Litter use by laying hens in a commercial aviary: dust bathing and piling

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1 Litter use by laying hens in a commercial aviary: dust bathing and piling D. L. M. Campbell, M. M. Makagon, J. C. Swanson, and J. M. Siegford,1 Animal Behavior and Welfare Group, Department of Animal Science, Michigan State University, East Lansing, MI 48824; and Department of Animal Sciences, Purdue University, West Lafayette, IN ABSTRACT The laying hen industry, including in the United States, is responding to social concerns about hen welfare by implementing alternative housing systems such as the aviary, to provide more space and resources to large groups of hens. Data detailing the behavior of hens in commercial aviaries is needed to determine hens use of the resources in order to understand their impact on hen welfare. The open litter area of aviaries provides additional space for hens during the day. Litter is also a substrate for dust bathing which is a strongly motivated natural behavior. Hens are often synchronous in their performance of dust bathing, which may lead to overcrowding in the litter area. Additionally, the open litter area can facilitate expression of unusual behavior such as flock piling (defined as the occurrence of densely grouped clusters of hens, resulting from no obvious cause and occurring randomly throughout the day and flock cycle) which may be a welfare concern. Therefore, we conducted observations of hen occupancy of the open litter area and the performance of dust bathing and flock piling across 3 production points (peak lay, mid lay and end of lay) for two flocks of Lohmann White laying hens housed in a commercial aviary. All areas of the open litter area were occupied to the same degree. Hens performed dust bathing throughout the day but showed peak dust bathing activity in the afternoon for Flock 1 (all P < 0.001) and in the late morning for Flock 2 (all P < 0.001). Overall, 174 incidents of piling behavior were observed between the 2 flocks, with piles varying in size, duration, and time of occurrence; however, no smothering was detected. Crowding on the open litter area sometimes occurred during peak periods of synchronous dust bathing and when hens piled. Further research is needed to understand the welfare implications of individual hen use of the open litter area and the causes and welfare implications of hen piling. Key words: behavior, welfare, aviary, laying hen, dust bathing 2016 Poultry Science 95: INTRODUCTION The North American laying hen industry is responding to changing legislation and consumer concerns for hen welfare with growing numbers of producers phasing out conventional cages in favor of alternative housing systems such as the aviary. These indoor tiered systems house large groups of hens together, providing more space and resources designed to meet hen behavioral needs in comparison to conventional cages (reviewed in Cooper and Albentosa, 2003). Though aviaries come in many configurations, all contain perches, nestboxes, and provide access to a litter area in which hens can forage and dust bathe. Observational research in commercial facilities shows hens use these resources (Hansen, 1994; Odén et al., 2002), suggesting this system allows hens to address biologically-driven behavioral needs, potentially improving their welfare (Moesta et al., 2008). However, we do not yet have detailed C 2015 Poultry Science Association Inc. Received January 15, Accepted June 3, Corresponding author: siegford@msu.edu information on hens behavior in the commercial aviaries currently being installed in the United States. Determining hens use of the available space and resources is essential for making recommendations for modifications in system design if required, and to determine optimal practices for long-term sustainable management, ensuring these systems provide the improved welfare as intended. The open litter area is a distinct feature of the aviary alternative system (cf. furnished cages), but we do not know how fully hens occupy the available space or what type of behavior is performed in this resource. Previous observations in percheries show individual hens spend approximately 23% of their time on the open litter (Carmichael et al., 1999; Channing et al., 2001), with more birds using the litter in the afternoon for behavior such as foraging and dust bathing (Channing et al., 2001). We focused on dust bathing in this study as it was easily distinguished from video recordings by multiple observers and has previously been shown to be subject to over crowding though other uses of the open litter, including foraging are also important. Dust bathing is important 164

