Evaluation of an Active LF Tracking System and Data Processing Methods for Livestock Precision Farming in the Poultry Sector.

Tracking technologies offer a way to monitor movement of many individuals over long time periods with minimal disturbances and could become a helpful tool for a variety of uses in animal agriculture, including health monitoring or selection of breeding traits that benefit welfare within intensive cage-free poultry farming. Herein, we present an active, low-frequency tracking system that distinguishes between five predefined zones within a commercial aviary. We aimed to evaluate both the processed and unprocessed datasets against a "ground truth" based on video observations. The two data processing methods aimed to filter false registrations, one with a simple deterministic approach and one with a tree-based classifier. We found the unprocessed data accurately determined birds' presence/absence in each zone with an accuracy of 99% but overestimated the number of transitions taken by birds per zone, explaining only 23% of the actual variation. However, the two processed datasets were found to be suitable to monitor the number of transitions per individual, accounting for 91% and 99% of the actual variation, respectively. To further evaluate the tracking system, we estimated the error rate of registrations (by applying the classifier) in relation to three factors, which suggested a higher number of false registrations towards specific areas, periods with reduced humidity, and periods with reduced temperature. We concluded that the presented tracking system is well suited for commercial aviaries to measure individuals' transitions and individuals' presence/absence in predefined zones. Nonetheless, under these settings, data processing remains a necessary step in obtaining reliable data. For future work, we recommend the use of automatic calibration to improve the system's performance and to envision finer movements.

Biomechanical comparison of canine fascia lata and thoracolumbar fascia: an in vitro evaluation of replacement tissues for body wall reconstruction.

OBJECTIVES: To compare the suitability of thoracolumbar fascia (TLF) and fascia lata (FL) for body wall defect repair in dogs, by examining their biomechanical properties and useable surface area. STUDY DESIGN: Experimental. ANIMALS: Dogs (n = 8). METHODS: Fresh TLF and FL grafts were obtained, surface area was calculated before testing to failure in 2 different modes: tensile testing and resistance to suture pullout, in 2 perpendicular orientations. RESULTS: Useable TLF surface area was significantly greater than for FL. Maximum load, energy to break, and elastic modulus of FL was significantly greater than that of TLF in tensile testing, but no apparent difference in the ultimate stress or strain was identified. There was no overall difference in suture pullout load between TLF and FL. During tensile testing, tissue orientation had a significant influence on ultimate load, stress, and elastic modulus for both tissue types, with strain and energy to break only having significant effects for TLF and FL, respectively. CONCLUSIONS: The greater tensile strength and stiffness of FL compared to TLF was not reflected in its material properties, implying any difference was a consequence of greater thickness. Suture pullout was not significantly different between the 2 tissues, perhaps limiting the clinical significance of the tissue mechanics. Tissues were anisotropic with respect to mechanical properties, thus orientation may be an important factor.

Evaluation of osteotomy accuracy and rotational and angular alignment for cranial closing wedge ostectomy performed with and without alignment aids.

OBJECTIVE: To (1) investigate the use of K-wires as alignment aids for cranial closing wedge ostectomy (CCWO) and (2) evaluate their effect on osteotomy accuracy and angular/rotational alignment. STUDY DESIGN: Cadaveric study. ANIMALS: Cadaveric pelvic limbs (n = 20). METHODS: CCWO was performed with and without alignment aids on 20 cadaveric pelvic limbs. CT scans were performed pre- and post-operatively to evaluate tibial torsion and valgus/varus deformity. Digital photographs of the ostectomized bone wedges were used to assess divergence of the 2 osteotomies, and the area of the medial and lateral aspects measured to assess osteotomy angulation within the dorsal plane. RESULTS: Osteotomy divergence angle, the difference between the area of the medial and lateral aspects of the ostectomized wedges, and the difference between the pre- and post-operative angles of valgus/varus deformity were all significantly smaller for the alignment aid group. The difference in pre- and post-operative tibial torsion was not significantly different between groups. CONCLUSIONS: K-wires can be used successfully as alignment aids during CCWO and help to create a significantly more orthogonal osteotomy. This allows a significant reduction in the difference between the preoperative and postoperative angles of valgus or varus, which may reduce the risk of developing a clinically important iatrogenically introduced valgus/varus deformity postoperatively.

Keel bone fractures affect laying hens' mobility, but no evidence for reciprocal effects.

