objects (anything other than feed, drink, and other birds) Hen pecking at cage mates Hen has fully extended wing or leg, flapping wings, or ruffling feathers Hen in process of laying egg Hen performs behaviors associated with dust bathing on the cage floor or in the nest box

Analysis of Corticosterone in all Extracted Samples Laboratory 1. The assay used in the first laboratory was a Corticosterone HS Enzyme Immunoassay (EIA) (IDS Ltd., Boldon, UK). Laboratory 2. The assay included one standard curve and unknown extracted samples in duplicate. The standard curve included triplicate tubes for total counts (TC) and non-specific binding (NSB), 9 replicates of the zero standard, 3 replicates of each standard, and 6 replicates each of 2 quality control pools containing 0.71 and 2.22 ng/mL which were used to estimate the intra-assay coefficients of variation (5 and 5.7). A total of 100 μL of H3 -corticosterone tracer (1,2,6,7H3 ; Amersham Biosciences Pty. Ltd., Castle Hill, NSW, Australia) and 150 μL first antibody (B3-163; Endocrine Sciences, Calabasas, CA) were added to tubes containing extracted samples. Buffer (150 μL) was added to the NSB tubes. Tubes were vortexed and incubated at 4◦ C for 24 h. On day 2, normal rabbit serum (100 μL, 1:800) was added, followed by 100 μL second antibody (anti-rabbit serum; 1:60 in PBS). The tubes were mixed and incubated overnight at 4◦ C. On day 3, 1 mL of 6% polyethylene glycol (PEG 6000) in PBS was added to all tubes (except TCs). The tubes were centrifuged in a refrigerated (5◦ C) centrifuge at 1,500 × g for 25 min, the supernatant aspirated, and the pellet was redissolved in 500 μL of HCL (0.05 M). The solution was dispensed into counting vials and then mixed with 2 mL of scintillant (Starcint, Packard Chemicals Operations, Australia). The vials were capped, shaken, and left in the dark for 2 h before counting in a liquid scintillation counter (Packard Tri Carb 1500).

Behavior For all video recordings, DC powered black and white cameras equipped with infrared (IR) lighting were

placed above and below each cage and at the front of each nest box. Two cameras (each with 12 forwardfacing IR light-emitting diodes (LED) for illumination), one placed above and one below the cage, provided views of both the top and bottom of each small cage, while 4 cameras, 2 placed above and 2 below, provided views of both the top and bottom of each large cage. Furthermore, a camera (measuring 52 mm wide × 42 mm high × 16 mm deep with 6 IR LED embedded in the front of the black plastic camera case) was placed at the front of each nest box. Each hen in a cage was given a unique marking to her back using a carbon-based ink and a unique combination of black and white leg bands so she could be easily identified on video footage. Behavioral Time Budgets. The behavioral time budgets were conducted for 2 d prior to the start of the physiological measurements at 26 wk of age during each first 5 min of each light hour (13 h/day) using instantaneous point sampling (Martin and Bateson, 1993) at 30-s intervals. The postures and behaviors recorded are displayed in Table 2. For each time point, the total number of hens in the cage performing each behavior was recorded and the average proportion of hens exhibiting each behavior in each cage at each 30 s sample point was calculated for the 2 study days. Y Maze Preference Testing. Video records were observed daily from 22 to 29 wk of age to record the times of oviposition (time the egg touched the floor of the cage) for each hen. Hen selection. At 29 wk of age, 32 of the 48 experimental hens were selected for preference testing. Sixteen hens (2 from each cage) were selected (Nest Box hens) to be tested for nest box preference over food. These hens were selected based on the regularity of their laying pattern, and therefore, the ease at which their time of oviposition could be predicted. Two of these hens in the first replicate were consistent floor layers; however, all other selected hens were consistent nest box layers. Each of the nest box-tested hens was then randomly paired with a hen (Space hens) to be tested for