2 LITTER USE BY AVIARY LAYING HENS 165 for maintaining feather condition (van Liere, 1992) and is a behavioral need of hens (Cooper and Albentosa, 2003; but see Widowski and Duncan, 2000). Hens express strong motivation to access substrates in which to perform dust bathing (Wichman and Keeling, 2008) and show stress when substrate access is removed (Vestergaard et al., 1997). Observations in commercial systems with litter, namely aviaries and percheries, confirm hens use the litter area for dust bathing (Carmichael et al., 1999; Channing et al., 2001; Odén et al., 2002), thus satisfying their dust bathing motivation (Colson et al., 2007). However, the quality of the litter material can influence the amount of dust bathing (Odén et al., 2002). The daily internal behavioral rhythm of hens typically leads to dust bathing in the afternoon every second day (Vestergaard, 1982; but see Carmichael et al., 1999) and the probability of hens dust bathing increases when other hens are observed dust bathing through a process called social facilitation (Duncan et al., 1998 but see Olsson et al., 2002). These factors combined may lead to behavioral synchrony or simultaneous dust bathing behavior in large groups of hens (Odén et al., 2002) which can potentially overcrowd the litter areas or prevent litter access to some individuals (Odén et al., 2002). The provision of open areas and group housing of large numbers of birds can also lead to unusual behavior that might otherwise not be prevalent or problematic in a less complex space housing small numbers of hens, such as the conventional cage. Population pressure within large groups of hens can lead to flock hysteria/panic, typically manifested as hens flying wildly about, running around, or crowding and piling together (Hansen, 1976; Richards et al., 2012; Barrett et al., 2014). Flock panic can cause hen injury and smothering as well as a decrease in egg production, which constitute welfare and economic concerns (Hansen, 1976; Laycock and Ball, 1990; Hegelund et al., 2006; Bright and Johnson, 2011; but see Barrett et al., 2014). To date, there is little information available on the behaviors that lead to flock panic and the relationship of panic to piling and smothering. Over 50% of surveyed free-range producers in the United Kingdom reported smothering at some point in their flocks (Barrett et al., 2014) that was attributed to flock panic, nestbox crowding, or creeping/recurring piling, with piling defined as the random occurrence of densely grouped clusters of hens for no obvious reason (Bright and Johnson, 2011; Barrett et al., 2014). These reports of undesirable flock behavior suggest that panic can lead to piling, but that not all piling results from flock panic and not all panic or piling causes smothering. Thus, further research is warranted to understand the causes and extent of any welfare problems resulting from such piling behavior. The objective of this research was to examine hens spatial use of the open litter area in a commercial laying hen aviary facility in order to better understand the impacts of providing access to litter covered open floor areas on hen behavior and welfare. We report the percentage of the open litter area that was occupied by hens at different times across the day. We also report the proportion of hens on the open litter that were using the floor substrate for dust bathing, a behavior pattern that may improve hen welfare, and whether dust bathing proportions peaked at specific times of day. Finally, we documented all occurrences of piling during recording days - behavior that may negatively impact hen welfare - detailing piling size, duration, possible causes, and smothering outcomes. Housing MATERIALS AND METHODS The commercial aviary housed 49,842 (Flock 1) and 49,677 (Flock 2) Lohmann White laying hens which were placed at 19 weeks and 17 weeks into Flock 1 and Flock 2, respectively, and depopulated at 77 or 78 weeks of age for Flock 1 and Flock 2, respectively. Experimental data were collected over 2 flock cycles: Flock 1 data collection spanned June 2011 to May 2012, and Flock 2 data collection spanned August 2012 to August (For additional details on provision of other resources, see Jones et al., 2014; Zhao et al., 2015). The aviary system contained 6 rows of 3-tiered enclosures with internal perches, water, feed, and nestboxes (Figure 1). There were 2 outer rows of enclosures that each faced a house wall across an open litter area (called single rows), and 2 central pairs of enclosure rows that faced each other across a shared open litter area (called double rows; Figure 2). Each single or double row and associated litter area was divided into 10 sections along its length (each 1,440 cm long) by wire gates. Each single row section was initially populated with 852 laying hens and each double row section with 1,704 hens. The tiered enclosures within each section were further divided into 6 units by internal wire dividers (Figure 2). Hens could enter and exit each unit through an opening locted on the lowest tier of the enclosure, but the dividers prohibited hens from moving directly between enclosures within an aviary section. A perch, which ran the length of the enclosure, was located outside the enclosure opening to facilitate movement of hens between the enclosure and the floor area. The floor area accessible to hens comprised open litter in front of the tiered enclosures (40% of total litter area) and the litter area underneath the enclosures (60% of total litter area Figure 1; for additional details on available space per bird, see Jones et al., 2014; Zhao et al., 2015). The litter area itself did not have dividers and thus the hens could reenter any of the 6 tiered enclosures within a section from the open litter area. Video observations for dust bathing and spatial litter use were conducted at the level of the unit (6 units within each focal section), though no physical dividers were present in the litter area. Piling was observed wherever it occurred within the entire section.

