Use of dynamic and rewarding environmental enrichment to alleviate feather pecking in non-cage laying hens

Applied Animal Behaviour Science 161 (2014) 75–85

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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim

Use of dynamic and rewarding environmental enrichment to alleviate feather pecking in non-cage laying hens Courtney L. Daigle a , T. Bas Rodenburg b , J. Elizabeth Bolhuis c , Janice C. Swanson a , Janice M. Siegford a,∗ a b c

Animal Behavior and Welfare Group, Department of Animal Science, Michigan State University, East Lansing, MI, USA Behavioural Ecology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands Adaptation Physiology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands

a r t i c l e

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Article history: Accepted 8 October 2014 Available online 18 October 2014 Keywords: Feather pecking Laying hen Environmental enrichment

a b s t r a c t Feather pecking (FP) can cause feather loss, resulting in physical injuries, which may lead to cannibalism. FP appears to be a redirection of foraging behavior, which intensifies when hens have difficulty coping with stress and fear. Dynamic environmental enrichment (EE) may allow expression of natural foraging behavior thus reducing conspecific pecking behavior and alleviating hen injury. Three treatments (plastic box: BOX; hay bale: HAY; and no enrichment: CON) were randomly applied to 30 identical floor pens (10 hens/pen; 10 pens/trt). At the pen level, hen behavior, and the number of severe FP (SFP), gentle FP (GFP), aggressive pecks (AP), and enrichment pecks (EP) were recorded from video prior to (21 wk) and after (24 wk) treatment implementation, and when hens were 27, 32, and 37 wk of age. A manual restraint test (MR) was performed immediately after behavioral observations and levels of blood serotonin (5-HT) and glucocorticoids (GC) measured. Short-term (ST) and long-term (LT) analyses identified the impact of EE over the ST (21 vs. 24 wk of age) and LT (21 vs. all other ages) at the pen level. At the pen level, HAY (3.18 ± 0.33) tended to reduce GFP compared to CON (4.10 ± 0.34) over the ST (P = 0.15) and LT (P = 0.09), but did not impact the number of SFP, or AP over the ST or LT. More EP was observed in HAY (3.56 ± 0.14) than BOX (1.61 ± 0.18) throughout the study (P < 0.0001). More HAY hens perched (P = 0.05) at 24 wk (0.28 ± 0.12) compared to 21 wk (0.19 ± 0.11), and more HAY hens (3.69 ± 0.25) performed dust bathing compared to CON (4.14 ± 0.22, P = 0.05) throughout the study. CON performed more struggles (1.13 ± 0.04, P = 0.04) and were quicker to vocalize (4.87 ± 0.07 s, P = 0.05) during MR than HAY (latency to vocalize(s): 5.16 ± 0.05; number of struggles: 0.96 ± 0.05), counter-intuitively suggesting CON were less fearful. Treatment did not affect 5-HT or GC. HAY appears to be a promising EE for mitigating GFP in non-cage laying hens. Future studies should examine the impact of EE on individual, rather than group-level responses. These results suggest that the presence of a hay bale is stimulating and may reduce GFP while encouraging hens to redirect pecking towards a dynamic and manipulable EE. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ∗ Correspondence to: 474 S. Shaw Lane, Anthony Hall, Room 1290, Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA. Tel.: +1 517 432 1388; fax: +1 517 355 1688. E-mail address: [email protected] (J.M. Siegford). http://dx.doi.org/10.1016/j.applanim.2014.10.001 0168-1591/© 2014 Elsevier B.V. All rights reserved.

Feather pecking (FP) is a common and serious problem for laying hens that can be influenced by multiple factors (e.g., genetics, environment, and rearing experiences). Nicol et al. (2013) indicates that FP can develop as early

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as one day of age, and gentle feather pecking (GFP) and severe feather pecking (SFP) have been observed in up to 94% and 65% of flocks, respectively, at 35 wk. Hens with feather damage have less insulation, and reduced plumage cover has been linked to poor food conversion ratios as bald chickens may need up to 40% more feed to maintain their body temperature (Blokhuis et al., 2007). Since research efforts and genetic selection take time to address problematic FP from an ultimate, internally-driven state, there is a need to identify proximal, effective management practices to alleviate the damage caused by FP in the interim. Feather pecking appears to be redirected foraging behavior that may be intensified by fear and stress (Rodenburg et al., 2013), and FP possesses characteristics similar to obsessive-compulsive disorders seen in humans and mice (van Zeeland et al., 2009). Different genetic lines of hens have different propensities for developing FP and express different levels of fearfulness and whole blood serotonin (5-HT). Hens selected for divergent levels of mortality due to injurious pecking and tendency to develop FP (Bolhuis et al., 2009; Uitdehaag et al., 2011), have different peripheral levels of 5-HT and behave differently during manual restraint tests (MR). These observations suggest that these differences in FP behavior are mirrored by differences in physiological responses. Environmental enrichment (EE) has been widely utilized by zoos to provide stimulation to animals unable to fulfill inherent drives due to environmental limitations (Shepherdson et al., 1998). Furthermore, considerable effort has been made to recreate the animal’s natural habitat in captive settings that provides a pleasurable aesthetic for visitors while complementing the animal’s natural history. This same approach should be made for agricultural animals where environments are not only efficient, but are designed around the animal’s biology, including EE (Newberry, 1995; Swaisgood and Shepherdson, 2005). Furthermore, EE should continue to stimulate the performance of natural behaviors over time as animals can quickly become habituated to their presence (Tarou and Bashaw, 2007). For laying hens in caged environments, EE has been employed to reduce fear and trauma during depopulation of caged hens (Reed et al., 1993), decrease aggressive pecking (Gvaryahu et al., 1994), and increase feeder use (Sherwin, 1995). Even though non-cage hens are provided with more opportunities, they face different challenges than their caged counterparts, and subsequently will need different EE. Successful EE must be both beneficial for the animal and practical for the producer. String has shown to be effective in reducing feather pecking in pen housed hens (Jones et al., 2002). However, these devices require manufacturing and installation by the producer, which could impact profit margins, and is impractical to implement on a commercial scale. Litter material is an important resource for hens, and litter availability is an important component of basic hen husbandry. Commercial rearing flocks that experienced a lapse in litter availability exhibited an increase in FP and a change 5-HT levels (de Haas et al., 2014) illustrating that hens responded strongly and negatively to litter removal. Non-cage hens housed with access to cut straw