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ensure they were being introduced to the nest box while space preference over food. As hens were being tested they were motivated to use it. Once all nest box hens for increased space preference, the y-maze apparatus were tested, their corresponding space hens were tested was specifically designed for this experiment to offer as in the same order. little extra space as possible. The space attachment was Y-maze testing. Hens were divided into 2 testing equivalent to the higher space allowance of 1,648 cm2 . groups (consisting of 1 pair from each cage). Groups The feed attachment was designed to resemble the front were tested on alternate days and hens were ordered of a cage and the nest box attachment was designed depending on the time the nest box-tested hen was preto resemble the nest boxes present in the experimendicted to lay her egg, but whether the nest box-tested tal cages, but were slightly smaller. The floor of the hen or the space-tested hen was tested first was rannest box attachment was lined with the same Astroturf domized for each testing day. A hen was placed in the present in the nest boxes in the cages. SB of the Y-maze and a similar protocol as in trainOviposition prediction. The recorded times of oviposiing phases 1 and 2 was used to allow each hen to enter tion of each hen were used to predict the time of oviposithe CA. If, after 30 s, she had not chosen either of tion on the days of testing for their nest box preference. the resources (nest box vs. food or space vs. food), she If the hen showed a clear temporal pattern, the averwas given a gentle nudge (not toward either particular age amount of time between successive eggs was used resource) to encourage her to make a choice. Once a to predict the time she would be expected to lay her resource was chosen, the gate to the other resource was next egg. However, if a hen failed to lay an egg (pause closed so the hen could not change her choice. The hen day) or laid an egg at a time that varied significantly was then given 2 min with the resource unless she chose from her usual pattern, the records were checked and the nest box. Should a hen choose the nest box, she was the predicted time of oviposition for the next day was promptly enclosed in the nest box and the attachment predicted to be approximately the time at which she set aside. She remained in the nest box until she laid laid her next egg following the previous pause day or her egg or for 30 min (whichever occurred first) before the unexpected oviposition. Once the time of oviposibeing returned to her cage. tion was predicted, the hen was assigned a testing time approximately 30 to 40 min prior to the expected time of oviposition so that she was tested during the period Productivity and other Measurements when she was likely to be exhibiting pre-laying behavior Body Weights. Birds were weighed individually on and most motivated to access a nest box. a weekly basis from 7 to 15 wk of age to monitor growth Training phase 1. The purpose of the first training and health. Animals recorded to have lost weight were phase was to familiarize hens with the testing apparare-weighed to check the reading, and monitored closely tus. This phase of y-maze testing occurred over 5 conthereafter if they had actually lost weight. Once reachsecutive days. A hen was placed into the start box (SB) ing adulthood, birds were weighed individually at 16, of the y-maze. After 10 s, the gate of the SB was opened 18, 19, 20, 21, 22, 26, 30, and 34 wk of age. Weights and the hen was given 30 s to leave the SB (defined were averaged for each cage for statistical analysis. as the hen’s entire head and neck being outside of the Hen-Day Production. The average percent of days area). If at 30 s a hen did not leave the SB, she was in which hens laid an egg (hen-day production) was given a gentle nudge into the choice area (CA) and the calculated on a cage basis. gate to the SB was closed. At this point, she was given Feather Condition Scores. The methodology for 2 min to explore the rest of the apparatus. Pairs of hens feather condition scoring was adapted from Tauson were randomly ordered prior to training and were also et al. (2005). Feather condition was assessed using a randomized for whether the nest box hen or space hen subjective 4-point scoring system applied to the neck, would be trained first. breast, cloaca/belly, back, wings, and tail. Once scores Training phase 2. The purpose of the second training were given, the scores for each of the 6 body areas were phase was for hens to associate each arm of the y-maze added together to create the following scores: >20 = with a given resource. This phase of testing occurred good – very good; 15 20 = average; 13 - 15 = rather over 5 consecutive days. Prior to testing, the side at poor; and < 13 = poor. which the hen was first exposed was randomized for each hen and each testing day. The gate of the arm the hen was first exposed to was left open, while the Statistical Analysis gate of the other arm was closed. A hen was placed Each measurement was analyzed using a multi-strata into the SB of the y-maze and a similar protocol as in factorial analysis of variance, corresponding to the training phase 1 was used to allow each hen to enter the sources of variation (Table 1), with the unit of analysis CA. If, after 30 s, she had not entered the open arm, being a cage of 6 chickens. Prior to analysis, the H:L she was given a gentle nudge to enter the arm. She ratio was logarithmically transformed. The percent of was then given 2 min with the resource. This was then times the next box or space was chosen were angularly repeated for the opposite arm. This phase of y-maze transformed. Mean feather scores at 26, 30, and 34 wk testing took approximately 10 min per bird. Therefore, of age were angularly transformed after linearly transthe nest box hens were tested in the order in which they forming the scores to a 0 to 100 scale. One unusual were predicted to lay their egg (as described above) to Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey378/5079179 by University of the Western Cape user on 25 August 2018

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BEHAVIOR AND WELFARE OF CAGED LAYING HENS Table 3. Main effect of treatments on physiology at 26 to 29 wk of age. Variables Corticosterone

Treatments Rearing (R) Small Large SED P-value Adulthood (A) Small Large SED P-value Nest box (NB) No Yes SED P-value

Basal plasma (ng/mL)

Fecal (ng/g)

Albumen (ng/g)

Yolk (ng/g)

Adrenocorticotropic hormone (ACTH) (ng/mL)

Hematology Heterophil: lymphocyte ratio1

1.23 1.14 0.090 0.40

49.1 52.2 2.73 0.34

0.29 0.31 0.057 0.70

2.69 2.79 0.243 0.71

17.6 16.4 1.68 0.53

0.03 (1.08) –0.02 (0.95) 0.088 0.60

1.20 1.17 0.096 0.72

51.4 49.9 2.91 0.61

0.31 0.30 0.027 0.76

2.68 2.80 0.192 0.53

18.1 15.8 2.74 0.43

–0.01 (0.98) 0.02 (1.04) 0.040 0.61

1.27 1.11 0.093 0.13

50.4 50.9 4.97 0.91

0.32 0.29 0.033 0.36

2.83 2.65 0.099 0.12

16.8 17.1 3.33 0.92

–0.02 (0.96) 0.03 (1.06) 0.067 0.56

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size and presence of a nest box are calculated on 1, 6 degrees of freedom. 1 Heterophil:lymphocyte ratios were log10 transformed prior to statistical analysis. Back transformed means presented in parentheses.

cage, with a much greater amount of feather damage than the other cages in its time replicate, was treated as a missing value for the mean feather score measurements (Payne, 2010).

