The effect of perch access during pullet rearing and egg laying on physiological measures of stress in White Leghorns at 71 weeks of age

The effect of perch access during pullet rearing and egg laying on physiological measures of stress in White Leghorns at 71 weeks of age F. F. Yan,* P. Y. Hester,* and H. W. Cheng†1 *Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; and †USDA-ARS, Livestock Behavior Research Unit, West Lafayette, IN 47907 life cycle. Treatment 2 chickens had access to perches only from 17 to 71 wk of age (laying phase). Treatment 3 chickens had access to perches only from hatch to 16.9 wk of age (pullet phase). Treatment 4 chickens always had access to perches during their life cycle. At 71 wk of age, chickens were sampled for measurement of plasma catecholamines (epinephrine, norepinephrine, and dopamine) and corticosterone; blood serotonin and Trp; fluctuating asymmetry of shank length and width; and adrenal weight. Only shank width differed among treatments. Chickens with previous exposure to perches during the pullet phase had wider shanks than chickens without access to perches (P = 0.006), suggesting that early perching promoted skeletal development. These results suggest that a stress response was not elicited in 71-wk-old White Leghorn hens that always had access to perches compared with hens that never had access to perches during all or part of their life cycle.

Key words: perch, catecholamine, serotonin, corticosterone, White Leghorn 2014 Poultry Science 93:1318–1326 http://dx.doi.org/10.3382/ps.2013-03572

INTRODUCTION Perching is an important natural behavior of domestic chickens derived from their ancestor, the jungle fowl, which sought out perches to provide protection from ground predators (Thorsten et al., 2010). Perches are used extensively by domesticated chickens to escape from dominant conspecifics during the daytime (Braastad, 1990; Cordiner and Savory, 2001). Chickens have a greater sense of security when they rest on roosts at elevated levels, even in the absence of nonhuman predators (Keeling, 1997), and they exhibit a strong preference for the highest perch in various indoor housing environments (Newberry et al., 2001; Struelens et al., 2008). Chickens display signs of frustration if access to a perch is denied (Olsson and Keeling, 2000). Dawkins ©2014 Poultry Science Association Inc. Received August 20, 2013. Accepted February 11, 2014. 1 Corresponding author: [email protected]

(1990) suggested that domestic animals are motivated to exhibit similar behaviors in artificial environments as they would display under natural conditions. In a companion study, Enneking et al. (2012b) reported that caged White Leghorn pullets given access to perches at hatch displayed perching behavior as early as 2 wk of age, although the incidence was rare at this age. Perching frequency increased as the pullets aged, reaching a peak at 12 wk of age with 31 to 37% of the pullets using 1 of the 2 perches at night (Enneking et al., 2012b). Other studies have reported that more than 90% of egg laying chickens in flocks roost on perches at night (Appleby, 1998; Olsson and Keeling, 2000). The use of conventional cages, which are the main housing system for laying hens worldwide, have been criticized for their negative effect on hen welfare over the past several decades (Lay et al., 2011; Silversides et al., 2012). Actions have been taken globally to increase hen welfare by allowing them to exhibit certain natural behaviors through providing furnishings such as perches. In Switzerland, conventional cage production

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ABSTRACT Egg laying strains of chickens have a strong motivation to perch. Providing caged chickens with perches allows them to perform their natural perching behavior and also improves their musculoskeletal health due to exercise. Little is known about the effect of perch access for hens on physiological measures of stress. Our hypothesis was that denying chickens access to perches would elicit a stress response. The objective of this study was to determine the effect of perch access during all or part of life cycle on physiological homeostasis in caged 71-wk-old White Leghorn hens. A total of 1,064 chicks were assigned randomly to cages with and without perches (n = 14 pullet cages/perch treatment) on day of hatch. As pullets aged, chicks were removed from cages to provide more space. At 17 wk of age, 324 chickens in total were assigned to laying cages consisting of 4 treatments with 9 replicates per treatment. Treatment 1 chickens never had access to perches during their