3 166 CAMPBELL ET AL. Figure 1. Representation of the tiered aviary enclosures as seen from the end of a unit showing the outer perch, open litter, underneath litter areas, the inner perches and ledges on each numbered tier and location of the nestbox. Figure 2. A schematic top-down diagram of the aviary house showing the tiered enclosure and open litter area, focal video-recorded sections, unit dividers, and single and double rows. System Management Hens remained confined to the tiered enclosures until they reached peak lay, which in this study was defined as 95% production. Once this criterion was reached for each flock (Flock 1 = 26 wk and Flock 2 = 24 wk), the doors in the lower tier of each unit opened daily after the majority of eggs were laid (at approximately 11:00) and closed again 30 min prior to lights coming on the following day (at approximately 06:00). As might be expected in a commercial facility that was adapting to a new housing system, adjustments to management practices occurred during the observation period of Flock 1 and several issues affected hen distributions and data collection. First, installation of video cameras was not complete prior to opening of the aviary enclosure doors allowing hens to access the open litter, thus peak lay recording for Flock 1 occurred 2 wk after the hens initial exposure to litter. Second, the gates between sections remained open during the peak lay video recording period of Flock 1, allowing birds to travel the length of the row, affecting absolute hen numbers per section. Section gates were closed and hens were redistributed evenly between sections prior to mid lay video recording. Finally, unit doors of aviary enclosures remained open continuously during the mid lay video recording period, giving birds 24 h access to litter. Doors began closing again following the schedule above prior to end lay recording. All management issues were resolved by the observation period of Flock 2, and hen disturbances were minimized with no personnel entry after morning egg collection during all recordings. Litter Assessment We visually scored floor litter coverage and dryness and measured litter depth to document how floor cover might affect litter area use (see Table 1 for scoring descriptions). Assessments were made in open litter

4 LITTER USE BY AVIARY LAYING HENS 167 Table 1. The definitions for scoring litter coverage and litter dryness in the open floor litter area, as well as how depth in each section was assessed. Litter cover Litter dryness Litter depth 0 Less than 25% of area is covered 0 Dry and flaky Measured in cm from floor to surface of litter 1 25 to 49.9% of area is covered 1 Dry and clumped, not easy to move 2 50 to 74.9% of area is covered 2 Foot leaves imprint in litter 3 75 to 99.9% of area is covered 4 100% of area is covered areas under each of the 3 ceiling-mounted cameras in each section immediately prior to peak, mid, and end lay video recording, with the exception of Flock 1 peak lay, where litter dryness and depth were not measured. At all time points, litter dryness was scored as zero, so this parameter was not analyzed further. Video Data Collection Video recordings of the outer perch of the tiered enclosure and of the open litter area were made in 8 of the 40 sections within the house (4 sections in singlerows and 4 in double-rows) using ceiling-mounted highresolution digital video cameras (VF450, Clinton Electronics, Loves Park, IL). Three cameras were installed per section with each camera capturing the outer perch and litter area in front of 2 units. For peak lay of Flock 1 (the first data collection point), cameras were installed in the 4 single-row sections only; thereafter additional cameras were installed to include an additional 4 double-row sections (8 focal sections total). Video data were collected at 3 points in the laying hen production cycle commencing at peak lay (Flock 1: 95.93% production, 27 wk; Flock 2: 96.5% production, 24 wk), mid lay (Flock 1: 93.19% production, 52 wk; Flock 2: 89.33% production, 55 wk) and end lay (Flock 1: 82.86% production, 77 wk; Flock 2: 76.85% production, 76 wk). With the exception of the Flock 1 mid lay period (see System Management), behavioral observations began at approximately 11:00 AM, once tiered enclosure doors opened and hens had access to the open litter. Video recordings were decoded to document 3 aspects of behavior across the day (interobserver reliability 85% between 5 observers for dust bathing, 7 observers for pixel analysis, and 4 observers for piling). First, we used the software program ImageJ (National Institutes of Health, Bethesda, MD; Schneider et al., 2012) to complete pixel analysis of video snapshots sampled every 15 min across three 2 h periods for overall hen occupancy of the visible litter area (which included the area underneath the outer perch (Figure 1). We used ImageJ (National Institutes of Health, Bethesda, MD; Schneider et al., 2012) to convert the images to binary (black/white, hens/litter) and to calculate the percentage of litter floor space occupied by hens. Second, for observing specific behavior patterns performed on the open litter area (but not