Keel bone fractures (KBF) are prevalent in commercial laying hens and are considered one of the greatest welfare concerns in the egg-production industry. While clear associations exist between KBF and animal mobility, suggesting that KBF impair mobility, the effect of mobility on KBF remains unclear. We combined data from three studies that assessed keel bone fracture severity through radiographs and monitored hens' transitions between different zones of a multi-tier aviary system (the three tiers, a littered floor, and a winter garden) the week prior to radiograph. For each hen, we extracted two daily movement behaviours: the vertical distance travelled and the mean number of zones crossed within one transition; and two daily space-use behaviours: the time spent in the top tier and the unevenness of time spent across zones. We used hierarchical Bayesian continuous time dynamic modelling to estimate how a change in a behaviour predicted a later change in keel bone fracture severity, and vice versa. Increased fracture severity did not predict later changes in space-use behaviours, but it did predict changes in movement behaviours. Specifically, increased fracture severity led to decreased vertical travelled distance and a tendency to cross more zones within one transition, suggesting impaired mobility in hens with increased fracture severity. In contrast, we found no evidence that movement or space-use behaviours predict later change in fracture severity, challenging previous literature suggesting that vertical locomotion through jumping and flying may exacerbate keel bone fractures in complex three-dimensional systems due to increased risk of collisions. However, similar efforts accounting for the location of fractures on the keel could unveil the potential influence of movement and space-use behaviours in the formation and change (healing or worsening) of KBF and increase our ability to mitigate their effects.

Research Note: A validation of an image-based method to estimate chicken comb size.

Chicken combs carry important information about the individual, especially the size has been related to sexual reproduction, health, and social signaling. Comb size is usually estimated by weighing removed combs or by calculating the product of the comb's longest and highest dimensions (LHA) to approximate comb area based on measures of a ruler or caliper. These methods have several shortcomings including invasiveness or imprecision. As a result, more recent efforts have employed pixel-based approximations of comb area (PBA) from images. However, the validity of PBA to estimate comb area and how the approximation compares to previous approximation methods, such as LHA, is unknown. Therefore, we developed an apparatus for taking standardized images of the head position of the hens and then applied PBA using the software ImageJ. The hens were each photographed 3 times by 2 different handlers. We first tested the accuracy of the pixel-based area approximation on 3 geometric shapes of known area. Second, we tested the precision of PBA of 15 hens (Dekalb White), evaluated as within-image and within-individual hen precision. Furthermore, we compared the PBA with the LHA based on measures of a caliper. The PBA was both accurate and precise, whereas the LHA overestimated comb area with increasing overestimation for larger combs. Due to the greater accuracy of the PBA, as well as future possibilities of automation and inclusion of further measures, we suggest PBA as a more reliable approach to estimate comb area than LHA. Additionally, our results demonstrate that the outcomes of LHA should be evaluated on an ordinal scale level only.

Mind the ramp: Association between early life ramp use and spatial cognition in laying hen pullets.

Ramps facilitate earlier access to complex environments and increase early life voluntary exercise, which may positively affect the cognitive development of chickens. This study focused on quantifying individual differences in ramp use and its impact on spatial cognition of laying hen pullets. Sixteen identical pens were housed with Lohmann Selected Leghorn (LSL) chicks of which eight chicks from each pen were colour marked from one day of age (DoA) to serve as focal birds. We quantified overall ramp use (walk/run, wing-assisted incline running, and jump/fly to and from ramps) by scan sampling recorded videos for 6, 10, 12, 20, 27, 41, and 55 DoA for all focal birds. From 56 to 95 DoA, long and short-term spatial memory of three focal birds per pen were assessed in a holeboard test in three consecutive phases: cued, uncued and reversal. Mixed model analysis showed that the spatial cognitive abilities of the birds were linked to differences in ramp use frequency averaged across all observation days. Birds with higher ramp use made fewer reference (Estimate ± Confidence Interval = 0.94 [0.88, 0.99], p = 0.08) and working memory errors (Est ± CI = 0.77 [0.59, 1.00], p = 0.06) in the cued phase than birds with lower ramp use. In contrast, birds with higher ramp use made more reference memory errors (Est ± CI = 1.10 [1.01, 1.20], p = 0.05) in the reversal phase. Birds with higher ramp use also made more reference memory errors compared to birds with lower ramp use as the phases changed from cued to uncued (p = 0.001). Our results indicate that there might be a relationship between early life ramp use and spatial cognition of laying hens.