or a polystyrene block were observed to perform fewer FP than hens housed with polystyrene pellets or chopped straw (Huber-Eicher and Wechsler, 1998). This highlights the importance of providing an interactive environment in which hens can engage pecking. Such EE would allow them to perform behaviors they are strongly motivated to perform, without harming conspecifics. Many EE devices have been passive, meaning the bird was responsible for making the EE move or change, and these devices did not change the physical configuration of the room, These EE have included suet holders filled with peanut butter, seeds or cabbage (Dixon et al., 2010), wooden beads, and chrome chain (Jones et al., 2000), or plastic rings with spinning objects hung from the top of the cage (Bell and Adams, 1998). These EE have been unsuccessful in reducing FP, and in one case, unintentionally stimulated the development of FP (Lindberg and Nicol, 1994). Environmental enrichment can also change the spatial configuration of the room. Changing the hen’s space can impact hen perception, alter how they use the space, and may influence the social dynamics. Chickens will use a larger proportion of the pen when provided with vertical barriers (Cornetto and Estevez, 2001), and hens are more likely to use and perform comfort behaviors in areas with cover (Newberry and Shackleton, 1997). Hens are evolved to perch in the branches of a bush, and increasing vertical space by providing a hay bale could stimulate a sense of comfort for the hens similar to what they would seek in the wild. Our objective was to identify whether the a dynamic and rewarding (meaning the hen received a physical reward for her pecking efforts – in this case a piece of hay) EE (HAY) would reduce conspecific-directed pecking and exhibit a reduces stress response during manual restraint test via corticosterone, 5-HT, and behavior, compared to a plastic box (BOX, similar in size to HAY but static and nonrewarding) or to a negative control (CON). Specifically, we hypothesized that HAY hens would have reduced SFP and GFP, lower stress-induced corticosterone levels, higher 5HT levels, and would be less fearful during MR than BOX or CON hens. Furthermore, we anticipated that HAY hens would have better feather cover scores than BOX or CON hens. We anticipated that BOX and HAY would have a positive short-term effect on behavior, but only HAY would be long-lasting. 2. Materials and methods All procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (AUF 04/12-068-00). 2.1. Animals and housing Thirty identical pens (1.5 × 2.7 m) were constructed at the Michigan State University Poultry Teaching and Research Center. Pens were separated by floor to ceiling wire mesh, and temperature was regulated with forced heating and fan ventilation. Each pen was furnished with a commercial tube feeder, a water line containing three

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nipples, two nest boxes, and two wooden perches providing 1.5 m of available perch space. The floor was covered with 3 cm of litter (wood shavings). Litter depth was monitored weekly, and excess litter was removed when the depth surpassed 3 cm. Hens were exposed to incandescent lighting for 13.5 h/day (05:30 h–19:00 h) which measured 21.1 ± 0.9 lux at hen level. Each pen housed 10 White Shaver infrared beaktrimmed laying hens which were placed at 16 wk. At 18 wk, the back of each hen was marked with livestock marker in different color combinations to facilitate individual recognition on video recordings. Livestock marker (LA-CO® Industries, Inc., Elk Grove Village, IL, USA) was reapplied prior to each video recording session. After test completion, hens that were not tested were gently handled and had livestock marker reapplied, as hens are less likely to identify a social target for aggression if all hens are marked equally (Dennis et al., 2008), and is particularly important for hens housed in small groups (Marin et al., 2014). Furthermore, each hen was fitted with a plastic leg band to ensure that they were individually identifiable for the duration of the study, including when blood was collected and feathers were scored. 2.2. Treatments When the hens were 22 wk, three treatments (10 pens/trt) were randomly applied to the 30 pens. The three treatments were as follows. (1) HAY—one 5 kg hay bale consisting of approximately three flakes of hay held together with twine and measuring approximately 0.38 m long × 0.38 m wide × 0.38 m tall. (2) BOX—a clear plastic box (Rubbermaid® Roughneck® Clear 17.9 L, Rubbermaid, High Point, NC, USA) measuring 0.42 m long × 0.27 m wide × 0.27 m tall, filled with loose hay that was visible to hens through the clear sides and bottom of the box and a cinderblock to ensure the hens did not turn the box over. The box was placed upside down in the litter of each BOX pen so the lid was not accessible to the hens. (3) CON–a negative control where no treatment was applied to the pen. Hay bales were checked bi-weekly to ensure they remained intact. As needed, loose hay was removed from a pen and replaced with a new bale. Partially destroyed bales were assessed, and replaced as necessary on a caseby-case basis. Each HAY pen had the hay bale replaced a minimum of three times throughout the duration of the study. At 24 wk, a gap was found in the wire separating two adjoining HAY and BOX pens, and the hens were observed moving between the two pens. Therefore, both pens were removed from the study. 2.3. Manual restraint test and blood sampling At 21, 24, 27, 32, and 37 wk, 120 hens were randomly selected, balanced across treatments and pen location within the barn, and subjected to a MR. Birds were tested in random order and were not taken from the same or neighboring pens in consecutive tests. The sampled hens selected were balanced across treatments and trials so that each hen was selected a minimum of two times and a maximum