including the area directly under the outer perch), we counted the percentage of hens on the open litter that were dust bathing by sampling every 15 min within three 2 h periods starting from aviary opening ( 11:00 to 13:00 morning ; with an extra 2 h time period starting at lights on ( 6:30 to 8:30) for the mid lay period of Flock 1 only), during the mid-afternoon ( 15:00 to 17:00 afternoon ) and before lights out ( 19:00 to 21:00 evening ). At each 15 min interval, hens were observed for 1 min to determine if they were actively dust bathing versus sitting, standing, walking, or foraging. The video images used for the pixel analysis coincided with the start of the 1 min observations of hens to determine if they were dust bathing. Third, at each age, we observed all piling behavior occurring on the open litter from aviary opening until lights off. Based on preliminary observations, and for consistency between observers, we defined a pile as a minimum of 10 hens pressed against each other for at least 1 min, their heads facing the same direction and not performing any other discernible behavior (e.g., dust bathing). All piles were further classified by whether they occurred against a section gate or outer wall (for single-row sections only) or in a central area of the litter. We noted the time of day piles occurred, total time the pile lasted (duration), and how many hens were present at visually estimated peak pile size. Hens in piles typically had their heads up and were facing the same direction, thus counts of combs were made. However, in very large piles there may have been some hens ( 10 hens/pile) hidden from view during counting as the clustering could be very tight. We also discerned the cause of the pile to the best of our ability (Table 2). Ethics All research was approved by the Michigan State University Institutional Animal Care and Use Committee prior to the start of data collection. Data and Statistical Analyses Due to substantial differences in the management occurring between the periods of observation of the flocks, and because we recorded 2 weeks after aviary opening for Flock 1 but the day of aviary opening for Flock 2, data from each flock were analyzed separately. All analyses were conducted in JMP 11.0 (SAS Institute Inc.,

5 168 CAMPBELL ET AL. Table 2. The possible causes of pile formation for all observed piles as determined from video observations and the percentage of total observed piles for each cause. Behavioral cause of piling % of total observed piles Hens on other side of gate, appeared to interest hens in focal section No discernible cause: >5 hens begin interacting with each other and a pile formed >5 hens pecking at something and 8.62 others join Sudden mass movement resulting from 6.89 unknown flock disturbance (but not hysterical in nature; Hansen, 1976) Rooster accidentally present in enclosure 2.29 Aggression between 2 hens attracted 0.60 others Cary, NC) with α set at A t-test was applied to the litter depth data for Flock 1 (n = 48 units), and one-way ANOVAs were applied to the litter coverage data for both flocks (Flock 1 n = 60; Flock 2 n = 72 units) and litter depth data for Flock 2 (n = 72 units) for comparisons between different time points (peak, mid, and end of lay). ImageJ measurements of spatial occupancy of the litter in each sampled unit were averaged for all 15 min counts within each 2 h time period (lights on, morning, afternoon, and evening) for each age (peak, mid, and end of lay) of the 2 flocks. Kruskal-Wallis tests were applied to the non-normal data to assess differences across the time of day within each age for each flock (Flock 1 peak lay: n = 72 units, mid lay: n = 192 units, end lay: n = 144 units; Flock 2: all n = 144 units). To assess perferences for litter areas within a section, all 15 min counts occurring within the 2 h time periods were averaged between 2 units for each time point. The studentized residuals were checked for normality and a general linear model was applied to confirm no interaction between time of day and unit location (all P > 0.09). Data from the lights on (mid lay Flock 1 only), morning, afternoon, and evening time periods were pooled and comparisons were then made using Kruskal-Wallis tests between each of the outer 2 units (averaged) of the litter area (by the gates) and the central 2 units (Flock 1 peak lay: n = 36, mid lay: n = 94, end lay: n = 72; Flock 2: all n = 72). A previous partial dataset from Flock 1 showed no difference in spatial use between single and double rows (Makagon et al., 2012) therefore we did not distinguish between row type in our analysis. Wilcoxon signed-rank tests were applied to duration and size of Flock 2 piles only to assess for differences between time points (peak lay: n = 59, mid lay: n = 20, end lay: n = 29). The possible causes of all piles were described and tallied. Box plots for Figures 3 7 were generated by JMP, and unless otherwise stated, lines within the boxes Figure 3. The percentage of open litter area occupied by hens during each 2 h observational time period during the day at each time point for both flocks. Dissimilar letters indicate significant differences.