CAGED WHITE LEGHORNS WITH PERCH ACCESS

MATERIALS AND METHODS Chickens and Management One-day-old White Leghorn female chicks of the HyLine W36 strain (n = 1,064) were transported from a commercial hatchery to the Purdue University Poultry Research Farm. The pullets were reared using stan-

dard management and vaccination practices under the guidelines approved by the Purdue University Animal Care and Use Committee (no. 10–082). Infrared beak trimming was performed at the hatchery. Chicks were assigned randomly to 28 pullet cages, half of which were equipped with and without perches, respectively. For the perch group, 2 smooth metal round perches, 32 mm in diameter, were arranged parallel to the feeder and were installed inside the cage at 8.9 cm height from the cage floor. The metal cage dimensions, perch placement, number of chickens per cage, as well as floor, perch, and feeder space per chicken during the pullet phase were described by Enneking et al. (2012a). As pullets aged, chickens were removed from pullet cages for tissue collection and to also allow for more floor, feeder, and drinker space (Enneking et al., 2012a; Yan et al., 2013). At 17 wk of age, 324 pullets were transferred to 36 laying cages with 9 chickens per cage, providing 439 cm2 of floor space per chicken. The cages were divided into 4 treatments. Treatment 1 chickens never had access to perches during their life cycle; treatment 2 chickens had access to perches only from 17 to 71 wk of age (laying phase); treatment 3 chickens had access to perches only from hatch to 16.9 wk of age (pullet phase); and treatment 4 chickens always had access to perches during their entire life cycle (hatch to 71wk of age). Out of the total of 36 laying cages, 8 of the laying cages (2 cages per treatment) received pullets from more than 1 pullet cage. The remaining 28 laying cages housed pullets from the same pullet cage. Perches were similar to the one used in the pullet cages except they were longer. Perch placement, cage dimensions, perch space per chicken, and feeder space per chicken were described by Hester et al. (2013). Chickens were housed in 1 room using temperature and ventilation regimens similar to the egg industry. Evaporative cooling was used during hot weather. A prelay diet (CP = 18.40%, Ca = 2.50%, and nonphytate P = 0.35%) was fed the first 5 d after pullets were transferred to laying cages followed by a laying hen diet (CP = 18.3%, Ca = 4.20%, and nonphytate P = 0.30%) until the end of the study. Each laying cage contained 2 nipple drinkers. Feed and water were provided for ad libitum consumption. Detailed information of additional management conditions used for the chickens was described by Hester et al. (2013). A total of 8 hens, all from control laying cages (1 dead hen/ cage), died between 17 and 71 wk of age, with 2 deaths caused by cannibalism (Hester et al., 2013).

Blood Sampling Two hens per cage were removed from each cage for blood sampling. To avoid oviposition-related peaks in circulating levels of stress hormones (Beuving and Vonder, 1978), hens were bled beginning 9 h before the end of photophase. All hens used for blood collection did not have an egg in the uterus as determined through palpation of the uterus. Sampling was done

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systems have been banned since 1992, and only the alternative systems, such as furnished cages with perches, are allowed (Van Horne and Achterbosch, 2008). According to marketing standards used in Scotland (Scottish Statutory Instruments, 2005), noncage housing systems must be “fitted with perches providing at least 15 cm of perch space for each hen.” In the United States, the United Egg Producers and the Humane Society of the United States have issued an agreement requesting egg producers to convert hen production systems from conventional cages to furnished cages equipped with nest, scratch pad, nail trimmers, and perches over a 16-yr period (Greene and Cowan, 2012). Most of the research on the effects of perch access in hens has focused on their behavior, productivity, and skeletal health (Tactacan et al., 2009; Donaldson and O’Connell, 2012; Enneking et al., 2012b; Hester et al., 2013; Tuyttens et al., 2013). Little is known about the influence of perch usage on the stress response of hens. In a parallel study, Yan et al. (2013) reported that pullets up to 12 wk of age without access to perches did not show a difference in neuroendocrine homeostasis compared with pullets raised with perches. The objective of this study was to investigate the effect of perch access during all or part of the life cycle on the physiological measures of stress of caged 71-wk-old White Leghorn hens. To reach our goal, several stress-related hormones, such as glucocorticoids [corticosterone (CORT)], catecholamines [epinephrine (EP), norepinephrine (NE), and dopamine (DA)], and serotonin (5-HT) were measured (Pohle and Cheng, 2009; Dennis and Cheng, 2011; Cronin et al., 2012). In addition, physical parameters, including adrenal weight and fluctuating asymmetry (FA), were also determined. Stress exposure commonly increases adrenal weight (Siegel, 1959). Asymmetry is used as a measure of instability as a result of stress (Møller et al., 1995, 1999; Bjórklund and Merilá, 1997; Møller and Swaddle, 1997). Animals unable to cope with stress will deviate from perfect symmetry (Kimball et al., 1997; Møller and Swaddle, 1997) resulting in abnormal locomotion and slightly abnormal posture (Cloutier and Newberry, 2002). Deviations are measured by calculating the difference between the size (e.g., length or width) of the structure on the left versus the right side of the animal. We hypothesized that 71-wk-old hens with access to perches during all or part of their life cycle would experience less stress compared with controls that never had access to perches.