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of three times throughout the duration of the study. The manual restraint procedure used in this study has been previously described in Uitdehaag et al. (2008). Briefly, a bird was taken out of its home pen and placed on its side on a flat surface for 5 min in a quiet room adjacent to the room where its home pen was located. The tester used one hand to loosely restrain the bird’s legs, while the other hand was placed over the upper part of the bird’s body. The hand restraining the legs mainly functioned to prevent the bird from escaping if it struggled or righted itself, whereas the mild pressure from the other hand encouraged the hen to remain recumbent. After any struggle, birds were gently brought back to their original position. Parameters measured during the manual restraint included latency to struggle, latency to vocalize, number of vocalizations, and number of struggles. Each MR was performed by one of two persons on two consecutive days between 9:00 h and 14:00 h. After the MR, each hen was placed individually in a plastic transportation crate located in a hallway adjacent to the testing room. The hen remained in the transportation crate until 15 min had elapsed following removal from her home pen, to allow the corticosterone response to reach its peak (Fraisse and Cockrem, 2006). Then a 2.5 mL blood sample was taken from a brachial vein using a 22-guage needle and 3-mL syringe. Prior to blood collection, the needle was flushed with 0.9% NaCl concentrated with EDTA to prevent clotting in the needle and syringe. A portion of the blood sample (∼1 mL) was separated and stored at −80 ◦ C until 5-HT analysis. The remainder was immediately centrifuged to separate phases for corticosterone analysis. After blood collection, the feather condition of multiple body parts (head, neck, back, rump, underneck, coverts, breast, legs, belly, wing-primary feathers, and tail feathers) was scored on a 0 to 5 scoring system as described in Bilˇcık´ and Keeling (2000). A high score represented poor feather condition/cover while a low score represented good feather condition/cover. After feather scoring, hens were returned to their home pen. Feather scores from all body parts were summed to provide a whole body score. 2.4. 5-HT analysis Most 5-HT in avian blood is localized in platelets (Sorimachi et al., 1970), and 5-HT concentrations in whole blood correlate (r = 0.34 − 0.57) with platelet 5-HT concentrations (Uitdehaag et al., 2011). Therefore we analyzed whole blood samples following a previously validated protocol (Bolhuis et al., 2009). Briefly, 5-HT levels in whole blood (1 mL) were determined by a fluorescence assay. Whole blood was pipetted into 50 mL centrifuge tubes to which was added 2 mL of 0.9% NaCl solution, 1 mL of an ascorbic acid solution (3% in deionized water saturated with KCl and EDTA) and 5 mL of a phosphate buffer (2 M K2 HPO4, saturated with KCl, and adjusted to pH 10 with KOH), followed by 20 mL of n-butanol. Tubes were shaken thoroughly for 5 min and centrifuged (Allegra X-15R, Beckman Coulter Inc., Indianapolis, IN, USA) at 895g for 15 min. Fifteen mililiters of the butanol layer were transferred to a second tube containing 2 mL of 0.1 M HCl and 25 mL of cyclohexane, and tubes were shaken for 20 s then

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centrifuged for 4 min at 895g. The butanol-cyclohexane layer was removed, and 1 mL of the acidic phase was pipetted in a tube containing 0.3 mL of 12 M HCl that was then vortexed for 3 s. Samples were pipetted in triplicate at a volume of 250 ␮L into a 96-well plate, and fluorescence was determined using a fluorometer (SpectraMax Gemini EM, Molecular Devices, Sunnyvale, CA, USA) set at an excitation of 295 nm and an emission of 540 nm. A standard curve was prepared by taking 0.1, 0.2, 0.3, 0.4, and 0.5 mL of serotonin hydrochloride (Sigma-Aldrich) dissolved in Krebs–Ringer-phosphate buffer (0.2755 ␮mol/mL) then diluted to a volume of 1 mL with 0.9% NaCl solution. Each dilution was subjected to the procedure as described above.