6 LITTER USE BY AVIARY LAYING HENS 169 Figure 4. The average percentage of open litter area occupied by hens with respect to their distribution across the section s open litter area at each time point of both flocks. Dissimilar letters indicate significant differences. represent the median while the lower and upper boundaries of the box represent the interquartile range (i.e., difference between the first and third quartiles.) The whiskers extending from the boxes are drawn to the outermost data point that falls within the distances computed as follows: upper whisker = third quartile (interquartile range) and lower whisker = first quartile (interquartile range). If the data points did not reach the computed ranges, then the whiskers were determined by the upper and lower data point values (not including outliers). The disconnected points are potential outliers. For litter area spatial use and dust bathing data, we display the raw unit sample values before averaging to accurately illustrate the variability of the individual observations. Data on piles were compiled to display their location within single and double rows, timeframes in which piles occurred and the average duration and average peak size at each time point. Litter Assessment RESULTS Litter substrate covered the smallest amount of the open floor area at peak lay for both Flock 1 (F 2, 57 = , P < 0.001) and Flock 2 (F 2, 69 = 101.4, P < 0.001; Table 3), and by mid lay in both flocks the open floor area was fully covered by litter. There was an increase in litter depth over time for both Flock 1 (t(42.38) = 6.72, P < 0.001) and Flock 2 (F 2, 69 = 90.39, P < 0.001; Table 3). Litter depth in the open area never exceeded 6.6 cm despite the fact that litter was not removed from the system until after hen depopulation. Open Litter Area Spatial Occupancy More open litter space was occupied in the afternoon (15:00 to 17:00) across flocks, time points and sections, except during peak lay in Flock 2, when hens first gained access to litter and occupied it most in the morning (11:00 to 13:00). The highest average amount of open litter space occupied at peak lay of Flock 1 was in the afternoon (H = 17.73, df = 2, P < 0.001; Figure 3), which was similar to the pattern observed at end lay (H = 68.35, df = 2, P < 0.001; Figure 3). At mid lay in Flock 1, the highest average amount of open litter space was occupied in the afternoon and the least in the evening (19:00 to 21:00) (H = , df = 2, P < 0.001; Figure 3). For peak lay of Flock 2, the highest average amount of open litter space was occupied in the morning (H = 32.91, df = 2, P < 0.001), while at mid lay, the highest average amount of open litter space was occupied in the afternoon and the least in the evening (H = 68.61, df = 2, P < ), which was similar to what was observed at end lay (H = 75.63, df = 2, P < 0.001; Figure 3).

7 170 CAMPBELL ET AL. Figure 5. The percentages of hens/individual unit on the open litter that were dust bathing within each 2 h observational time period during the day for each time point of both flocks. Dissimilar letters indicate significant differences. In Flock 1, there were no significant differences in the occupancy of the different locations within a section s open litter area (peak lay: H = 5.36, df = 2, P = 0.068; mid lay: H = 0.79, df = 2, P = 0.675; end lay: H = 0.37, df = 2, P = 0.83; Figure 4). However, in Flock 2 at peak lay, when recording occurred immediately after the hens first received access to the litter, litter areas closest to section-dividing gates had higher occupancy than the center of the open litter area (H = 32.42, df = 2, P < 0.001). This preference had disappeared by mid lay (H = 0.72, df = 2, P = 0.699) and was also not present at end lay (H = 0.53, df = 2, P = 0.765; Figure 4). Dust Bathing More hens on the open litter in Flock 1 at peak lay were dust bathing in the afternoon compared to other times of the day (H = 19.91, df = 2, P < 0.001; Figure 5). At mid lay of Flock 1, again, the highest average percentage of hens on the open litter was observed dust bathing in the afternoon while the lowest average percentage was observed dust bathing when the lights first came on (H = , df = 2, P < 0.001; Figure 5). At end lay in Flock 1, the highest average percentage of hens dust bathed in the afternoon and the fewest in the evening (H = 70.15, df = 2, P < 0.001; Figure 5). In Flock 2, at peak lay, the highest average percentage of hens dust bathed in the morning (H = 61.33, df = 2, P < 0.001; Figure 5), while the highest average percentage of hens on the open litter area were observed dust bathing in the morning and afternoon at mid lay (H = 24.35, df = 2, P < 0.001; Figure 5). Finally, at end lay in Flock 2, the highest average percentage of hens dust bathed in the morning and the lowest percentage in the evening (H = 60.49, df = 2, P < 0.001; Figure 5). Piling Overall, 174 piles were observed during data collection dates with 66 piles observed in Flock 1 and 108 in Flock 2. At least 1 pile was observed in each focal section across all data collection points. In the single rows, for both flocks, piles formed by the gate (Flock 1: n = 36, Flock 2: n = 28), against the wall (Flock 1: n = 12, Flock 2: n = 58), or started by the gate and moved to the wall (Flock 1: n = 4; Flock 2: n = 1). In Flock 1, 2 piles also formed in the center of the litter in the single rows. In the double rows, piles formed in the center of the open litter area (Flock 1: n = 12; Flock 2: n = 10) with 11 piles occurring against a gate in Flock 2 (NB: there was no wall in the double rows). Commencing with when the first pile began and the last pile ended, piles occurred throughout the day when hens had litter access for all time points of both flocks