RESULTS There was no mortality in the experiment; however, 2 birds were removed due to illness from the small rearing cages (1 each in replicates 1 and 2) and each replaced with a bird from a larger rearing cage. Another bird in a small rearing cage suffered an injury in replicate 2 when she became trapped between the feeder and egg tray. She was also removed from the study and replaced with a bird from a large rearing cage. None of the birds used as replacements in the small cages were used in the adult component of the experiment. In analyzing the 43 measurements reported in this paper, 9 2- and 3-factor interactions were statistically significant at the 5% level (P < 0.05), and none at the 1% level (P < 0.01). This is less than the number of interactions that would be expected to be significant (P < 0.05) by chance (number expected = (1/20) × number of measurements reported × number of interaction tests per measurement = (1/20) × 43 × 4 = 8.6). Thus, there is no meaningful evidence of any interaction between the 3 treatments (rearing cage size, adulthood cage size, and nest box), and hence only main effects are reported.

Physiology Despite good precision, there was no evidence (P > 0.05) of any effects of rearing cage size, adult cage size, or provision of a nest box on any of the 4 corticosteronerelated measurements, or on the H:L ratio (Table 3).

Behavior Behavioral Time Budgets. There was little evidence of space allowance during rearing affecting adult hen behavior at 26 wk of age, with only drinking being statistically significant at P = 0.05 (Tables 4 and 5). Space allowance during adulthood, however, affected numerous postures and behaviors in the hens at 26 wk of age. Hens in small cages were observed less frequently in an erect posture, mobile, preening and pecking inedible, and more frequently sitting, resting and feed pecking, sham dust bathing (P < 0.05; Tables 4 and 5). The absence of the nest box during adulthood had little, if any, effect on bird behaviors at 26 wk of age, with only 2 behaviors being significant at the 5% level. Hens in cages without access to a nest box may have rested less frequently (P = 0.046) but may have more frequently exhibited sham dust bathing (P = 0.044). Choice Behavior in Y Maze Tests. There were no effects of space allowance during rearing or during adulthood on the choice behavior in Y maze tests for a nest box over food or for space over food (Table 6). Furthermore, there were no effects of space allowance during rearing or during adulthood on the latencies to choose the nest or space when these 2 resources were chosen. In contrast, there was an effect (P = 0.005) of nest box access, in which hens with access to a nest box more often chose a nest box and an indication (P = 0.077) for hens with nest box access to choose a nest box in less time than hens without nest box access (Table 6).

Productivity and other Measures Body Weights and Egg Production. There were no material treatment effects on body weight, egg weight,

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Table 4. Main effects of treatment on postures and behaviors of mobile and resting in behavioral time budgets at 26 wk of age.1 Rearing (R) Variable Postures Sitting Erect Behaviors Mobile Resting

Adulthood (A)

Nest box (NB)

Small

Large

SED

P value

Small

Large

SED

P value

No

Yes

SED

0.07 0.93

0.07 0.92

0.011 0.011

0.86 0.87

0.08 0.92

0.06 0.93

0.005 0.005

0.0082 0.011

0.07 0.92

0.07 0.93

0.011 0.009

0.91 0.93

0.07 0.32

0.08 0.33

0.008 0.02

0.60 0.80

0.04 0.37

0.11 0.28

0.004 0.01

0.08 0.31

0.07 0.33

0.006 0.008

0.54 0.046

<0.0001 <0.0001

P value

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size and presence of a nest box are calculated on 1, 6 degrees of freedom. 1 Data presented as proportion of hens exhibiting each individual posture or behavior (average proportion of hens exhibiting each behavior in each cage at each 30 s sample point). Squatting was seldom recorded and data not presented.

