Effects of outdoor ranging on external and internal health parameters for hens from different rearing enrichments

Ethical statement

All research was approved by the University of New England Animal Ethics Committee (AEC17-092).

Animals and Housing

A total of 1386 Hy-Line^® Brown layers were used for this study. The chicks and pullets were reared indoors at the Kirby poultry facility and the adults were housed in the Laureldale free-range facility at the University of New England, Armidale, NSW, Australia. Day-old chicks (including additional chicks delivered by the hatchery but not transferred to the laying facility) were reared across 16 weeks within 9 pens (6.2 m L × 3.2 m W) distributed across three separate rooms. The pullets were exposed to 3 separate rearing enrichment treatments. These included a control group having no extra materials over the pen standard of rice hulls as floor litter, a novelty group where novel objects were added and changed at weekly intervals (e.g., balls, bottles, bricks, brooms, brushes, buckets, containers, pet toys, plastic pipes, strings) and a structural group where four custom-designed H-shaped perching structures (L, W, H = 0.60 m) with two solid panels and one open-framed side were provided. Each of the rooms had one pen replicate per treatment, balanced for location within rooms. Shade cloth hung on the wire pen dividers visually isolating birds from each other. At 16 weeks of age, bird density was approximately 15 kg/m² or 9 pullets/m² (average 174 –190 pullets/pen). Round feeders provided ad libitum access to commercially-formulated mash appropriate for the developmental stage and nipples supplied ad libitum water access. These resources were provided as per the current Australian Model Code of Practice for the Welfare of Animals –Domestic Poultry (Primary Industries Standing Committee, 2002). Artificial lighting and temperature schedules followed the recommended Hy-Line^® Brown alternative management guidelines (Hy-Line, 2016) but the LED lighting was maintained at 100 lux as the pullets were destined for outdoor access (no natural light was present during rearing). Mechanical ventilation with heating operated as needed but no cooling system was present. Chicks were infra-red beak-trimmed at the hatchery with a vaccination schedule as per regulatory requirements and standard recommendations including vaccination against Newcastle disease, Marek’s disease, fowl pox, fowl cholera, egg drop syndrome, Mycoplasma gallisepticum, Mycoplasma synoviae, infectious bronchitis, infectious laryngotracheitis, and avian encephalomyelitis.

At 16 weeks, 1386 pullets were transferred to the Laureldale free-range facility and remixed within pen replicates (extra delivered chicks that were grown out were rehomed). The hens were housed within their rearing treatments across 9 pens located in a single shed (n = 154 hens/pen, indoor density approximately 9 hens/m²). The indoor pens were visually separated via shade cloth and included nest boxes (2 small and 1 large nest box), perches, round hanging feeders and water nipples to fulfil the requirements of the Australian Model Code of Practice for the Welfare of Animals –Domestic Poultry (Primary Industries Standing Committee, 2002). Rice hulls were used as floor litter material with one complete litter replacement mid-way through the flock cycle. The LED lighting schedule gradually increased to 16 h light and 8 h dark by 30 weeks of age with an average pen intensity of 10.0 (± 0.84 SE) lux (Lutron Light Meter, LX-112850; Lutron Electronic Enterprise CO., Ltd, Taipei, Taiwan) as measured at birds’ eye height from 3 pen locations (front, middle, back) when the pop-holes were closed. This lux was the highest that could be achieved with the shed lighting system. The shed was fan-ventilated with no temperature or humidity control.

Each of the 9 pens was connected to an outdoor range area (31 m L × 3.6 m W for each pen, density approximately 1.4 hens/m²) which was accessed via two pop-hole openings (18 cm W × 36 cm H). The range area immediately after the pop-holes was 1.1 m of concrete path, then 1.6 m of river rock followed by a grassed area with no additional trees or shelter. The grassed area became denuded following both hen access and the winter season. Each range was visually divided by shade cloth hung along the wire fences. Hens were provided access to the outdoor area from 25 weeks of age (May 2018) for most of the day time via automatic opening and closing of the pop-holes. The pop-holes opened at 9:15 am and closed after sunset daily. This equated to approximately 9 h of available ranging time across winter followed by approximately 11 h of available ranging time after daylight saving time started (October 2018).