Genetic parameter estimates for the use of an aviary with winter garden by laying hens.

The behavioral activity of laying hens in an aviary is indicative of their welfare and health. Furthermore, hens' usage of the different locations within an aviary has been shown to influence laying performance and egg quality. For example, hens that spent a longer duration of time in the nest during laying were observed to have lower laying performance. Therefore, understanding genetics of laying hens' usage of the aviary could be important for predicting egg quality, production traits and health and welfare. The objectives of this study were to estimate genetic parameters for duration of time spent at different locations within the aviary and an adjacent winter garden using a multivariate repeatability model and to compare correlations between time spent in these locations. For this study, a total of 1,106 Dekalb white laying hens (Hendrix Genetics) were genotyped using a proprietary 60K SNP array. These hens had access to 5 different zones within the aviary, which included the top level tier, nest box tier, lower level tier, floor littered area and a winter garden. Hens were in the aviary for a total of 290 d and daily records of duration were collected for each hen visit to any location in the aviary, culminating in a total of 937,740 records. Heritability estimates ranged from 0.05 (0.01) to 0.28 (0.03) for the duration of time spent in the different zones. The lowest heritability was estimated for time spent at the lower level tier, while a higher heritability was estimated for time spent in the floor littered area. A moderately high negative genetic correlation of -0.59 (0.08) was observed between time spent in the top level tier and time spent in the floor littered area, while a favorable correlation of 0.37 (0.14) was found between time spent in the lower level tier and time spent in the winter garden. The findings of this study show that the duration of time spent at different zones within an aviary has genetic basis and could be used for selecting animals for better performance and higher welfare.

Influence of keel impacts and laying hen behavior on keel bone damage.

Keel bone damage, which presents as fractures and/or deviations of the keel, has been detected in laying hens housed in all types of systems. Factors leading to keel bone damage in hens housed with limited vertical space, such as those housed in furnished systems, are not well understood, and are the topic of this study. Ten focal hens from each of 12 furnished cages (4 rooms of 3 cages) were fitted with keel mounted tri-axial accelerometers. Their behavior was video recorded continuously over two 3-wk trials: the first when the hens were between 52 and 60 wk of age, and the second approximately 20 wk later. The integrity of each hen's keel was evaluated at the start and end of each 3-wk trial using digital computed tomography. We identified predominant behaviors associated with acceleration events sustained at the keel (collisions, aggressive interactions and grooming) by pairing accelerometer outputs with video data. For each recorded acceleration event we calculated the acceleration magnitudes as the maximum summed acceleration recorded during the event, and by calculating the area under the acceleration curve. A principle components analysis, which was used as a data reduction technique, resulted in the identification of 4 components that were used in a subsequent regression analysis. A key finding is that the number of collisions a hen has with structures in her environment, and the number of aggressive interactions that a hen is involved, each affect the likelihood that she will develop 1 or more fractures within a 3-wk time span. This relationship between hen behavior and keel fracture formation was independent of the magnitude of acceleration involved in the event. Observed behavior did not have an impact on the formation of keel bone deviations, further supporting reports that the mechanisms underlying the 2 types of keel bone damage are different.

Providing elevated structures in the pullet rearing environment affects behavior during initial acclimation to a layer aviary.

Spatial abilities of hens are particularly sensitive to development during early life. Experiences in pullet housing may have lasting consequences on adult hens' movements in cage-free environments. We tested whether opportunities to access elevated spaces during rearing improved hens' use of a multitiered aviary. Female Dekalb White pullets were reared in either floor pens (FL), single-tiered aviaries (ST), or 2-tiered aviaries (TT; n = 5 pens/environment) through 16 wk of age. Rearing structures were replaced with identical multitiered aviaries at 17 wk. The distribution of the flock within the aviary and the vertical transitions of 10 focal hens/pen across the aviary were determined from videos recorded during their first (D1) and seventh (D7) day of aviary access, as well as at 19, 23, and 27 wk of age. Prevalence of floor eggs was recorded weekly from 17 to 28 wk of age. On D1, more ST and TT hens utilized the aviary during the daytime (P = 0.0077), made more vertical transitions when searching for a roosting spot in the evening (P = 0.0021), and maintained a consistent distance traveled during transitions compared to FL hens (P = 0.02). These differences disappeared by D7, except that ST and TT hens continued to roost on the highest perches of the aviary more (P < 0.0001) than FL hens through 27 wk of age. FL hens laid more floor eggs than ST and TT hens for the first 2 wk of lay (P < 0.0001). The majority (97.9%) of vertical transitions was controlled. Uncontrolled transitions were highest at D1 and decreased by D7 (P = 0.0009) and were not affected by rearing (P = 0.33). The results suggest that hens reared with minimal height are hesitant to use the laying hen aviaries when they are first transferred. They acclimate within 1 to 2 wk, but continue to roost less in the highest accessible level.