Table 5. Main effect of treatments on active behaviors in behavioral time budgets at 26 wk of age.1 Rearing (R) Variable

Adulthood (A)

Nest box (NB)

Small

Large

SED

P value

Small

Large

SED

P value

No

Yes

SED

P value

0.11 0.24 0.08 0.12 0.03 0.01

0.11 0.26 0.06 0.11 0.04 0.01

0.009 0.018 0.006 0.011 0.015 0.003

0.35 0.43 0.048 0.51 0.72 0.98

0.10 0.28 0.07 0.03 0.03 0.02

0.12 0.22 0.08 0.04 0.04 0.01

0.003 0.010 0.004 0.015 0.008 0.002

0.00039 0.0013 0.78 0.021 0.19 0.15

0.11 0.26 0.070 0.13 0.03 0.02

0.11 0.25 0.08 0.10 0.04 0.01

0.006 0.016 0.004 0.012 0.010 0.003

0.24 0.60 0.39 0.086 0.29 0.044

Behaviors Preening Feed pecking Drinking Inedible pecking Cohort pecking Sham dust bathing

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size and presence of a nest box are calculated on 1, 6 degrees of freedom. 1 Data presented as proportion of hens exhibiting each individual posture or behavior (average proportion of hens exhibiting each behavior in each cage at each 30 s sample point). Stretching and oviposition were seldom recorded and data not presented.

Table 6. Main effect of treatments on choice behavior in Y maze tests at 32 to 34 wk of age. Variables

Treatments Rearing (R) Small Large SED P value Adulthood (A) Small Large SED P value Nest box (NB) No Yes SED P value

Nest box rather than food chosen1 (%)

Latency to choose the nest box (s)

18.8 (10.4) 15.9 (7.5) 3.77 0.49

23.9

15.9 (7.5) 18.8 (10.4) 2.92 0.37

22.2

6.9 (1.4) 27.8 (21.8) 4.90 0.0053

25.0

21.5 4.40 0.62

23.2 2.78 0.74

20.4 2.14 0.077

Space rather than food chosen1 (%)

Latency to choose space (s)

or hen day production (P < 0.01; Tables 7 and 8). Three of the 39 P values presented are statistically significant at the 5% level, but these results are only for specific times that are associated with lower estimated standard errors, and thus are possibly chance effects. Extra-cuticular Calcium There were no treatment effects (P < 0.05) on extracuticular calcium (Table 8). Feather Condition Score. While there were no effects of rearing cage on feather condition score (Table 7), there were effects (P < 0.05) of both adult cage space allowance and access to a nest box on feather condition score, especially later in the study. Feather condition score was worse (P = 0.011) in small cages at 34 wk of age, and there was a similar indication (P = 0.091) at 30 wk. Feather condition score was worse (P = 0.037) in cages with access to a nest box at 34 wk of age, and there was also a similar indication (P = 0.10) at 30 wk.

42.5 (6.0) 34.4 (31.9) 6.01 0.27

21.9

32.3 (6.9) 44.6 (49.3) 6.93 0.13

20.4

35.9 (34.4) 41.1 (43.2) 7.55 0.52

22.8

DISCUSSION

17.9

Hen welfare in this experiment was assessed in 2 ways (Broom, 1986; Broom and Johnson, 1993; Barnett and Hemsworth, 2003; Hemsworth et al., 2015; Tilbrook and Ralph, 2017). First, by measuring the normality of biological functioning, on the basis of both the magnitude of the behavioral and physiological responses that animals utilize to assist them in coping with challenges and the consequent significant biological costs, leading to growth, reproductive, health, and

18.8 5.21 0.59

20.3 5.07 0.98

3.03 0.16

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size and presence of a nest box are calculated on 1, 6 degrees of freedom. 1 Y maze preferences were angularly transformed, prior to statistical analysis, after the scores were scaled between 0 and 1. Back transformed means presented in parentheses.

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BEHAVIOR AND WELFARE OF CAGED LAYING HENS Table 7. Main effect of treatments on body weight and feather condition score. Rearing (R) Variable

Adulthood (A)

Small

Large

SED

P value

Body weight (kg) 16 wk 1.53 18 wk 1.66 19 wk 1.76 21 wk 1.87 22 wk 1.88 26 wk 1.95 30 wk 2.02 34 wk 2.05

1.52 1.69 1.79 1.86 1.89 1.97 2.01 2.07

0.011 0.013 0.006 0.013 0.016 0.024 0.028 0.048

0.85 0.069 0.015 0.27 0.42 0.42 0.92 0.67

1.26

0.21

1.87

0.69

0.71

0.61

Feather condition score1 26 wk 84.7 (23.8) 30 wk 79.5 (23.4) 34 wk 77.5 (23.2)

82.7 (23.7) 78.7 (23.3) 77.0 (23.0)

Nest box (NB)

Small

Large

SED

P value

1.53 1.68 1.79 1.87 1.89 1.96 2.01 2.06

1.52 1.66 1.76 1.86 1.88 1.96 2.02 2.07

0.011 0.013 0.007 0.008 0.010 0.014 0.019 0.028

0.42 0.16 0.013 0.23 0.38 0.89 0.73 0.63

84.2 (23.8) 78.1 (23.2) 74.6 (22.7)

83.2 (23.7) 80.2 (23.5) 79.9 (23.4)

0.90

0.29

1.03

0.091

1.46

0.011

No

Yes

1.52 1.68 1.78 1.86 1.89 1.95 2.00 2.05

1.53 1.67 1.76 1.86 1.88 1.97 2.03 2.07

84.5 (23.8) 80.3 (23.5) 78.0 (23.2)

82.9 (23.7) 77.9 (23.2) 76.5 (23.0)

SED

P value

0.016 0.015 0.011 0.015 0.017 0.018 0.019 0.020

0.53 0.84 0.15 0.87 0.46 0.31 0.29 0.25

1.18

0.25

1.23

0.10

0.54

0.037

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size 1, 6 degrees of freedom, and those for presence of a nest box are 1, 6 degrees of freedom for body weight measurements and 1, 5 degrees of freedom for feather condition score measurements. 1 Feather condition scores were angularly transformed prior to statistical analysis. Back transformed means presented in parentheses.