Radio-frequency identification (RFID) system

All birds were banded with microchips (Trovan^® Unique ID 100 (FDX-A operating frequency 128 kHz) glued into adjustable leg bands (Roxan Developments Ltd, Selkirk, Scotland) to track their movement in and out of the range pop-holes via radio-frequency identification (RFID) systems. The RFID systems were designed and supported by Microchips Australia Pty Ltd (Keysborough, VIC, Australia) with equipment developed and manufactured by Dorset Identification B.V. (Aalten, the Netherlands) using Trovan^® technology. For a schematic of the RFID system, see Campbell, Horton & Hinch (2018b). The system recorded the date and time of each tagged bird passing through and in which direction (onto the range, or into the pen) with a precision of 0.024 s (maximum detection velocity 9.3 m/s). Individual ranging data were collected daily from 25 until 65 weeks of age.

RFID data and selection of hens

RFID data from 56 until 64 weeks of age (54 days of data) were initially run through a custom-designed software program written in the ‘Delphi’ language (Bryce Little, Agriculture and Food, CSIRO, St Lucia, QLD, Australia) that filtered out any unpaired or ‘false’ readings that may occur if, for example, a hen sits inside the pop hole but does not complete a full transition onto the range or back into the pen. Once screened in this way, the data were used to select a sample of birds from each pen to conduct post-mortem examinations on. A total of 307 hens were selected across all of the 9 pens at 64 weeks of age. The selected hens were categorized as ‘indoor’—accessed the outdoors on one or zero of the 54 days, ‘low outdoor—accessed the range on 53 or 54 of the 54 days but only for 1 h 24 mins or less, and ‘high outdoor’—accessed the range for 54 of 54 days for 5 h 12 mins to 9 h. Based on these criteria, a total of 95 indoor, 109 low outdoor and 104 high outdoor hens were selected from the flock. Maximum ranging times differed between pens, thus the high outdoor birds were selected as the highest for their pen, resulting in the variation in hours. Where possible, hen selection was balanced across pens within treatments, but some pen replicates did show higher numbers of the extreme ranging patterns than others.

Pre-mortem welfare assessment

All the selected hens were weighed 4 to 5 days before dissection using electronic hanging scales (BAT1; VEIT Electronics, Moravany, Czech Republic). The external health and welfare parameters including feather loss at different body parts (neck, chest, back, wing, vent, tail) and footpad lesions were assessed using the scoring system described by Tauson et al. (2005). In this scoring system, 4 scores were available for feather coverage with a score 4 indicating minimal feather damage, whereas a score 1 indicated bare skin. The back of the neck was scored separately from the front of the neck that was not included in the analyses as the majority of damage on the neck front was believed to have resulted from rubbing on the feeder rims rather than pecking. A maximum score of 24 could be obtained for feather condition across 6 body parts. Footpad lesions were scored as a 4 for a normal footpad with no lesions (an additional category to the Tauson et al. (2005) system) and a score 1 for swollen, infected bumble foot. The exact number of fresh or healing comb wounds were counted, and toenail length was measured in mm using a seamstress tape measure. Beaks were scored as 0, 1, or 2 indicating no, mild, or moderate damage respectively. The birds were also examined for any other external signs of injury or illness, such as body wounds, bleeding, abnormal respiratory sound etc., but none were observed in these hens. All selected hens were tagged with an additional leg band for identification in their home pen to enable later capture.