Table 1 Description of hen behaviors recorded from video observation of the home pen. Posture

Description

Stand

Bird is upright and supported off of the ground by legs Bird takes at least three consecutive steps causing the body to move in the same direction while head moves laterally forward and backwards in same direction as body. Bird is upright with body touching the ground

Walk

Sit/lie Behavior Eat Drink

2.5. Corticosterone analysis Forage

Immediately after blood collection, blood samples were centrifuged at 930g for 6 min at 4 ◦ C. Plasma was transferred to a 1.7 mL mini-tube with a transfer pipette and stored at −80 ◦ C until analysis. Hormone measurements were carried out according to manufacturer instructions in triplicate for each sample using a micro plate enzyme-immunoassay (Cayman Chemical, Grand Rapids, Michigan). All samples were diluted to 1:3 with assay dilutant prior to analysis. 2.6. Behavioral observations in the home pen Twenty-four hours prior to each MR (at 21, 24, 27, 32, and 37 wk), ceiling-mounted video cameras (VF-540 Bullet Camera, Clinton Electronics Corp., Loves Park, IL, USA) recorded (30 frames/s) hen behavior during two 30-min periods (7:30–8:00 h and 15:30–16:00 h) during the light period (05:30 h–17:00 h), similar to the recording protocol used by Rodenburg and Koene (2003). Instantaneous scans were performed every 5 min during the same 30-min periods to determine the number of hens feeding, drinking, foraging, resting, dust bathing, perching, and in the nest box (Table 1). These observations were averaged to determine the average number of hens performing each behavior during each 30-min observation period. Subsequently, the number of hens performing each behavior was divided by the total number of hens in the pen to identify the proportion of the pen performing each behavior. Furthermore, the number of pecks given to the enrichment (EP; BOX, and HAY only), the number of aggressive pecks (AP), and the number of severe and gentle feather pecks (SFP and GFP) were recorded from the video (Table 2).

Preening

Dust bathing

Rest

Perching

Table 2 Description of pecking behaviors observed in the home pen. Pecking behavior

Description

Gentle feather pecking (GFP)

Hen uses beak to gently peck at feathers of conspecific. This pecking is normally ignored by the recipient and usually does not result in the removal of a feather. Usually occurs in bouts where the hens will GFP several times in a single bout. Normally directed at the back or tail, but may be directed at the head. Count total number of pecks. Hen uses beak to forcefully peck at victim. Victim will usually respond to pecking by moving away or retaliating. May result in removal of a feather. Usually occurs as a single event, but may happen twice in a row. Will not occur in bouts. Usually directed towards the back, rump, or tail, but may be directed at the head. Count total number of pecks. Occurs when one hen raises her head and forcefully stabs beak either once or multiple times at another hen. Aggressive pecks will usually be directed at the head, but may also be directed at the body. The recipient will usually show avoidance behavior by ducking or moving away from aggressive bird. May be associated with a chase, standoff, or leap. Count total number of pecks. Hen uses beak to peck at top or sides of hay bale or plastic box (HAY and BOX rooms only). Count total number of pecks.

Severe feather pecking (SFP)

2.7. Statistical analysis All analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, South Carolina, USA). Data were first averaged per week and per pen, with the pen as the experimental unit. Each response parameter was tested for normality and heterogeneity of variance prior to analysis. Proportions of behavioral observations from scan samples were arc sin square root transformed, with the exception of foraging and dust bathing which were log-transformed; GC concentration, the number of AP, SFP, and latency to struggle were log transformed; and GFP, EP, number of

Bird has head in feeder and is using beak to consume grain Head is turned upwards towards water source and bird uses beak to consume water from nipple drinker Hen pecks at substrate while standing or stepping forward with head below rump level. Starts when hen makes >3 successive pecks at substrate, or when foraging hen has not been standing or walking with head up, or feeding, for the previous 5 s. Bird may be sitting or standing and uses beak to manipulate, rearrange, pull, or clean body feathers on self Bird is lying on ground while moving head and body vigorously while causing substrate to rise into ruffled feathers. May be accompanied by ground pecking. Starts when hen lies down (sternum resting on substrate) from an upright position or when lying bird has made no dust bathing or preening movements for the previous 8 s Hen is sitting or standing on either the perch attached to the wall, the perch in front of the nest box or on the water line

Aggressive peck (AP)

Enrichment peck (EP)