Table 8. Main effect of treatments on egg weight, average hen day production, and extracuticular calcium. Rearing (R) Variable

Small

Egg weight (g) 26 wk 58.3 27 wk 59.5 28 wk 60.1 29 wk 60.7 Hen day production (%) 95.8 Extracuticular calcium 26 wk 2.3 27 wk 2.1 28 wk 2.1 29 wk 2.0

Adulthood (A)

Nest box (NB)

Large

SED

P value

Small

Large

SED

P value

No

Yes

SED

P value

58.7 59.3 60.0 59.8

0.54 0.32 0.45 0.20

0.44 0.66 0.86 0.028

58.3 59.2 59.8 59.6

58.7 59.5 60.2 60.9

0.31 0.32 0.41 0.97

0.18 0.38 0.37 0.24

58.8 59.9 60.2 60.4

58.2 58.8 59.8 60.1

0.51 0.66 0.47 0.61

0.25 0.15 0.42 0.60

95.0

1.23

0.58

95.0

95.7

0.61

0.30

95.7

95.1

0.80

0.50

2.3 2.2 2.1 2.0

0.13 0.14 0.11 0.11

0.93 0.42 0.87 0.79

2.1 2.2 2.0 2.0

2.4 2.1 2.1 2.0

0.17 0.13 0.17 0.14

0.21 0.82 0.55 0.93

2.3 2.2 2.1 2.0

2.2 2.1 2.1 1.9

0.23 0.11 0.09 0.10

0.85 0.69 0.40 0.57

P values for rearing cage size are calculated on 1, 3 degrees of freedom, while those for adult cage size and presence of a nest box are calculated on 1, 6 degrees of freedom.

other impairments. Second, measuring what resources the animal chooses and are thus presumably perceived to be important (i.e., animal preferences). Overall, the physiological and production measurements were not affected by the space and nest box access treatments; however, there were treatment effects on behavioral time budgets and choice behavior of laying hens. There were no effects of space allowance during rearing, space allowance during adulthood, or access to a nest box during adulthood on corticosterone concentrations in plasma, feces, and egg albumen or egg yolk, or on corticosterone response to an ACTH challenge at 26 to 29 wk of age. There are conflicting reports regarding stress (corticosterone concentrations) in hens kept in increasingly greater spaces above 542–697 cm2 /hen (see review Widowski et al., 2016). While there is considerable evidence of stress arising from spatial restriction on other species, there is also evidence of animals adapting over time to spatial restriction in mice, poultry, and pigs (Craig, 1982; Craig et al., 1988; Peng et al., 1989;

Faure, 1991; Hemsworth et al., 2013, 2016). Therefore, measurements on both the behavioral and physiological responses early in the adult period of the current experiment would have been valuable in examining temporal spatial treatment effects. There is little evidence in the literature that a lack of a nest box results in either an acute or a chronic stress response (Nicol, 2015). In an experiment with hens in cages with and without nest boxes, Cronin et al. (2008) found that hens in cages with a nest box had 33% higher plasma corticosterone concentrations than hens without nest boxes early in lay at 23 wk of age and suggested that the elevated stress response in cages with nest boxes was likely associated with social factors, such as competition for the nest box. However, there were no longer-term effects of treatment on corticosterone concentrations. Furthermore, when hens that were accustomed to a nest box were denied access to the nest box at 39 wk, egg albumen corticosterone concentrations were not different from controls during the