8 LITTER USE BY AVIARY LAYING HENS 171 Figure 6. The total duration (min) of piles at each time point for both flocks. Dissimilar letters indicate significant differences for Flock 2. (Flock 1 peak lay: 11:00 to 18:50; mid lay: 08:20 to 18:45; end lay: 11:04 to 18:45; Flock 2 peak lay: 11:21 to 20:45; mid lay: 11:19 to 19:05; end lay: 10:52 to 19:51). Over both flocks and all time points, the durations of piles ranged from 1 min to 359 min. The longest lasting piles occurred at mid lay in both flocks, and more long-lasting piles (>1 h)occurred in Flock 2. For Flock 2, mid lay piles had the significantly longest duration and those at peak lay the shortest (H = 46.71, df = 2, P < 0.001; Figure 6). Piles were always localized and never included all hens in a section, with peak size varying across time points and flocks ranging from 10 (minimum requirement for a pile) to 229 hens (Figure 7). On average, piles were larger in the double rows, (Flock 1: mean ± SE 6.82; Flock 2: ± 14.69) compared to single rows (Flock 1: ± 2.71; Flock 2: ± 3.42), likely a consequence of differences in population size as there were twice as many hens in double sections and twice as much open litter space. Analyses of pile size revealed Flock 2 piles had the fewest hens at peak lay (H = 57.72, df = 2, P < 0.001; Figure 7). Behavioral observations of the formation of piles showed 5 possible causes, but the study was only observational in nature. We took no measurements of environmental parameters such as changes in lighting or temperature and had no acoustic information, which may have affected piling behavior. Possible causes of pile formation and descriptions are listed in Table 2. Overall, the piles were dynamic, shrinking and growing throughout their duration as hens constantly left and joined piles. Some hens appeared motivated to reach the center of the pile and would walk over the top of other hens to squeeze themselves into the middle (personal observation). The piles in the center of the open litter area were typically circular in shape and in all piles, the majority of hens had their heads facing in the same direction and their bodies were tightly squeezed together. However, no hen death was observed following piling, suggesting that smothering was not occurring. Finally, all piles, with the exception of 3 that appeared to end due to a sudden disturbance, eventually just dissolved, with hens leaving one by one for no discernible reason. DISCUSSION The results from our observations of laying hen behavior on the open litter area of a commercial aviary showed hens were using the entire open litter area. Hens performed dust bathing, fulfilling a behavioral need that has been determined to be important for improving their welfare (Cooper and Albentosa, 2003; but see Widowski and Duncan, 2000). However, the hens also exhibited unusual piling activity, which might be detrimental to welfare although the causes and implications of this behavior in this study are inconclusive. Spatial analysis of hens on the open litter area showed great variability in the amount of floor space occupied

9 172 CAMPBELL ET AL. Figure 7. The peak number of hens in a pile for each time point of both flocks. Dissimilar letters indicate significant differences for Flock 2. Table 3. The mean (± SE) litter depth (cm) measurements and litter coverage scores describing the mean (± SE) percentage of the open floor area covered by litter at 3 time points for both flocks. A litter coverage score of 0 was defined as < 25% of the area was covered by litter; a score of 4 was defined as 100% of the open litter area was covered by litter. (See Table 1 for definitions of all score categories.) No litter depth measurements were taken for peak lay of Flock 1. Flock Time point Depth (cm) % litter coverage scores One Peak lay N/A 3 ± 0.12 One Mid lay 3.10 ± ± 0 One End lay 5.40 ± ± 0 Two Peak lay 0.71 ± ± 0 Two Mid lay 1.27 ± ± 0.09 Two End lay 3.57 ± ± 0.04 by the hens with higher percentages at certain times of day, typically in the afternoon. Hens also occupied all open litter areas to the same degree, suggesting they did not have a preference for areas closer to gates dividing sections or the more open central areas. The only exception was observed at peak lay in Flock 2 when there was higher litter occupancy near the gates. This system provided approximately 196 cm 2 open litter area per hen in the double rows and 200 cm 2 open litter area per hen in the single rows (see Jones et al., 2014 for full details). This open litter area, in combination with the underneath litter area and available cage area, exceeded the minimum per hen space requirements set by United Egg Producers (UEP, 2010) although there has been little research which definitively determines how much space a hen actually needs or prefers. Recent kinematic analysis of Hyline W-36 hens indicates different static and dynamic physical space requirements for a range of behavior. At a minimum, a single standing hen requires approximately 563 cm 2, but the amount of physical space needed increases to approximately 1,693 cm 2 for wing flapping (Mench and Blatchford, 2014). However, group housing is suggested to affect these requirements as, for example, not all hens will wing-flap at the same time. Further, spatial distribution in open areas is not expected to be uniform (as hens cluster in groups rather than disperse evenly; Channing et al., 2001). Thus, modeling for group size, hens in flocks of 100 individuals or greater are predicted to need approximately 600 cm 2 of space to perform both static postures and dynamic behaviors (Mench and Blatchford, 2014). Based on these numbers, we calculated optimal maximum occupancy of the open litter area to occur when hens bodies take up approximately 33% of the available space. On average, our results indicate hens are using percentages of the open litter area close to this figure, suggesting even distribution of birds within the system and adaptation to the available space. There were also times of higher litter occupancy, which may or may not be indicative of crowding depending on the types of behavior being performed, such as piling, when the hens chose to be in extremely close proximity