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on 2 different days when hens were 71 wk of age with 1 hen per cage sampled on each day for a total of 72 hens. The brachial vein of the chicken was injected with 30 mg of sodium pentobarbital/kg of BW to sedate hens before blood collection. A 5-mL blood sample was collected from each chicken via cardiac puncture and was placed into an EDTA-coated tube and iced. All chickens, including those that were sampled for blood, were weighed individually and euthanized by cervical dislocation. A 500-µL aliquot of whole blood was retained for the analysis of 5-HT and Trp. Plasma was collected by centrifuging the remaining blood at 700 × g for 15 min at 4°C for analysis of CORT, EP, NE, and DA. Plasma and whole blood were frozen at −80°C until assayed.

HPLC Assay

RIA The concentrations of plasma corticosterone were measured in duplicate by RIA using a commercial 125I CORT RIA kit (MP Biomedicals, Orangeburg, NY) as previously described by Cheng et al. (2001).

Adrenal Weights The right adrenal gland was dissected from 8 to 9 hens per cage. The right adrenal gland was cleaned of connective tissue, and weighed.

The length and width of the left and right shanks of 2 chickens per cage were measured in duplicate using a digital caliper to the nearest 0.01 mm (Dennis et al., 2008). The width was recorded at the metatarsal spur. The measurements of the right and left shank of each chicken were averaged to determine mean length and width. The asymmetry was determined as the absolute difference between the left and right legs [i.e., |left length (or width) − right length (or width)|]. Relative asymmetry was calculated for shank length and width as symmetry divided by mean shank length or width (Møller et al., 1995; Cloutier and Newberry, 2002). Asymmetries are categorized by the distribution of the data (left-right) and whether the mean was different from zero. Specifically, bilateral asymmetry can be divided into 3 categories: 1) fluctuating asymmetry: normal distribution and mean zero; 2) directional asymmetry: normal distribution and mean different from zero; and 3) anti-symmetry: nonnormal distribution and mean zero (Yang et al., 1997; Cloutier and Newberry, 2002).

Statistical Analysis The asymmetry data were examined to determine fluctuating, directional, or anti-symmetry by using the Shapiro-Wilks and Kolmogorov-Smirnov tests to evaluate normality followed by the t-test to determine if the mean differed from zero using the UNIVARIATE procedure of the SAS Institute (2008) as described previously (Cloutier and Newberry, 2002, Table 3). Data were analyzed using an ANOVA (Steel et al., 1997). The MIXED model procedure of the SAS Institute (2008) was used in the data analysis. A 2 × 2 factorial arrangement was used in which the 2 fixed factors were the presence or absence of perches within the pullet or laying cages. The interaction of the 2 fixed factors was included in the statistical model. The covariate of day of sampling was used for analyzing all stress-related hormones except CORT, and BW was used as a covariate for right adrenal weight and shank length. Both day of sampling and BW were used as covariates for shank width analysis. Transformation of data was performed for normality when variances were not homogeneous (Steel et al., 1997). Statistical trends were similar for both transformed and untransformed data; therefore, the untransformed least squares means and SEM are presented. Significance was set at a P < 0.05 for all statistical analyses.

RESULTS There were no interactions between the presence or absence of perches during the pullet or laying phases for any parameter measured in the study (Tables 1 and 2).

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A commercial catecholamine analysis kit (Thermo Scientific, Sunnyvale, CA) was used for detecting plasma EP, NE, and DA following a previously described protocol (Cheng et al., 2001). Perchloric acid was added to plasma to acidify and deproteinize each sample followed by centrifugation. The acid supernatants and dihydroxybenzylamine (the internal standard) were added and absorbed onto an alumina mini-column to bind the catecholamine. Washing solutions provided by the company were used to rinse the columns. Eluents were added to the reverse-phase columns. Coulochem II electrochemical detectors were used to detect catecholamines by liquid chromatography. Concentrations of EP, NE, and DA of each sample were calculated by a reference curve developed using a serial diluted standard (Sigma-Aldrich Corporation, St. Louis, MO). The EP:NE ratio was calculated. To measure blood 5-HT and Trp levels, whole blood was acidified with a fresh preparation of 3% ascorbic acid followed by deproteination with 4 M perchloric acid. After centrifugation, the supernatants were filtered through a syringe filter (0.22 μm). The final samples were injected onto the column and analyzed using HPLC. The concentrations of 5-HT and Trp were calculated from a reference curve that was developed by using standards provided by the company (SigmaAldrich Corporation).