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first 2 days, were significantly higher on day 3 but returned to control concentrations by day 7 (Cronin et al., 2008). Alm et al. (2016) found that while denying hens access to nests increased their interest toward the nests near the time of oviposition, there were no differences between hens deprived of their nest and control hens on fecal corticosterone metabolites levels, corticosterone levels in yolk and plasma, irregularities of eggshells, and H:L ratios. In an experiment examining effects of perches, dust baths, and nest boxes, either alone or in combination with group size and floor space, Barnett et al. (2009) concluded that any effects of nest boxes on the HPA axis and immune function were smaller than effects of group size and space allowance. The results of the present experiment suggest that when a nest box is present, hens generally use them well; however, there was no evidence based on corticosterone concentrations and H:L ratios that hens that were not provided with nest boxes experienced increased stress. Nicol (2015) suggests that although behavioral evidence indicates that hens may be frustrated in the absence of a nest access, this experience may be severe but short-lived; most of the physiological indicators of stress such as the non-invasive measures depend on stress levels being elevated for some time. The HPA axis response has also been measured noninvasively in the eggs (Rubolini et al., 2005; Royo et al., 2008) and excreta (Ludders et al., 2001; Dehnhard et al., 2003) of laying hens. Non-invasive sampling represents a different period than the time-point samples gathered during blood collection; however, if they are to be used to estimate effects on plasma corticosterone concentrations, these measures need to be validated (Tilbrook and Ralph, 2017). Nevertheless, the lack of measureable treatment differences in egg yolk and albumen and fecal corticosterone concentrations as well as corticosterone concentrations in plasma and corticosterone response to an ACTH challenge at 26 to 29 wk of age in the present experiment indicate no longterm effects of the treatments on corticosterone concentrations during the period sampled. Long-term exposure to a stressful event has been shown to result in an increase in the H:L ratios in chickens. This has been exhibited through long-term addition of corticosterone to the diet (Gross, 1980) and water for broilers (Post et al., 2003b). OnbasIlar and Aksoy (2005) reported higher H:L ratios in hens housed 5 to a cage at 393.8 cm2 /hen than 1 or 3 to a cage at 1,968 and 656 cm2 /hen, respectively. There were no treatment effects on H:L ratios in the present experiment. and several studies have also found no effects of spatial restriction on H:L ratios. Patterson and Siegel (1998) reported that there were no density-dependent differences in H:L ratios when pullets were reared at densities varying from 20 to 38 pullets per cage (185.8 down to 97.8 cm2 /bird). Mench et al. (1986) reported no differences in the H:L ratios in hens housed 1 or 2 to a cage at 1394 cm2 /hen or housed 2 to a cage at 697 cm2 /hen. Similarly, Barnett et al. (2009) found no differences in white cell counts

and the differential white cell counts (ratio of granulocytes to lymphocytes plus monocytes) in hens housed in groups of 16 with 750 cm2 /hen or groups of 8 or 16 with 1,500 cm2 /hen. While floor space is confounded by group size in some of these studies, there were no effects of space on the H:L ratios in the 3 studies in which there was no confounding (Mench et al. 1986; Barnett et al., 2009; the present study). There were numerous effects of space allowance during adulthood on the time budgets of behavior of hens at 26 wk of age in the present experiment. Housing adult hens with a floor space allowance of 542 cm2 /hen rather than 1,650 cm2 /hen resulted in marked changes in the frequency of many behaviors such as locomotion, preening, and feed pecking. Less floor space and thus, increased opportunity for physical contact with other birds clearly may restrict locomotion, as well as comfort behaviors such as preening, and investigation such as pecking cage features. With a reduction in locomotion, an increase in sitting and resting are expected. Hens with less floor space in adulthood were observed to more frequently peck at feed. Displacement behavior is often defined as occurring when there is motivational conflict or frustration, and is a normal behavior that is exhibited in a different and apparently inappropriate situation (Taylor, 2010). For example, excessive feeding (Meijsser and Hughes, 1989) and preening (Duncan and Wood-Gush, 1972) have been suggested as displacement behaviors exhibited in the absence of a suitable nest site. However, there is no evidence from the current experiment that reduced floor space in adulthood was associated with increased stress. The increase in sham dust bathing observed in the absence of a nest box during adulthood is also difficult to explain and, considering its level of significance (P = 0.044), may just be a chance effect. Dust bathing occurs more frequently at midday (Hogan, 2008), so it is unclear how the absence of access to a nest box would lead to an increase in sham dust bathing unless there was social facilitation of the behavior when hens were in the nest box during the mid-day period (Duncan et al., 1998). Keeling (1994) studied the effects of decreasing floor space allowances of 5,630, 3,000, 1,200, and 600 cm2 /hen on groups of 4 hens kept on litter and found that as space decreased, frequency of walking and ground pecking decreased, frequency of standing increased, but the frequency of preening remained unchanged. While aviary systems vary markedly from cage systems, Hansen (1994) examined the time budgets of hens in aviaries (2,953 cm2 of ground area/hen) and conventional cages (720 cm2 of ground area/hen). The author found that hens in cages spent less time lying, walking, and object pecking, and more time standing/sitting, food pecking, and drinking from 25 to 26, 40 to 41, and 60 to 61 wk of age. No differences were found between the 2 housing systems in comfort behaviors such as preening, scratching, stretching, and beak cleaning. Birds in cages performed less wing flapping and fleeing but there was no difference between the 2