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vocalizations, number of struggles, latency to vocalize, and feather scores were square root transformed prior to analysis. All values presented in the text and figures are of transformed means and transformed standard errors of the mean. Significance was determined as P < 0.05. Least squared means with a Tukey–Kramer adjustment were used to identify differences among treatments and ages as appropriate. The proportion of the pen performing each behavior (Table 1) was analyzed using a separate Linear Mixed Model (PROC MIXED). The model included the fixed effects of treatment, age, and the interaction of treatment and age. Age was treated as a repeated factor, and pen nested within treatment was treated as a random factor. With the pen as the experimental unit, Linear Mixed Models (PROC MIXED) identified whether pecking behavior in the home pen (AP, GFP, and SFP), behavior during the MR (latency to vocalize, latency to struggle, number of vocalizations, and number of struggles), feather scores, 5-HT, and corticosterone levels differed among the three treatments. At the pen level, the number of enrichment pecks (EP) was analyzed for BOX and HAY only using a similar model. The short-term analysis used data from 21 wk and 24 wk only, while the long-term analysis included data from all time points. 3. Results 3.1. Behavior in the home pen When behavioral observations from before (21 wk) and after (24 wk) the start of the treatments (which were applied at 22 wk) were analyzed to understand the short-term effect of the treatments on behavior, an interaction between age and treatment was observed for the proportion of the pen performing perching (Fig. 1a; F2, 24 = 3.68, P = 0.04). A larger proportion of the pens in the HAY treatment perched (transformed mean ± transformed S.E.M.; 21 wk: 0.19 ± 0.04, 24 wk: 0.28 ± 0.04) after the presence of the hay bale (t24 = −3.02, P = 0.05) while the proportion of hens perching in BOX (21 wk: 0.15 ± 0.03, 24 wk: 0.18 ± 0.05) and CON (21 wk: 0.20 ± 0.04, 24 wk: 0.17 ± 0.03) remained stable. An interaction between age and treatment was observed for preening (Fig. 1b; F2, 24 = 3.75, P = 0.04) where both CON (t24 = 4.01, P < 0.01) and BOX (t24 = 3.88, P < 0.01) exhibited a decrease in the proportion of hens performing preening behavior after (24 wk) treatment implementation (BOX: 0.27 ± 0.02, CON: 0.30 ± 0.02) compared to before (21 wk) treatment (BOX: 0.36 ± 0.02, CON: 0.40 ± 0.02), while the proportion of hens observed preening in HAY remained stable (21 wk: 0.32 ± 0.01, 24 wk: 0.31 ± 0.02). A larger proportion of the pen was observed foraging at 24 wk (−1.48 ± 0.11) compared to 21 wk (−2.32 ± 0.20; F1, 24 = 20.86, P = 0.0001) but no differences were observed among the treatments (F2, 25 = 0.36, P = 0.70). No short-term effects on other behaviors were found. Over the long term, treatment influenced the proportion of hens dust bathing (Fig. 1c; F2, 23 = 3.26, P = 0.05) across all time points. A larger proportion of hens were observed dust bathing in the HAY (−3.69 ± 0.25) compared to CON

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(−4.14 ± 0.22, t23 = −2.43, P = 0.05) with intermediate levels of dust bathing in BOX hens (−3.86 ± 0.21). No differences were observed among the three treatments for the proportion of hens observed walking (F2, 25 = 1.43, P = 0.26), sitting (F2, 25 = 1.14, P = 0.34), standing (F2, 25 = 0.28, P = 0.76), eating (F2, 25 = 0.69, P = 0.51), drinking (F2, 25 = 0.05, P = 0.96), foraging (F2, 25 = 0.42, P = 0.66), preening (F2, 25 = 0.96, P = 0.40), resting (F2, 25 = 1.76, P = 0.19), or perching (F2, 25 = 1.10, P = 0.35) across all ages.

3.2. Pecking behavior 3.2.1. Short-term effects Treatment tended to have an effect on the average number of GFP performed per pen before (21 wk) and after (24 wk) treatment implementation (Fig. 2a; F2, 25 = 2.48, P = 0.10); more GFP tended to be performed in CON pens (4.10 ± 0.34) compared to HAY pens (3.18 ± 0.33; t25 = 1.93, P = 0.15) and BOX pens (3.21 ± 0.34; t25 = −1.94, P = 0.15). No differences were observed among the three treatments for the average number of AP (Fig. 2b; F2, 25 = 0.61, P = 0.55) or SFP (Fig. 2c; F2, 25 = 0.60, P = 0.56) per pen performed. However, age did impact the average number of AP (F1, 24 = 7.05, P = 0.014) and SFP (F1, 24 = 8.65, P = 0.007) per pen performed. A higher pen average of AP were observed at 24 wk (2.15 ± 0.15) compared to 21 wk (1.62 ± 0.13; t24 = −2.65, P = 0.01) and a lower pen average of SFP were observed at 24 wk (4.73 ± 0.16) compared to 21 wk (5.21 ± 0.11; t24 = 2.94, P < 0.01). Although the differences among the three treatments were not statistically significant (AP: F2, 25 = 0.61, P = 0.55; SFP: F2, 25 = 0.60, P = 0.56), numerically, more AP and SFP were performed by CON hens compared to HAY hens at 21 wk and 24 wk.

3.2.2. Long-term effect Throughout the duration of the study, treatment tended to have an effect on the pen average of GFP performed (F2, 25 = 2.66, P = 0.09), where CON pens (4.10 ± 0.20) tended to perform more GFP than HAY pens (3.43 ± 0.21; t25 = 2.16, P = 0.09). The pen average of AP was influenced by age (F4, 94 = 3.78, P = 0.007) where more AP were observed at 24 wk (2.15 ± 0.15) compared to 21 wk (1.62 ± 0.13; t94 = −2.78, P = 0.05) and 32 wk (1.56 ± 0.11; t94 = 3.05, P = 0.02). The pen average of SFP was influenced by age (F4, 95 = 4.91, P = 0.001) where more SFP were performed at 21 wk (5.21 ± 0.11) compared to 24 wk (4.73 ± 0.16; t95 = 2.98, P = 0.03) and 32 wk (4.50 ± 0.15; t95 = 4.08, P < 0.01). Differences were observed between BOX (1.61 ± 0.18) and HAY (3.56 ± 0.14) for the average number of EP per pen performed (Fig. 2d; F1, 17 = 48.86, P < 0.0001). An interaction between treatment and age was also observed for EP (F3 , 47 = 3.34, P = 0.03); at the pen level, more EP were performed in HAY compared to BOX at 24 wk (BOX: 1.82 ± 0.36; HAY: 3.92 ± 0.26; t47 = −4.93, P < 0.01), 32 wk (BOX: 1.24 ± 0.38; HAY: 3.05 ± 0.23; t47 = −4.19, P < 0.01), and 37 wk (BOX: 1.19 ± 0.29; HAY: 3.97 ± 0.10; t47 = −6.41, P < 0.0001).