Post-mortem health examination

Post-mortem examinations of the selected 307 hens were carried out across two days at 65 weeks of age at a separate post-mortem facility located 5.5km from the free-range facility. The selected hens of a single pen were transported to the post-mortem lab up to 2 h prior to the dissection using plastic carrier crates. Hens were killed using CO₂ to maintain hen physical structure for later scanning. Immediately after the CO₂ administration and cessation of all hen movement, the birds were opened by a veterinarian to examine the health condition of the hens (presence/absence of diseases) by inspecting the visceral organs for any abnormalities including haemorrhage, tumours, caseous necrosis and/or other exudates, the respiratory system (nares and trachea) for any haemorrhage, inflammation or exudates and the reproductive system for signs of salpingitis, being egg bound, or other abnormalities. Whether the hen was in production or not was determined by examination of the ovary and presence of active or regressing follicles. The spleen, gizzard, and right adrenal gland were removed and weighed (to the nearest mg) from each of the selected birds. The gizzard was emptied prior to weighing with surrounding connective tissue and fat removed. The jejunum, duodenum and ileum were opened longitudinally and the number of Ascaridia galli worms in each bird recorded. This is one of the most prevalent nematode parasites present within Australian commercial free-range systems (Dao et al., 2018). The carcass weight of each bird was taken after post-mortem examination (minus the removed organs and small intestines) prior to the Computed Tomography (CT) scanning.

CT scanning and image analysis

Following post-mortem examination, all birds were scanned using a HiSpeed QX/I (2003) CT scanner (GE Medical Systems). Scans were performed using a voltage of 100 kV, a current of 80 mA, a pitch of 0.75:1, a field of view of 500 × 500 mm, a thickness of 2.5 mm, and a spacing of 3.75 mm per rotation. Resultant images had a pixel matrix of 512 × 512, and hence a pixel resolution of 2.384 mm³.

CT image stacks of each individual hen were analysed using ImageJ (Rasband W.S., ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1887–2018) to generate pixel frequency plots against greyscale pixel values (0–255). Greyscale value density was defined using a linear regression parameterized to known carcass weights (g) recorded immediately prior to scanning. As such, the linear regression between density (ρ, µg/mm³) and greyscale value (x) was given as: (1)${\displaystyle \rho =10.19\left(\pm 0.4546\sigma \right)\cdot \mathrm{x}}$

where σ is a standard deviation.

The weight of individual chicken (w, g) was therefore given as: (2)${\displaystyle \mathrm{w}=\sum _{\mathrm{x}=0}^{255}\mathrm{y}\cdot \mathrm{\alpha }\cdot \mathrm{\rho }\cdot 1000000}$

where y is the frequency of each greyscale value; α is the pixel resolution (2.384 mm³); and ρ is density (µg/mm³, Eq. (1)).

Body composition of each chicken was estimated assuming that the density of raw chicken fat was ≤865.64 µg/mm³ (https://www.aqua-calc.com/page/density-table/substance/chicken-coma-and-blank-broilers-blank-or-blank-fryers-coma-and-blank-separable-blank-fat-coma-and-blank-raw), and that bone was identified as having a greyscale value of 255 (2598.45 µg ± 0.4546 σ), with the remaining mass being attributed to muscle. Some hens contained an egg which was not removed prior to scanning and was classified the same as bone in the images. All hens with an egg present (1/0) were later identified (n = 87) and removed from the analysis.

Assessment of the keel bone

Following CT scanning, the keel bone was excised from each hen to assess deformities and damage. All keel bones were stored at −20 ° C until thawed for processing. For processing, the fleshes on the keels were removed using a knife and scissors. The defects on the keels were examined by two experimenters using a visual scoring system for bending of the spine that was classified as low, moderate, or high based on the extent (Fig. 1). The two observers scored each bone independently and then immediately confirmed scores to ensure agreement for each bone. The dorsal surface of the tip of the keels were also observed for the presence of calluses to indicate healed fractures and the number of calluses were counted and recorded by two observers in agreement (Fig. 2). No fresh pre-mortem fractures were observed.