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BEHAVIOR AND WELFARE OF CAGED LAYING HENS

housing systems in the frequency of feather pecking. Reduced floor space in the present experiment also reduced frequency of mobility and pecking at inedible objects and increased frequency of sitting and food pecking. While many of the effects of reduced space were similar, in contrast to the observations of Keeling (1994) and Hansen (1994), hens in the present experiment with less space spent more time drinking and less time preening but did not display increased cohort pecking, which included feather and body pecking. Buijs et al. (2010) reported decreased preening in broilers as stocking density increased. They suggested that preening may be interrupted when there is less space or more birds to disrupt the behavior. Generally, preening can be described as a comfort or maintenance behavior (Tinbergen, 1952; Hansen, 1994; Duncan, 1998; Cheng and Jefferson, 2008). It may be performed as a displacement behavior when there is motivational conflict or frustration since Duncan and Wood-Gush (1972) found that preening in hens increased when they were thwarted from accessing feed. Keeling (1994) considered that preening is a resilient behavior, one that will still be performed when the cost is high, since there was no decrease in frequency as the pen size decreased. A behavior that many authors have proposed that is important to hen welfare is nesting in an enclosed nest site away from the feeding area. Using preference and behavioral demand tests, research has shown that most hens prefer and are highly motivated to access an enclosed nest site. Hens have been shown to be willing to squeeze through narrow gaps (Cooper and Appleby, 1997), push open weighted doors (Follensbee et al., 1992), and pass through cages occupied by unfamiliar or dominant hens in order to gain access to a nest box (Freire et al., 1997). In the present experiment, hens that had access to a nest box more often chose a nest box during their expected pre-laying period over food in the Y-maze tests than hens that were denied access to a nest box (22% of tests vs. 1%). It should be recognized that procedural differences in tests of preference and motivation can influence the results and therefore the interpretation of these tests (Kirkden and Pajor, 2006), but the correlation between specific methodologies has rarely been investigated (Browne et al., 2011). When comparing hens’ preferences for 3 environments in pairs of tests using either a discrete choice method (T-maze; a hen’s choice is followed by a short period of confinement with that choice) or a free-access method (hens can freely move between choices), Browne et al. (2011) found that the discrete choice method was more sensitive for detecting a mild aversion to one of the environments and was more consistent in the preference rankings for the 3 environments. In the present study, a discrete choice test was used based on some previous studies (Laine, 2011; Arnold and Hemsworth, 2013), but if and how the results might have differed if we had used a free-access method is unknown. Using the discrete

11

choice methodology, we were able to detect differences among individual hens in preferences for nest boxes. It is possible that one specific type of test methodology may be more or less appropriate for a given type of stimulus (e.g., space vs. nest box). Another consideration in interpreting these results is that the handling and novelty of the testing procedure and variability in the predictions of the oviposition time may have underestimated the hens’ motivation to access the nest box in the present Y maze tests. Hens were all handled considerably in the training phases and consequently were reasonably familiar with both the Y maze apparatus and handling. Food is generally considered as the “gold standard” in preference testing (Matthews and Ladewig, 1994), and Lam and Hemsworth (unpublished data) used a similar Y-maze apparatus and observed that the choice of food over peat moss was high (78%) and that level of feed deprivation (0, 2, 3, 4, and 5 h) had no effect on choice of food. Thus, the present findings suggest that hens with experience with nest boxes are at least moderately motivated to choose a nest box over food near the time of oviposition (nest box chosen in 22% of tests). Hens without experience with nest boxes from 16 to 29 wk of age chose a nest box infrequently over food (1% of tests). Furthermore, if there was sustained frustration without a suitable nest site, stereotypies such as pacing and excessive drinking, in addition to elevated glucocorticoids, may occur. There was no evidence of these behavioral and physiological changes in hens without access to a nest box in this experiment. The literature on the effects of space allowance in layer cages suggests that, in general, as floor space decreases, within a range of 650 to 300 cm2 per caged laying hen, mortality increases and egg production, body weight, and efficiency of feed utilization decrease (Hill, 1977; Hughes, 1983; Adams and Craig, 1985; Sohail et al., 2004). It is generally accepted that stress impairs growth and reproduction (Moberg, 2000), although a proportion of animals appear to be resistant to the effects of chronic stress or sustained, increased corticosteroids (Turner et al., 2005). In the present experiment, there was little or no treatment effect on body weight or egg production. Similar to the results reported here, Widowski et al. (2017) found no effects of housing hens at 520 vs. 748 cm2 /hen in large (enriched colony) furnished cages on any measures of egg production, egg quality, or mortality, although feather condition deteriorated faster in the hens at the lower space allowance. Thus, the space allowances in these studies may have been above the threshold for causing a chronic stress response. There is a host of purported stressors that have been shown to cause delays in the expected time of oviposition with subsequent effects on egg shell quality (Mills et al., 1987; Reynard and Savory, 1997, 1999). Delayed oviposition is generally due to retention of the egg in the shell gland (uterus) which is caused by the release of adrenaline. A change in egg shell color or quality,