Asymmetry of Shanks

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CAGED WHITE LEGHORNS WITH PERCH ACCESS Table 1. The effect of perch availability on physiological measures of stress in 71-wk-old caged White Leghorn hens1 Treatment

DA2 (ng/mL)

EP2 (ng/mL)

NE2 (ng/mL)

EP:NE

5-HT2 (μg/mL)

Trp2 (μg/mL)

Right adrenal weight3 (mg)

  6.97 5.42 0.90 26 0.23   5.85 6.54 0.90 26 0.59   5.93 8.01 5.77 5.08 1.27 13 0.29

  1.04 0.74 0.21 35 0.33   0.82 0.95 0.21 35 0.67   1.03 1.04 0.61 0.87 0.30 18 0.67

  3.20 4.80 0.98 35 0.25   3.73 4.27 0.98 35 0.69   3.14 3.25 4.31 5.29 1.38 18 0.75

  1.55 1.71 0.41 35 0.78   1.58 1.68 0.41 35 0.86   1.47 1.64 1.70 1.72 0.57 18 0.90

  2.67 2.86 0.23 35 0.54   2.75 2.78 0.23 35 0.94   2.76 2.57 2.74 2.98 0.32 18 0.51

  15.05 15.45 0.56 26 0.62   14.66 15.85 0.56 26 0.13   15.21 14.90 14.10 16.79 0.78 13 0.06

  10.04 10.42 0.26 26 0.31   10.38 10.08 0.26 26 0.42   10.11 9.96 10.64 10.19 0.37 13 0.68

  66.37 66.08 1.00 147 0.84   66.20 65.25 1.00 147 0.97   65.94 66.80 66.46 65.70 1.39 74 0.55

1CORT:

corticosterone; DA: dopamine; EP: epinephrine; NE: norepinephrine; 5-HT: serotonin. squares means were adjusted using day of sampling as a covariate. 3Least squares means were adjusted using BW as a covariate. 4Average number of observations per least squares means during the pullet or laying phases or pullet-laying interaction. 5Control-control chickens never had access to perches during their life cycle; control-perch chickens had access to perches only during the egg laying phase of the life cycle (17 to 71 wk of age); perch-control chickens had access to perches only during the pullet phase (0 to 16.9 wk of age); and perchperch chickens always had access to perches (0 to 71 wk of age). 2Least

Table 2. The effect of perch availability on asymmetry, and relative asymmetry of shank length and width of 71-wk-old caged White Leghorn hens Shank length

Item Pullet phase  Control  Perch  SEM  n3   P-value Laying phase  Control  Perch  SEM  n3   P-value Interaction (pullet-laying)4  Control-control  Control-perch  Perch-control  Perch-perch  SEM  n3   P-value a,bLeast

Shank width

Length1 (mm)

Asymmetry (mm)

Relative asymmetry (mm/mm)

Width2 (mm)

Asymmetry (mm)

Relative asymmetry (mm/mm)

  83.75 83.45 0.36 36 0.56   83.43 83.77 0.36 36 0.52   83.54 83.96 83.33 83.58 0.50 18 0.87

  0.45 0.56 0.07 36 0.27   0.48 0.53 0.07 36 0.58   0.38 0.52 0.57 0.54 0.10 18 0.38

  0.0054 0.0067 0.0008 36 0.28   0.0057 0.0064 0.0008 36 0.59   0.0046 0.0062 0.0069 0.0065 0.0012 18 0.39

  8.87b 8.98a 0.07 36 0.006   8.85 8.83 0.07 36 0.77   8.71 8.69 9.00 8.96 0.09 18 0.90

  0.18 0.26 0.03 36 0.06   0.25 0.19 0.03 36 0.20   0.19 0.17 0.31 0.21 0.04 18 0.37