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Fig. 1. Proportion of the pen performing perching (a) and preening (b) before and after treatment implementation, at 21 wk, and 24 wk respectively. The proportion of the pen performing dust bathing (c) was compared across treatments, irrespective of hen age. Lower case letters are used to indicate differences (P < 0.05) among ages or treatments, respectively.

3.3. Behavior during the manual restraint test 3.3.1. Short-term effect of enrichment No differences were observed among the three treatments for the number of vocalizations (Fig. 3a; F2, 25 = 0.33, P = 0.72) performed during a MR. Age influenced the duration of time that passed before the hen performed its first struggle (F1, 25 = 41.98, P < 0.0001), where, at the pen level, hens took longer to struggle during a MR conducted when the hens were 21 wk (13.52 ± 0.45) compared to the test conducted when the hens were 24 wk (9.05 ± 0.53; t25 = 6.48, P < 0.0001). Treatment weakly tended to have an effect on the number of struggles (Fig. 3b; F2, 25 = 2.30, P = 0.12) and tended to have an effect on the latency to vocalize (Fig. 3c; F2, 25 = 2.65, P = 0.09) during a MR. More struggles tended to be performed during a MR by CON hens (1.15 ± 0.07) than HAY hens (0.89 ± 0.08; t25 = 2.13, P = 0.10), and CON hens (4.62 ± 0.18) tended to be quicker to vocalize than HAY hens (5.01 ± 0.10; t25 = −2.20, P = 0.09). Treatment did not influence the latency to struggle (Fig. 3d; F2, 25 = 0.28, P = 0.76) during a MR. 3.3.2. Long-term effect of enrichment Across the entire study, no differences were observed among the three treatments for the number of vocalizations during a MR (F2, 25 = 1.40, P = 0.26). Irrespective of treatment, age influenced the number of vocalizations performed during a MR (F4, 100 = 3.28, P = 0.01), where more vocalizations were performed at 24 wk (4.76 ± 0.66) compared to 32 wk (3.63 ± 0.68; t100 = 3.16, P = 0.02). Treatment (F2, 25 = 3.40, P = 0.05) and age (F4, 100 = 6.67, P < 0.0001)

affected the latency to vocalize during a MR, with CON hens (4.87 ± 0.07) being quicker to vocalize than HAY hens (5.16 ± 0.05; t25 = −2.56, P = 0.04). Hens were quicker to vocalize during a MR at 21 wk (4.90 ± 0.08) compared to 32 wk (5.15 ± 0.07; t100 = −2.76, P = 0.05) and 37 wk (5.27 ± 0.06; t100 = −4.09, P < 0.01). The number of struggles performed in a MR was impacted by treatment (F2, 25 = 3.69, P = 0.04) and age (F4, 99 = 5.34, P = 0.01). CON hens (1.13 ± 0.04) performed more struggles than HAY (0.96 ± 0.05; t25 = 2.67, P = 0.03) during a MR, and more struggles across all treatments were performed at 27 wk (1.23 ± 0.05) compared to 24 wk (0.98 ± 0.05; t100 = −3.33, P = 0.01), 32 wk (1.00 ± 0.05; t100 = 2.97, P = 0.03) and 37 wk (0.91 ± 0.05; t100 = 4.16, P < 0.01). The number of struggles performed at 21 wk did not differ from any of the subsequent ages [21 wk vs. 24 wk (t100 = 0.95, P = 0.88); 21 wk vs. 27 wk (t100 = −2.38, P = 0.13); 21 wk vs. 32 wk (t100 = 0.60, P = 0.98); 21 wk vs. 37 wk (t100 = 1.79, P = 0.39)]. Treatment did not affect latency to struggle (F2, 25 = 1.02, P = 0.38), yet hens took less time to perform their first struggle during a MR performed at 24 wk (9.05 ± 0.53) compared to 21 wk (13.52 ± 0.45; t100 = 7.40, P < 0.0001), 27 wk (12.91 ± 0.35; t100 = −6.40, P < 0.0001), 32 wk (13.69 ± 0.40; t100 = −7.67, P < 0.0001), and 37 wk (13.94 ± 0.38; t100 = −8.16, P < 0.0001). 3.4. Corticosterone and 5-HT 3.4.1. Short-term effect of enrichment When comparing 21 wk and 24 wk, no effect of treatment was observed on corticosterone concentrations

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Fig. 2. Average number (count) per pen of aggressive pecks (a), gentle feather pecks (b), severe feather pecks (c), and enrichment pecks (d) observed during two, 30-min periods (07:30–08:00 and 15:30–16:00) per day throughout the duration of the study. Differences (P < 0.05) are represented by different lowercase letters. Tendencies (0.05 < P < 0.10) are represented by symbols. Effects of treatment are presented in the legend.

(F2, 25 = 1.54, P = 0.23). Age tended to have an effect on the levels of corticosterone (Fig. 4a; F1, 25 = 4.07, P = 0.06), with higher levels measured from blood samples collected at 24 wk (6.82 ± 0.07) compared to those collected when the hens were 21 wk (6.64 ± 0.05; t25 = −2.02, P = 0.06). Neither treatment (F2, 25 = 0.00, P = 0.99), age (F1, 25 = 1.28, P = 0.27), nor the treatment by age interaction (F2, 25 = 0.94, P = 0.41) were significant in the short term for whole blood serotonin levels (Fig. 4b).