Data and statistical analyses

Statistical analyses were conducted in JMP^® 14.0 (SAS Institute, Cary, NC, USA) with α set at 0.05. Data were transformed where needed but the raw values are presented in the tables and graphs. Non-significant interactions were removed from the final models and post-hoc Student’s t-tests were applied to the least-squares means where significant differences were present. For the pre-mortem welfare assessment data, the per hen values (n = 307 hens) for the total feather score (up to 24) and the number of comb wounds were square-root transformed. The body weight and toenail length measurements per bird were compiled. General Linear Mixed Models (GLMM) were fitted to each welfare variable with rearing enrichment treatments and ranging patterns as fixed effects including their interaction and bird ID nested within pen, rearing treatment, and ranging as a random effect. Restricted maximum likelihood estimation methods were applied.

The feather scores for each body part and footpad damage scores were compiled per individual hen and the effects of rearing treatment, ranging, and their interaction were tested using ordinal logistic regressions.

The body composition data (muscle, fat, bone in grams) from the CT scans (n = 307), post-mortem parameters including organ weights (liver, spleen, right adrenal gland, and empty gizzard proportional to live body weight), and A. galli counts were compiled per individual bird (n = 306 hens, data from 1 bird were missing). The relative organ weight proportional data were logit-transformed for analysis but expressed as percentages in the table. The A. galli count data were square-root transformed. GLMMs were fitted to test the fixed effects of rearing treatment and ranging groups including their interaction, with bird ID nested within pen, rearing treatment, and ranging as a random effect. The bone composition data of hens with an egg present (n = 87) were excluded from the analysis and data from 220 hens only were used.

Although a total of 307 birds were dissected, only 300 keels were assessed due to labelling issues with 7 keels. The qualitative data for the keel bones were compiled per individual bird (n = 300) including the overall presence of damage to the keel bone (yes/no), the type of keel spine bending, the presence (yes/no) and the number of calluses on the tips of the keels. Pearson’s Chi-square tests for differences between rearing treatments and ranging patterns were conducted separately for each variable of each set of data. When conducting the Chi-square test with one treatment, the other treatment effect was blocked for each of the variables. The counts of calluses were square-root transformed and analysed using a GLMM to test the effects of rearing treatment and ranging groups including their interaction, with bird ID nested within pen, rearing treatment, and ranging as a random effect.

Pre-mortem welfare assessment

Enriched rearing treatments did not affect the live weight of hens (F_2,302 = 0.11, P = 0.90) but the high outdoor hens had the lowest body weight (F_(2,302) = 10.90, P < 0.0001, Table 1). The total feather score of hens was the lowest in the control rearing treatment (F_(2,302) = 31.41, P < 0.0001) indicating the most feather loss in these birds compared with the enriched hens (Table 1). The total feather score was highest in the high outdoor hens (F_(2,302) = 18.06, P < 0.0001) compared with low outdoor and indoor hens indicating the least feather loss in these birds (Table 1). The control hens had more comb wounds than the novelty hens but neither group differed from the structural group (F_(2,302) = 3.63, P = 0.03, Table 1). The high outdoor hens had fewer comb wounds compared with the indoor hens but neither ranging group differed from the low outdoor hens (F_(2,302) = 3.08, P = 0.04, Table 1). Rearing treatment did not affect toenail length (F_(2,302) = 2.19, P = 0.11), but the indoor hens had the longest nails and the high outdoor hens had the shortest (F_(2,302) = 86.84, P < 0.0001, Table 1). There was no interaction between rearing treatment and ranging patterns on any of the pre-mortem variables (P ≥ 0.11).

A higher percentage (95%) of hens from the structural group showed feet with no damage (score 4) compared with the control (93%) and novelty groups (93%). A higher percentage of hens (97%) from the low outdoor ranging groups showed no footpad damage (score 4) compared with the high outdoor (88%) and indoor hens (95%). Only 0.92% of hens from the novelty group and 0.96% hens from the high outdoor ranging group showed the worst footpad condition (score 1). Ordinal logistic regression analysis showed ranging patterns significantly affected the footpad damage of hens (χ2 = 7.07, df = 2, P = 0.03) with high ranging increasing damage but the rearing treatments did not (χ2 = 0.86, df = 2, P = 0.65). There was no interaction between rearing treatment and ranging patterns (P = 0.14).