  0.020 0.029 0.004 36 0.09   0.028 0.021 0.004 36 0.19   0.022 0.019 0.035 0.023 0.005 18 0.37

squares means within a column within the pullet phase lacking a common superscript differ (P < 0.05). squares means were adjusted using BW as a covariate. 2Least squares means were adjusted using BW and day of sampling as covariates. 3Average number of observations per least squares means during the pullet or laying phases or pullet-laying interaction. 4Control-control chickens never had access to perches during their life cycle; control-perch chickens had access to perches only during the egg laying phase of the life cycle (17 to 71 wk of age); perch-control chickens had access to perches only during the pullet phase (0 to 16.9 wk of age); and perchperch chickens always had access to perches (0 to 71 wk of age). 1Least

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Pullet phase  Control  Perch  SEM  n4   P-value Laying phase  Control  Perch  SEM  n4   P-value Interaction (pullet-laying)5  Control-control  Control-perch  Perch-control  Perch-perch  SEM  n4   P-value

CORT (ng/mL)

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Table 3. Summary statistics of the shank asymmetry measures and Pearson correlations between character size and signed asymmetry for shank length and width1 Item Shank length Shank width 1P-values 2Average

Skewness

Kurtosis

t-test

Shapiro-Wilks

Kolmogorow-Smirnov

Pearson correlation

n2

−0.68 −0.18

0.95 0.22

0.68 (0.50) −1.78 (0.08)

0.97 (0.05) 0.98 (0.21)

0.09 (0.15) 0.08 (0.15)

−0.12 (0.33) −0.22 (0.07)

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for the statistical tests are in parentheses. number of observations per least squares means during the pullet or laying phases.

DISCUSSION In contrast to our prediction, hens at 71 wk of age with perch availability during all or part of the life cycle experienced similar stress responses compared with controls that never had access to perches. The hormone levels of hens in the current study were within normal range similar to levels previously reported in our laboratories (Hester et al., 1996; Cheng et al., 2002, 2003; Cheng and Fahey, 2009; Felver-Gant et al., 2012) further indicating that hens were not stressed and were experiencing homeostasis. The possibility exists that pullets that had perch availability during rearing when placed in laying cages without perches could have experienced short-term stress, but by the time samples were collected at 71 wk of age, the hens had adapted. We did not collect blood samples or tissues immediately after placement of pullets in laying cages at 17 wk of age because we were collecting data on perching, feeding, and drinking behaviors (unpublished data), egg production, feed efficiency, and welfare parameters (Hester et al., 2013) and did not want to affect these outcomes. The results of the current study are in agreement with Barnett et al. (1997) who reported that heterophil to lymphocyte ratios, levels of circulating corticosterone (either at rest or in response to adrenocorticotropic hormone), and the cell-mediated immune response were similar between caged hens with and without wooden perches. In addition to hens experiencing homeostasis, perches in cages provide other welfare benefits such as improved bone strength, foot pad, toe, and nail health, and quality of the feather tracts on the back of the chicken. Egg production and other performance traits

were similar between hens with and without perches, although perches in cages can lead to more dirty and cracked eggs (see review of Hester, 2014). Epinephrine and NE are stress hormones that have important functions in regulating an animal’s emotions and ability to cope with both acute and chronic stress. Acute stress involves the fright, fight, or flight response where nerve impulses from the hypothalamus stimulate the adrenal medulla to release EP and NE. These neurohormones, when released into circulation, increase blood pressure, respiration rate, muscle tone, nerve sensitivity, and metabolism. Blood glucose levels increase as a result of glycogen breakdown (Siegel, 1995). Chronic stress also involves EP and NE. Chronic repeated stress in rats causes less turnover of brain catecholamine, leading to an increase in concentrations of hypothalamic EP and NE (Roth et al., 1982). Evidence suggests that both EP and NE stimulate adrenocorticotropic hormone release, leading to an increase in plasma corticosterone in rats subjected to chronic intermittent stress (Katz et al., 1981). Laying hens subjected to feed reduction (Fujita and Yamamoto, 1996) or genetically selected for antisocial behavior and aggression (Cheng et al., 2001), and rats experiencing cold stress (Bernuci et al., 2013) all demonstrated elevated plasma EP levels and EP:NE ratio. Therefore, EP and NE, as well as the EP:NE ratio, have been validated as chronic stress indicators in evaluating animal welfare (Cheng and Muir, 2005; Hara et al., 2013). Our results showing that circulating EP and NE as well as the EP:NE ratio in 71-wk-old hens were not affected by perch access during all or part of their life cycle were similar to Pohle and Cheng (2009) who reported no difference in plasma EP and NE levels between chickens housed in conventional compared with furnished cages equipped with perches, nest, claw trimmer, and sand bath from 19 to 60 wk of age. Increasing levels of DA are related to aggression in multiple species including chickens (Cheng et al., 2003), mice (Nikulina and Kapralova, 1992), and rats (Ferrari et al., 2003). Increased levels of brain or plasma DA have been reported in mice subjected to repeated aggressions (Barik et al., 2013), hens under strain intermingled social stress (Cheng et al., 2002), and female broiler breeders with food restriction (Kostál et al., 1999). The absence of perches in cages did not affect the circulating level of DA in 71-wk-old White Leghorns of the current study as levels were similar to hens with perches. Although Cordiner and Savory (2001) reported that the presence of perches in floor