Commercial laying hens exhibit long-term consistent individual differences and behavioural syndromes in spatial traits.

Past research has supported the importance of animal personalities for the productivity and welfare of farm animals. However, current assessments of personality traits are commonly conducted over short periods using standardized assays and may not reflect all important aspects of behaviours in commercial settings throughout the production period. This study aimed to evaluate consistent behavioural differences between 194 commercial laying hens within an aviary across most of the production period (eight months). We used five spatial behaviours related to various aspects of commercial hens' daily routine, including the sleeping, feeding, nesting, indoor movements and outdoor usage. All behaviours were repeatable over time and across contexts, with consistent differences between individuals explaining between 23% and 66% of the variation. These long-term consistencies revealed the potential applicability of the behaviours as personality traits of commercial hens. Moreover, we identified behavioural syndromes comprising all behaviours except the nesting-related behaviour, indicating two axes of spatial personalities that may be driven by different mechanisms. We discussed the significance of such individual differences in using personality traits to breed more resilient farm animals. Future research should evaluate associations of these behaviours with animal welfare and productivity to inform breeding efforts.

Coup in the coop: Rank changes in chicken dominance hierarchies over maturation.

Chicken dominance hierarchies or pecking orders are established before maturation and maintained by consistent submissive responses of subordinate individuals, leading to stable ranks within unchanged groups. We observed interactions of 418 laying hens (Gallus gallus domesticus) distributed across three small (20) and three large (∼120) groups. The observations were performed before sexual maturation (young period) and additionally after onset of maturation (mature period) to confirm stability of ranks. Dominance ranks were estimated via the Elo rating system across both observation periods. Diagnostics of the ranks revealed unexpected uncertainty and rank instability for the full dataset, although sampling appeared to be adequate. Subsequent evaluations of ranks based on the mature period only, showed more reliable ranks than across both observation periods. Furthermore, winning success during the young period did not directly predict high rank during the mature period. These results indicated rank changes between observation periods. The current study design could not discern whether ranks were stable in all pens before maturation. However, our data rather suggested active rank mobility after hierarchy establishment to be the cause for our findings. Once thought to be stable, chicken hierarchies may provide an excellent system to study causes and implications of active rank mobility.

Commercial hatchery practices have long-lasting effects on laying hens' spatial behaviour and health.

The commercial hatchery process is globally standardized and exposes billions of day-old layer chicks to stress every year. By alleviating this early stress, on-farm hatching is thought to improve animal welfare, yet little is known about its effects throughout production. This study compared welfare indicators and spatial behaviours during the laying period of hens hatched in an on-farm environment (OFH) to those hatched in a commercial hatchery and transferred at one day-old to a rearing barn (STAN). In particular, we assessed how OFH and TRAN hens differed in space-use and movement behaviours following the transfer to the laying barn at 17 weeks of age, a similar stressor encountered by STAN hens early in life, and determined whether effects aligned more with the 'silver-spoon' or 'environmental matching' hypothesis. We found that for the first three months post-transfer into the laying barn, OFH hens, on average, transitioned less between the aviary's tiers and spent less time on the littered floor. Because OFH hens became behaviourally more similar to STAN hens over time, these results suggest that OFH hens required a prolonged period to establish their daily behavioural patterns. Furthermore, OFH hens had more severe keel bone fractures throughout the laying period but similar feather damage and body mass to STAN hens. No differences were found in hen mortality or the number of eggs per live hen. These findings support the environmental matching hypothesis and suggest that early-life stressors may have prepared hens for later-life stressors, underscoring the importance of both early-life and adult environments in enhancing animal welfare throughout production.

Intra-individual variation of hen movements is associated with later keel bone fractures in a quasi-commercial aviary.