10 LITTER USE BY AVIARY LAYING HENS 173 to each other. To better understand what constitutes over-crowding and the welfare consequences of high occupancy on the open litter area, further research should investigate the interactions between space occupancy and physical contact between hens, specifically injurious contact (e.g., collisions with other hens following flight landings: D. L. M. Campbell, unpublished data) or frequency of aggressive contact or feather pecking. Consistent with previous studies in both aviaries and commercial percheries, hens used this litter resource for dust bathing (Hansen, 1994; Carmichael et al., 1999; Channing et al., 2001; Odén et al., 2002) displaying an expected diurnal rhythm (Vestergaard, 1982), although hens in Flock 2 showed peak dust bathing somewhat earlier in the day than those in Flock 1. We presented dust bathing data as percentages of hens on the litter using it for that purpose and found comparable results to those reported by Odén and colleagues (2002) when hens had good quality litter. As anticipated, the litter depth in our study system increased as the flock cycle progressed due to the accumulation of manure, feathers, and feed, but at each video recording time point, litter was deemed dry and flaky, which is generally considered to be optimal for dust bathing. We did not estimate absolute numbers of hens dust bathing in the system as we could not observe either the dust bathing occurring in the portion of the litter area under the tiered enclosures or sham dust bathing within the enclosures, which may still occur in the presence of litter (Lindberg and Nicol, 1997; but see Hansen, 1994). Additionally, hens typically dust bathe every 48 h (Vestergaard, 1982) and thus we would not expect to see all hens dust bathing in the course of a single day. Within our system, we had no method of tracking single hens to investigate dust bathing variability at the individual level (Vestergaard, 1982); an avenue warranting further investigation. Hens from our aviary system did show lower feather lipid levels in comparison to those of hens from conventional or enriched colony cages at the same facility (see Blatchford et al., 2014), which might be due to effective dust bathing activity (van Liere and Bokma, 1987). The percentages of hens dust bathing on the open litter, averaged across 2 h time periods, suggest that each hen had sufficient room to perform all the motions of the dust bathing sequence (if hens were distributing themselves evenly on the litter). However, Figure 3 illustrates the variability within our data. High occupancy of the litter area (e.g., Flock 2 at peak lay in Figure 3) combined with high percentages of hens dust bathing (e.g., Flock 2 at peak lay in Figure 5), indicate that at these points, large numbers of hens were synchronous in their behavior and that the majority of the floor area was covered. Thus under these conditions crowding (defined as minimal space between birds or compressed hens; Appleby, 2004) could occur as a result of high occupancy and simultaneous performance of a dynamic behavior pattern requiring space to execute. This potential for crowding is consistent with previous observations of hens in commercial facilities and may result in some hens experiencing restricted room available for dust bathing effectively, leading to interrupted dust bathing sequences and incomplete dust baths or prevention of litter access altogether (Odén et al., 2002), an avenue for future research. Alternatively, laying hens are gregarious and may be socially facilitated to dust bath together (Duncan et al., 1998), thus, flock synchrony could represent an expression of a preference rather than a forced choice resulting from inadequate resources. Additionally, the large groups we observed may have sometimes resulted from litter substrate distribution rather than lack of available space as initial litter coverage at peak lay of Flock 2 was patchy in comparison to mid and end lay coverage. It is also important to note that on occasion we observed a low total percentage of the open litter space occupied (e.g., evening of peak lay in Flock 2; Figure 3), but a high percentage of the hens on the litter dust bathing (e.g., scattered points evident in the evening of peak lay in Flock 2; Figure 5). In these cases, only a small group of hens were out on litter and their behavior was synchronized, without crowding, unless hens chose to cluster together. This situation should give all hens in these small groups the space needed to dust bathe and to regulate the distance between hens according to their preference. The above examples illustrate that it is important to consider both the number of hens present (or, here, the space taken up by the hens) as well as the percentage of hens present that are engaged in a behavior when determining if crowding is occurring. Future investigations might couple such measurements with individual tracking of hens to determine whether individuals are able to complete full dust bathing sequences or if hens dust bathing outside the peak time did not have litter space access earlier in the day and were thus forced to dust bathe in the evening. Furthermore, it will be important to understand whether these hens experience poorer welfare as a result of dust bathing at a time potentially different than that dictated by their internal rhythm. To the best of our knowledge, our detailed observations of unusual hen piling within United States aviaries are the first of this type to be reported in the scientific literature. Furthermore, the published accounts of piling in United Kingdom systems (Bright and Johnson, 2011; Barrett et al., 2014) suggest our piles are distinct, although more detailed published behavioral descriptions would enable stronger direct comparisons. Our observed piling might be similar to creeping/recurring piling defined by Bright and Johnson (2011) asthisalso occurred spontaneously at any time of day across all time points of the flock cycle and was most frequent on open litter and in corners (Barrett et al., 2014). But in contrast, we never observed smothering resulting from these piles. We are unsure if the piles we observed in our study represent a welfare concern, and further research would need to determine whether they cause stress or injury to the hens or affect egg production.