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Circulating levels of CORT, DA, EP, NE, 5-HT, and Trp as well as the EP:NE ratios of 71-wk-old hens were not affected by prior availability to perches as pullets or by perch access during the laying phase. Right adrenal weights were also not affected by the perch treatments (Table 1). Among the measured leg parameters, only shank width was affected during the pullet phase. Previous exposure to perches as pullets resulted in 71-wk-old hens with wider shanks compared with hens without perches during the pullet phase (P = 0.006, Table 2). The shank width was similar among chickens with and without access to perches during the laying phase (P = 0.77, Table 2). Shank width showed fluctuating asymmetry, and shank length showed anti-symmetry (Table 3).

CAGED WHITE LEGHORNS WITH PERCH ACCESS

by many factors such as the serotonin transporter (Kim et al., 2005) and CYP2D protein (Haduch et al., 2013). Elevated plasma CORT levels (Cockrem, 2007) and adrenal weights (Selye, 1937; Siegel, 1959) have been used as stress indicators in poultry. Plasma CORT levels and adrenal weights were unaffected by the perch treatment, providing further evidence that stress level in chickens was not affected by perch access during all or part of the life cycle. Similar results on plasma CORT concentrations have been reported in chickens kept in different housing systems (Pavlik et al., 2008; Tactacan et al., 2009). Barnett et al. (2009) reported that perch access as compared with no perch had no effect on plasma and egg CORT concentrations as well as the plasma CORT response to exogenous adrenocorticotropic hormone in caged Hy-Line brown chickens. Fluctuating asymmetry has been used to evaluate the stability of animals when coping with environmental (Parsons, 1990; Dennis et al., 2008) and social stressors (Cloutier and Newberry, 2002). Increased FA values are indicators of poor welfare because of higher degrees of asymmetry and occur in animals under stress (Leung and Forbes, 1996). Our results, like most other studies, showed that perches did not affect FA of the shank. As examples, the FA of shank length and width were similar among 44-d-old broilers (Bizeray et al., 2002) and 36-wk-old Black Menorca breed hens (Campo and Prieto, 2009) reared in pens with and without perches. However, with floor-reared broilers, Ventura et al. (2010) reported that FA and relative FA of the tibia length were lower with complex barrier perches, but not simple barrier perches, compared with controls, indicating that broilers with complex barriers had tibia with better symmetry suggesting improved welfare. The inconsistency between the study of Ventura et al. (2010) and other results, including our own, could be due to multiple factors such as the type of bone being evaluated for symmetry (shank vs. tibia), genetic and phenotypic background, age of the bird, previous stress experience, and housing conditions. The wider shanks of 71-wk-old hens with access to perches during the pullet phase compared with those hens without pullet perch access may be related to a higher level of activity as a result of mounting and dismounting the perch early in life when the skeleton undergoes rapid developmental growth. In a parallel study, access to perches as pullets had no effect on the width or length of the tibia, humerus, ulna, and radius up to 12 wk of age (Enneking et al., 2012a). Pullet shank length and width were not measured in this companion study (Enneking et al., 2012a). However, access to perches as pullets increased bone surface area when sampled at 3, 6, and 12 wk of age. In addition, the early perching activity of caged pullets increased musculoskeletal development, leading to greater leg muscle deposition and a larger skeletal frame at 12 wk of age as indicated by increased bone surface area and mineral content of the tibia, sternum, and humerus compared with pullets without perches (Enneking et al., 2012a).