3.4.2. Long-term effect of enrichment Across the entire study, age influenced the concentration of whole blood serotonin (F4, 100 = 3.67, P = 0.008). Serotonin concentrations were higher at 32 wk (54.71 ± 1.15) compared to 21 wk (49.10 ± 1.33; t100 = −3.21, P = 0.02) and 27 wk (49.53 ± 1.12; t100 = −2.96, P = 0.03). Neither treatment (F2, 25 = 0.93, P = 0.41) nor the treatment by age interaction (F8, 100 = 0.59, P = 0.78) had an effect on whole blood serotonin levels. Age also influenced the levels of log transformed corticosterone measured in blood plasma (F4, 100 = 6.12, P = 0.0002). Corticosterone levels were lower at 21 wk (6.64 ± 0.05) compared to 27 wk (7.01 ± 0.05; t100 = −4.52, P < 0.01)

and 37 wk (6.95 ± 0.06; t100 = −3.86, P < 0.01). Neither treatment (F2, 25 = 1.01, P = 0.38) nor the treatment by age interaction (F8, 100 = 0.80, P = 0.61) had an effect on blood plasma corticosterone levels. 3.5. Feather score Feather cover and condition decreased (represented by an increase in feather score) as the hens aged (F4, 100 = 236.89, P < 0.0001), and, in general, HAY feather scores remained lower (indicating better condition and coverage) than BOX and CON, although the differences were not significant (F2, 25 = 0.40, P = 0.68). 4. Discussion The aim of this study was to identify the effect of dynamic and rewarding environmental enrichment on conspecific-directed pecking, as well as behavioral and physiological responses to a MR, either in the short or long term at the group level. The propensity to develop feather pecking varies at the individual level, yet the consequences of this individual behavior are felt at the group level. Therefore, strategies must be identified that will be beneficial to

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Fig. 3. Hen behavior averaged at the pen level during a 5-min manual restraint test as represented by the number (count) of vocalizations (a), number (count) of struggles (b), latency (s) to vocalize (c) and latency (s) to struggle (d) at five different ages. Differences (P < 0.05) are represented by different lowercase letters. Tendencies (0.05 < P < 0.10) are represented by symbols. Effects of treatment are presented in the legend.

the group, regardless of an individual’s tendency to perform this unwanted behavior. Group selection for hens that experience low mortality from pecking behaviors and higher egg production has shown to be an effective strategy

for mitigating injurious pecking behavior (Craig and Muir, 1996) and hens from differently selected groups have also shown to exhibit differences in peripheral serotonin levels (Bolhuis et al., 2009). Just as genetic selection against

Fig. 4. Plasma corticosterone (pg/mL) (a) and whole blood serotonin (␮mol/mL) (b) levels measured from hens immediately after a manual restraint test at five different ages. Differences (P < 0.05) are represented by different lowercase letters.

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injurious pecking has been successful, the identification of environmental parameters that benefit the group is equally important. The enrichments provided in this study were designed to impact the group dynamic and influence group behavior. Therefore, these results suggest that the HAY treatment reduced GFP in the home pen in the long and short term, but increased the prevalence of fear-related behavior in a MR, albeit no changes in peripheral serotonin or corticosterone were observed. This suggests that dynamic and rewarding environmental enrichment, such as a hay bale, could reduce GFP in a flock of laying hens, regardless of an individual hen’s propensity to develop injurious pecking behavior. Contrary to our hypothesis, the presence of the HAY did not significantly decrease conspecific-directed AP or SFP, over either the short or long term, although numerically fewer SFP and AP were observed in HAY hens than in CON hens. In spite of this lack of a clear effect of HAY provision on injurious pecking behavior, it should be noted that HAY hens directed more pecks towards the environmental enrichment compared to BOX hens, an effect which persisted throughout the duration of the study. Foraging is a natural behavior that hens are motivated to perform (Browne et al., 2011). The HAY treatment provided an opportunity for hens to engage in more species-specific foraging behavior relative to the BOX treatment, which may have been rewarding. Therefore, this result – not wholly unexpected – supports the theory that providing speciesappropriate environmental enrichment can stimulate the performance of natural behaviors. HAY hens performed higher levels of EP soon after treatment implementation; however, their interest appeared to lessen the longer they were exposed to the stimulus. This suggests that the hens habituated to the presence of the HAY. However, since the HAY hens did not show a reduction in preening behavior and performed more dust bathing compared to the other treatments, HAY may still have had a positive effect on the hens. Therefore, future studies should investigate the effect of periodically removing and re-exposing hens to the environmental enrichment to help it maintain its novelty. The levels of AP and SFP observed in this study were low, which may also explain the lack of a significant treatment effect. However, our observations were comparable to observations from other studies (Huber-Eicher and Wechsler, 1997; Rodenburg and Koene, 2003; Gilani et al., 2013), and with infrequently occurring behaviors, like FP, identifying any statistical differences between groups is challenging. Providing foraging material is an important aspect of laying hen husbandry; however some breeds may be more sensitive than others to its presence or withdrawal. Therefore, consideration must also be made for the strain of hen observed in this study (White Shaver – a white leghorn variety). ISA Brown hens were recently observed to perform more SFP than Dekalb White hens after litter restriction (de Haas et al., 2014), suggesting that strain impacts the hen’s motivation to perform pecking behavior and engage with an environmental stimulus. Therefore, HAY may have been a more effective management strategy for a heavierbodied bird, as these types of hens appear to be more