For other health issues examined pre-mortem, 0.01% of hens showed a prolapse. Most of the hens (91.86%) had no defects on the beaks with a scoring of ‘0’, but 0.06% and 0.03% of hens had beaks with a score of ‘1’ for mild and a score of ‘2’ for moderate defects, respectively.

Feather scores of individual hen body parts

Analyses of the feather scores of separate hen body parts showed rearing treatments had significant effects on plumage damage to the back of the neck, chest, and back with control hens showing the poorest feather coverage (Table 2). Ranging patterns had significant effects on plumage damage to the chest with high outdoor hens showing the greatest plumage coverage (Table 2). There were no significant interactions between rearing treatments and ranging groups (all P ≥ 0.07).

Post-mortem parameters

Rearing treatments affected the relative weight of the spleen (F_(2,301) = 4.82, P = 0.01) with the spleens from the novelty hens having lower weight than the spleens from the control hens but neither group differed from the structural group (Table 3). The high outdoor birds’ relative spleen weight was the highest (F_(2,301) = 4.44, P = 0.01). The high outdoor hens had higher empty gizzard weights than the indoor hens (F_(2,301) = 3.22, P = 0.04) but there was no effect of rearing treatment (F_(2,301) = 0.85, P = 0.43). Both the rearing treatments and ranging had no significant effects on the relative liver and adrenal weights (Table 3). There was no overall significant effect of rearing treatments on the number of A. galli worms in the GI tract (F_(2,301) = 2.30, P = 0.10), but post-hoc tests (which are more focussed to differentiate the clear visual differences in means, (Hsu, 1996) showed the novelty hens had more A. galli worms than the control hens and neither group differed from the structural hens (Table 3). There was no effect of ranging group on the number of A. galli worms (F_(2,301) = 1.15, P = 0.32). There were no significant interactions between rearing treatment and ranging for any of the measured variables (all P ≥ 0.21).

Post-mortem examination also revealed all hens under study except one, were in production. There were no disease lesions observed on the respiratory system of the hens. One bird had a fatty liver, one peritonitis and one had keel adhesion. A cystic right oviduct was also present in 0.04% of hens.

Body composition

There was a trend for an effect of rearing treatment on body fat (F_(2,302) = 2.80, P = 0.06) with the novelty group showing lower body fat (LSM ± SEM = 122.39 ± 1.78) than the control group (LSM ± SEM = 128.15 ± 1.91) but neither differed from the structural group (LSM ± SEM = 126.99 ± 1.83). Ranging had a significant effect on body fat (F_(2,302) = 19.70, P < 0.0001) and muscle (F_(2,302) = 11.49, P < 0.0001) with the high outdoor birds showing the lowest amount of fat and muscle (Fig. 3). There was no effect of rearing treatment on muscle mass (F_(2,302) = 0.25, P = 0.78). There was a trend for an effect of rearing treatments (F_(2,215) = 2.84, P = 0.06) on bone mass with the post-hoc tests showing the novelty hens had higher bone mass than the control group but the structural group did not differ significantly from either (Fig. 4). The ranging patterns had no significant effect (F_2,215 = 1.95, P = 0.14) on bone mass of the hens. There were also no significant interactions of rearing treatments and ranging on any measured variable (all P ≥ 0.16).

Keel bone damage

The free-range hens from different rearing treatments and ranging patterns showed no significant differences in the overall presence of keel bone defects, presence of spine bending, spine bending types, and the presence of callus formation on the dorsal surface of keel tips (all P ≥ 0.19, Table 4). The rearing treatments (F_(2,295) = 0.40, P = 0.67), ranging patterns (F_(2,295) = 1.31, P = 0.27) and their interaction (P = 0.55) had no effect on the number of calluses on the tip of the dorsal surface of the keels.