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therefore, can be used as an indirect measure of delayed oviposition because additional time in the uterus after cuticle deposition on the egg can result in the deposition of extracuticular calcium. In the present experiment there were no effects of space allowance during rearing, space allowance during adulthood, or access to a nest box on extracuticular calcium. This supports the observations by Yue and Duncan (2003) who also found no difference in extracuticular calcium on shells from hens in cages with or without nest boxes, or from hens blocked from using their usual nest box, again suggesting that hens with reduced space in adulthood or no nest box access in the present experiment did not experience additional stress. There were no effects of space during rearing on feather condition score, but both space during adulthood and access to a nest box affected feather condition. It should be recognized that feather condition scores in all treatments were on average in the good to very good category but nevertheless, feather condition score was worse with a reduction in floor space at 34 wk of age. Feather condition score was also decreased in cages with access to a nest box at 34 wk of age. It should be noted that feather condition is likely to decrease over time. While feather pecking was low at 16 wk and there were no effects of space during rearing or adulthood or access to a nest box on cohort pecking at 26 wk of age, the reduction in feather condition score with reduced space may simply be a result of increased abrasion with the cage fittings (Hughes and Black, 1976; OnbasIlar and Aksoy, 2005). Similarly, the deterioration in feather condition score due to access to a nest box may be due to abrasion with the nest box, particularly during periods of high use. In the present experiment birds had 200 cm2 /bird of nest space and this is less than the optimal nest space of 300 cm2 /hen suggested by Appleby (2004). The latter recommendation was based on a theoretical model of nest area requirements but is similar or greater than the nest space allowance in many studies with furnished cages. For example, Wall et al. (2002) and Wall (2011) reported increased use of the nest box (>95%) when birds were provided 150 cm2 /bird of nest space in furnished cages which were similar to the models used in our study with 8 or 10 hens per cage. Hens that had access to a nest box in the present experiment laid 82.8% of eggs in the nest box. Responses to stressors are an integral part of an animal’s ability to cope with a variety of environmental challenges (Moberg, 2000; Barnett, 2003). For many stressors, the first and at times the most economically and effective biological response is a change in behavior. Thus, behavioral responses to spatial restriction in the birds in the present experiment may have been sufficient to allow adaptation, without the need to summon longer-term physiological responses and consequent fitness costs. As discussed earlier, there are some limited examples in the literature of animals adapting to spatial restriction. There is also some evidence that the rearing environment can affect an animal’s motivation for space

later in life (Faure, 1991; Nicol, 1986). In the present experiment, space during rearing had little or no effect on corticosterone concentrations, extra-cuticular calcium, or H:L ratios, and preference for additional space in adulthood, however there was considerable behavioral restriction. In relation to the effects of nest boxes, a suitable nest site is a resource that hens are highly motivated to access in preference and behavior demand tests (Follensbee et al., 1992; Cooper and Appleby, 1997; Freire et al., 1997). Nevertheless, in the present experiment, there was no evidence based on corticosterone concentrations, extra-cuticular calcium, or H:L ratios that depriving hens of access to a nest box resulted in a prolonged stress response. During the discrete choice test, hens with experience with nest boxes were at least moderately motivated to choose a nest box over food near the time of oviposition whereas hens with no previous experience with nest boxes rarely chose one over food. This suggests a low preference to access a nest box, perhaps because using a nest box may be a behavior learned through experience and may require the opportunity to perform searching or investigation of the nest before it is chosen. This is in contrast to Cooper and Appleby (1995) who reported no effect of previous experience with nest boxes on the hens’ willingness to access a pen with a nest or their nesting behavior. Overall, the results of this experiment suggest that while reduced floor space, particularly during adulthood, imposed considerable behavioral restrictions, these effects were not sufficient to elicit a sustained HPA axis response with adverse consequences on body weight and egg production. There are some limited examples in the literature of animals adapting to spatial restriction. Animals are equipped with a number of physiological and behavioral responses that are utilized in a coordinated fashion to assist them in their attempts to cope with challenges. Indeed, behavioral responses are often highly sensitive and can be seen in response to less severe challenges that may not provoke physiological responses (Coe et al., 2000). In relation to nest boxes, motivation for nesting has been studied extensively and a number of studies using preference and behavioral demand tests consistently show that most hens prefer and are highly motivated to access an enclosed nest site. While individual and strain differences in nesting motivation and nest box use are not well understood, there is no evidence from the present experiment that na¨ıve hens experience chronic stress, with adverse consequences on body weight and egg production, or are highly motivated to access a nest box when deprived of a nest box. Attitudes toward animal welfare have moved beyond whether the animal is suffering and there is an emerging shift in community values toward not merely minimizing suffering, but also enhancing positive affective experiences in animals (Mellor, 2012; Hemsworth et al., 2015). These developments, in turn, lead to questions such as the need to provide commercial laying

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hens with both increased space to allow more behavioral freedom and nest boxes for the opportunity to perform the motivated behavior of laying their eggs in a discrete enclosed nest box, both of which presumably provide hens with positive affective experiences.

ACKNOWLEDGMENTS This project was supported by the Australian Egg Corporation, Ltd. (AECL). The authors would like to acknowledge the technical support of Judy Nash, Tracie Storey, Samantha Borg, and Bruce Schirmer. Advice and support from Philip Szepe are gratefully acknowledged.

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