11 174 CAMPBELL ET AL. Hen panic or hysteria is an undesirable flock behavior which can cause piling (Richards et al., 2012; Barrett et al., 2014), but only 6.89% of our observed piles appeared to result from flock disturbance, though we were unable to account for the presence of sounds or other variables not discernable on film. There may have been other hysteria or panic episodes similar to previous reports with hens flying wildly about (e.g., Hansen, 1976), but in our study they did not lead to piling. We attempted to determine other causes for piles, but we do not know why certain events (e.g., activity of a small group of hens) would trigger piling or what might lead to their apparently spontaneous formation. Hen piling may fall under the principles of preferential social aggregation (Febrer et al., broilers), attraction to unfamiliar conspecifics (Lindberg and Nicol, 1996), or may provide some degree of reward to the hens through physical contact with other hens within the pile, similar to chicks huddling for warmth. With respect to why hens tried to access the center of the pile, we hypothesize they may have been trying to get closer to the apparent source of an attraction. As we only made visual recordings, it is possible there may have been auditory or olfactory cues that were attracting the hens to each other; future research could determine whether vocalizations change when a pile is forming and if there are other cues signaling the pile to dissolve. Additional research could investigate contributing roles of environmental parameters such as localized temperature, light, or drafts, including examination of pile formation in other areas of the aviary system such as under or within the aviary enclosures. Lastly, previous research indicates piling occurs in multiple laying hen strains but that possible differences in piling frequency exist between them (Bright and Johnson, 2011), an opportunity for further study. Overall, we saw differences between ages and flocks in the behavior we observed. This may in part be due to variation in our data collection and sampling protocol. There were management differences within Flock 1 that were resolved by Flock 2, and we were unable to sample within the same age week for both flocks as our collection points were production-related and based on when peak lay (95% production) occurred in each flock. This sampling difference may, for example, be apparent in the high proportions of hens dust bathing at peak lay of Flock 2. We recorded on the very first day that the aviaries opened and thus, may have captured a rebound dust bathing effect (Nørgaard-Nielsen, 1997) not seen in the recordings of Flock 1 peak lay. This sampling difference may also account for hens preferring to occupy the litter area closest to the gates at peak lay of Flock 2 as they may have been acclimating to the spatial configuration of this new area access. Previous studies have also found differences between time points and variation between flocks in distributions and behavior within commercial alternative systems (Carmichael et al., 1999; Barrett et al., 2014). We also do not have enough information on day-to-day variation of flock behavior, however, some degree of consistency is indicated as similar behavioral patterns are evident across our sampled time points (see Figures 3 5). In conclusion, hens used all areas of the open litter and displayed some synchronicity with respect to dust bathing and piling; however, on average, hens occupied all areas of the open litter area without preference for areas near walls or gates dividing sections. These results are similar to previous research findings (Odén et al., 2002) and our own observations of hens transitioning between the enclosure and the litter areas (Campbell et al., 2015). Further research should track individual use of the litter areas to determine if all hens use litter equally, as well as determine the impact of piling on hen welfare. ACKNOWLEDGMENTS We would like to thank H. Albeer, J. Bergen, L. Devoe, S. Dorey, K. Dunn, N. Fairfield, R. Gohier, S. Goodwin, Y. Guo, A. Hinson, L. Kim, A. Marsh, E. Stefansky, L. Turner and D. Voishich for assistance with video data collection and C. Daigle, K. Dunn, M. Erasmus, P. Regmi, L. Turner and K. 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