Measuring intra- and inter-individual variation in movement can bring important insights into the fundamental ecology of animals and their welfare. Although previous studies identified consistent differences in movements of laying hens within commercial aviaries, the level of consistency was not quantified, limiting our capacity to understand the importance of individual movements for welfare. We aimed to quantify the scope of intra- and inter-individual differences in movements of commercial laying hens and examined their associations with indicators of welfare at the end of production. We quantified individual differences in one composite daily movement score for 80 hens over 54 days post-transfer to a quasi-commercial aviary. Results showed consistent inter-individual differences in movement averages, explaining 44% of the variation, as well as individual variation in predictability and temporal plasticity (at the population-level, hens increased their movements for 39 days). Hens that were more predictable in their daily movements had more severe keel bone fractures at the end of production while we found no such correlation between daily movement averages (individual intercept) and welfare indicators. Our findings highlight the importance of inter-individual difference in intra-individual variation of movements to improve poultry welfare.

Conservative versus arthroscopic management for medial coronoid process disease in dogs: a prospective gait evaluation.

OBJECTIVE: To investigate, using objective gait analysis, the long-term outcome of dogs with medial coronoid process disease (MCPD) treated with conservative management (CM) versus arthroscopic treatment (AT). STUDY DESIGN: Prospective clinical trial. ANIMALS: Dogs (n = 20) with unilaterally confirmed MCPD. METHODS: Eleven dogs were treated arthroscopically with removal of coronoid fragments and burring of any associated chondromalacic cartridge and 9 dogs were managed conservatively. All dogs were administered a 6-week course of oral tepoxalin on enrollment. Inverse dynamics gait analysis was performed at initial presentation and at 4, 8, 26, and 52 weeks. The gait variables analyzed were elbow moment (EM), elbow power (EP), total support moment (TSM), and total support moment ratio (TSMR) as a measure of forelimb asymmetry. RESULTS: Affected peak EM increased from 0.58 to 0.76 Nm/kg in the AT dogs, and from 0.66 to 0.81 Nm/kg in the CM dogs and there was no significant difference between groups. Affected peak EP increased marginally in the AT dogs, but was unchanged in the CM dogs and there was no significant difference between groups. TSM increased from 1.49 to 1.92 Nm/kg in the AT dogs and from 1.52 to 2.06 Nm/kg in the CM dogs and there was no significant difference between groups. TSMR was statistically different between treatment groups at 1 (P = .003) and 2 months (P = .048) with the AT group more asymmetric and hence more lame. TSMR at 12 months was 0.83 (AT) and 0.86 (CM) implying a failure of return to soundness by either group. CONCLUSIONS: AT dogs had increased mechanical asymmetry at 4 and 8 weeks compared to the CM group revealing surgery worsened limb function. There was no significant difference in mechanical symmetry between groups at 26 and 52 weeks.

PCR-denaturing gradient gel electrophoresis of complex microbial communities: a two-step approach to address the effect of gel-to-gel variation and allow valid comparisons across a large dataset.

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.

Radiographic Evaluation of Keel Bone Damage in Laying Hens-Morphologic and Temporal Observations in a Longitudinal Study.

The keel bone of commercially kept laying hens is known to be frequently affected by morphologic changes such as fractures and deformations with important implications for animal welfare. To detect morphologic changes, various methods such as palpation, computed tomography, and ultrasound are available, though radiography allows for the greatest level of detail in combination with the most ease of use. To explore the benefits of radiography in providing objective data on keel fractures from the age of 22-61 weeks within a single laying period, the keel bones of 75 Lohmann Brown and 75 Lohmann Selected Leghorns were radiographed every 3 to 5 weeks. Type, location, angulation, dislocation, callus formation, and healing process were assessed descriptively for each lesion. Ninety-nine percent of the animals showed at least one keel bone lesion during the study and 97% of the animals had at least one keel bone fracture. In 77% of the cases, the caudal third of the keel bone was affected. The fracture types were transverse and oblique (88%), comminuted, and butterfly. Further lesions were sclerosis, new bone formation and angulation. For each keel bone, an average of three fractures (3.09 ± 1.80) was detected at the end of the study. The described radiographic protocol for keel bone lesions was suitable for longitudinal, on-site examinations in conscious laying hens. Our results also indicate that keel bone fractures are more frequent than reported in earlier studies. The described radiographic examination protocol can be used to perform comparative studies of palpatory findings, or to assess the clinical significance of different fracture types which require a high level of detail.

Perch Positioning Affects both Laying Hen Locomotion and Forces Experienced at the Keel.