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pens reduced the incidence of aggressive pecks in laying hens, aggression was not noted as a problem in the beak-trimmed flock of the current study (P. Y. Hester, unpublished data). Mortality due to cannibalism was low at 0.6% (2 of 324 hens) from 17 to 71 wk of age (Hester et al., 2013). Another function of DA is to regulate reproduction in animals (Dufour et al., 2010), including egg production in chickens (Xu et al., 2010). In a genetic selection study, higher peripheral DA concentrations were found in chickens with lower productivity (Cheng and Muir, 2005). In the brain, DA promotes prolactin secretion in the hypothalamus via D(1) DA receptors (Youngren et al., 2002), which plays a critical role in the onset and maintenance of incubation behavior in birds, causing them to stop laying eggs (March et al., 1994). In addition, it is well known that stress adversely affects hen productivity (Edwards, 2011; Miftakhutdinov et al., 2012). The current finding that DA concentration was not affected by the perch treatment is in agreement with hen productivity as reported in a parallel study in which egg production, egg weight, and egg quality (cracked eggs, shell weight, and shell thickness) did not differ between chickens that had access to perches throughout their life cycle compared with chickens that never had access to perches (Hester et al., 2013). The current results and previous findings on DA further indicate that the physiological homeostasis of chickens at 71 wk of age was not affected by the absence or presence of perches in cages. As a neurotransmitter in the brain, 5-HT regulates numerous pathophysiological processes including appetite, mood, behavior, cognition, and contributes to feeling of well-being and happiness (Shively and Bethea, 2004; Airan et al., 2007; Canli and Lesch, 2007; Young, 2007). Whole-blood 5-HT concentrations were positively related to good feelings or mood (Williams et al., 2006) and negatively correlated with variations in self-depreciation, depressive mood, and anger (Verkes et al., 1998). Stimulation induced changes in peripheral 5-HT concentrations in laying hens have shown increased (Rodenburg et al., 2009), decreased (Cheng et al., 2001), and unchanged responses (Cheng and Fahey, 2009; Uitdehaag et al., 2011; Felver-Gant et al., 2012). These inconsistent findings could be affected by multiple factors such as genetic background, the age of the laying hen, previous stress experience, housing conditions, and type of stressors used, as well as duration and frequency of the stressor. In the current study, blood concentrations of 5-HT and its precursor Trp were not affected by perch access in chickens. However, the correlation between peripheral 5-HT and brain 5-HT concentrations in laying chickens requires further study. Blood 5-HT levels may not reflect the changes of 5-HT and Trp levels in the brain because 5-HT cannot cross the blood-brain barrier (Mann et al., 1992). Brain and blood 5-HT are derived from different sources, brain serotonergic neurons and intestinal enterochromaffin cells, respectively, and are regulated

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ACKNOWLEDGMENTS Support of this project was from Agriculture and Food Research Initiative competitive grant no. 201167021-30114 from the USDA National Institute of Food and Agriculture. Perches were donated by T. L. Pollard of Big Dutchman (Holland, MI), and the chicks were provided by Hy-Line Hatchery (Warren, IN). Appreciation is extended to F. A. Haan and B. D. Little from the Poultry Research Farm at Purdue University (West Lafayette, IN) for the management and care of the hens. Also, gratitude is extended to G. L. Nowling, R. J. Lockridge, R. L. Dennis, S. Jiang, J. Y. Hu, and G. M. Morello of the USDA Livestock Behavior Research (West Lafayette, IN) for technical assistance. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement of the USDA. The USDA is an equal opportunity provider and employer.

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These perching-induced developmental changes in the musculoskeletal system may contribute to the wider shanks found at the late age of 71 wk. Similar to the current results, broilers reared with barriers had wider shanks than those without barriers perhaps due to increased exercise (Bizeray et al., 2002). In summary, similar to results from pullets (Yan et al., 2013), there were no effects of perch availability on the concentrations of Trp and stress related hormones in the blood (CORT, DA, EP, NE, 5-HT), right adrenal weights, and shank FA in 71-wk-old hens with prior experience to perches as pullets or with access to perches during the laying phase. Hens at 71 wk of age with previous exposure to perches during the pullet phase had wider shanks than chickens without access to perches, suggesting that early perching promoted skeletal development. These results imply that 71-wk-old White Leghorn hens that never had access to perches showed no evidence of eliciting a stress response at the end of lay.

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