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responsive to the presence or absence of litter (de Haas et al., 2014), and may be more intrinsically motivated to perform FP. Furthermore, this analysis was performed at the pen level, so even though HAY was not effective in reducing group levels of SFP, specific individuals may have responded favorably to this stimulus, and future studies should seek to elucidate differences at the individual level. Changes in spatial configuration of an environment can influence habitat use by birds in non-cage environments (Cornetto and Estevez, 2001), and the presence of canopy cover for free range hens has been associated with an increase in feather condition and quality (Bright et al., 2011). More dust bathing was performed in HAY compared to CON, which could have been stimulated by either the increase in the amount of dust available for dust bathing or the presence of more vertical structures in the pen. Hens engage in comfort behaviors, dust bathing and preening, with either their eyes closed or obscured by plumage. However, performing such behaviors could make the hen vulnerable to predators or aggressive conspecifics, and for protection, hens may choose to perform these behaviors close to a vertical structure or among conspecifics (Keeling and Duncan, 1991; Newberry and Shackleton, 1997). Therefore, hens may have dust bathed more in the HAY because the hen may have felt safe enough to dust bathe in a perceived protected area and the litter in the HAY treatment contained an increased number of hay particles in the litter, a substrate that hens have been observed to prefer for dust bathing (Sanotra et al., 1995; Gunnarsson et al., 2000). Alternatively, even though BOX and HAY changed the birds’ environment in a similar fashion, the hens interacted more with the HAY than the BOX and this could have stimulated dust bathing behavior. Furthermore, the consistently higher preening levels in HAY may have reflected a positive emotional state (Nicol, 1989). This suggests that not only is changing the spatial environment important for maximizing space use, but, providing structures with which the hens can interact may be beneficial to welfare through promoting the expression of rewarding natural behaviors. Surprisingly, hens in the HAY treatment exhibited behaviors in alignment with an increased fear response when removed from the home pen. HAY hens took longer to vocalize and performed fewer struggles during a MR in the short term and throughout the duration of the study. Conversely, CON hens were quicker to vocalize and performed more struggles than HAY. Typically, birds with a greater fear response take longer to vocalize and longer to struggle and perform fewer vocalizations and fewer struggles during the MR (Bolhuis et al., 2009; Rodenburg et al., 2009; Uitdehaag et al., 2011). However, since these hens were subjected to repeated restraint tests, they may have become habituated to the test. Such habituation has been documented previously in domestic chicks that have been repeatedly handled and exposed to environmental enrichment and then shown a reduced fear response (Jones and Waddington, 1992). Alternatively, corticosterone concentrations remained high throughout the study, suggesting that the MR remained a powerful stressor for the hens even after

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repeated exposure. It is possible that hens may have expressed fear in the form of learned helplessness based upon their previous experiences. Hens exposed to an inescapable electric shock had longer tonic immobility durations and required fewer inductions compared to hens that had the opportunity to either escape the shock or were not shocked, suggesting that hens have the capacity to develop learned helplessness based upon their prior exposure to uncontrollable events (Rodd et al., 1997). However, CON hens may have been less fearful of humans because they did not have the structure (either BOX or HAY) in the middle of their home pen, which may have allowed them to have more visual contact with humans during the daily husbandry routine and they were more habituated to their presence. Environmental enrichment can be beneficial for animal welfare. Animals are motivated to perform natural behaviors, and their environment should provide them with the opportunity to perform a wide repertoire of behaviors. The results from this study suggest that the presence of a hay bale may be an effective long-term strategy for alleviating conspecific-directed GFP in cage free hens. One factor to keep in mind is that pecking behavior and fear responses vary at the individual level and this analysis was conducted at the pen level. This approach was taken because we were interested in how the group of hens would respond to the environmental stimulus, however, future studies should address the impact of the environmental enrichment on the individual response. 5. Conclusion As solutions to mitigate the problems associated with and the development of damaging FP are still under investigation, management strategies that will benefit the entire flock and reduce the damage caused by FP should be explored further. Understanding what environmental stimulations motivate hens to redirect unwanted pecking behavior away from conspecifics is important, and FP should continue to be studied until the problem is resolved. Providing hay as a regular part of hen husbandry may prove to be an effective management strategy that will benefit the individual and the group. In addition, hay bales provide an additional benefit of creating micro-environments that may be perceived as more safe, thus stimulating the performance of comfort behaviors, and ultimately increase welfare. By creating visual barriers at the hen level, we may be able to alter the spatial configuration of hen housing, thus creating more opportunities for hens to escape from individuals performing this undesirable feather pecking behavior, and provide them with environments that are in alignment with their inherent preferences. Acknowledgments We are thankful to the Animal Agricultural Initiative and the Department of Animal Science for the financial support of this project. We are grateful to the Adaptation Physiology Group, the Animal Breeding and Genetics Group and the Wageningen Institute of Animal Sciences (WIAS) of Wageningen University for providing the

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