The aim of this study was to assess the effect of perch positioning on laying hens' locomotion and the resulting energy experienced at the keel. Twenty Nick Chick and 20 Brown Nick hens were trained to transition from a platform to a perch in several configurations. Three variables of perch positioning were tested in a 2 × 2 × 2 factorial design: direction (upward vs. downward), angle (flat vs. steep), and distance (50 cm vs. 100 cm). All hens were tested for five jumps of each treatment combination at 27-28 weeks of age. As predicted, we found steep angles and long distances to result in higher peak forces and impulse during take-off, flight, and landing; longer latency to jump; a higher likelihood to perform balancing movements; and a longer latency to peck at the provided food reward. The effect of perch positioning on locomotion and force at the keel during downwards jumps and flight was more pronounced in Brown Nick hens than in Nick Chick hens. Although we cannot state how the observed forces at the keel relate to the risk for keel bone fractures, our results indicated that optimizing perch positioning can reduce accumulated forced at the keel and consequent risk for fracture due to unsuccessful transitions.

Explanations for keel bone fractures in laying hens: are there explanations in addition to elevated egg production?

The current article served to provide the most up-to-date information regarding the causes of keel bone fracture. Although elevated and sustained egg production is likely a major contributing factor toward fractures, new information resulting from the development of novel methodologies suggests complementary causes that should be investigated. We identified 4 broad areas that could explain variation and increased fractures independent of or complementing elevated and sustained egg production: the age at first egg, late ossification of the keel, predisposing bone diseases, and inactivity leading to poor bone health. We also specified several topics that future research should target, which include continued efforts to link egg production and bone health, examination of noncommercial aves and traditional breeds, manipulating of age at first egg, a detailed histological and structural analysis of the keel, assessment of prefracture bone condition, and the relationship between individual activity patterns and bone health.

Evaluation of Poultry Stunning with Low Atmospheric Pressure, Carbon Dioxide or Nitrogen Using a Single Aversion Testing Paradigm.

Low atmospheric pressure stunning (LAPS) has been suggested for use in poultry under 4 kg in the abattoir as a more humane alternative to carbon dioxide (CO2). However, there are currently no studies offering a direct comparison of the aversion between methods. Here, we trained adult female broiler breeders to relinquish a food reward by moving to another area of the gas chamber in response to aversive stimuli. They were then stunned and subsequently killed using single exposure to either CO2, N2, LAPS or medical air as a control. Birds exposed to CO2 relinquished the food reward the quickest and exhibited gasping and headshaking more than the other groups. LAPS resulted in the quickest time to loss of posture (LOP) and birds in the N2 group took the longest. Birds exposed to N2 displayed the longest duration of ataxia of any group; however, they did not show any wing-flapping prior to LOP, unlike the LAPS and CO2. Collectively these data demonstrate that both LAPS and N2 are less aversive to poultry than CO2 and may offer a significant welfare refinement for poultry killed for meat production.

DNA methylation variation in the brain of laying hens in relation to differential behavioral patterns.

Domesticated animals are unique to investigate the contribution of genetic and non-genetic factors to specific phenotypes. Among non-genetic factors involved in phenotype formation are epigenetic mechanisms. Here we aimed to identify whether relative DNA methylation differences in the nidopallium between groups of individuals are among the non-genetic factors involved in the emergence of differential behavioral patterns in hens. The nidopallium was selected due to its important role in complex cognitive function (i.e., decision making) in birds. Behavioral patterns that spontaneously emerge in hens living in a highly controlled environment were identified with a unique tracking system that recorded their transitions between pen zones. Behavioral activity patterns were characterized through three classification schemes: (i) daily specific features of behavioral routines (Entropy), (ii) daily spatio-temporal activity patterns (Dynamic Time Warping), and (iii) social leading behavior (Leading Index). Unique differentially methylated regions (DMRs) were identified between behavioral patterns emerging within classification schemes, with entropy having the higher number. Functionally, DTW had double the proportion of affected promoters and half of the distal intergenic regions. Pathway enrichment analysis of DMR-associated genes revealed that Entropy relates mainly to cell cycle checkpoints, Leading Index to mitochondrial function, and DTW to gene expression regulation. Our study suggests that different biological functions within neurons (particularly in the nidopallium) could be responsible for the emergence of distinct behavior patterns and that epigenetic variation within brain tissues would be an important factor to explain behavioral variation.