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.

Similarity in Temporal Movement Patterns in Laying Hens Increases with Time and Social Association.

We explored the relationship between social associations and individual activity patterns in domestic hens. Out of 1420 laying hens, 421 hens were equipped with RFID tags attached to RFID-specific leg bands (leg bands from Company Roxan, Selkirk, Scotland) to continuously track their change in location across four different areas (one indoor and three outdoor areas). Using a combination of social network analysis for quantifying social relationships and dynamic time warping for characterizing the movement patterns of hens, we found that hens were consistent in their individual variation in temporal activity and maintained stable social relationships in terms of preferred association partners. In addition to being consistent, social associations correlated with movement patterns and this correlation strengthened over the period of observation, suggesting that the animals aligned their activity patterns with those of their social affiliates. These results demonstrate the importance of social relationships when considering the expression of individual behaviour. Notably, differences in temporal patterns emerge despite rather homogeneous rearing conditions, same environment, and low genetic diversity. Thus, while variation in behavioural phenotypes can be observed across isolated individuals, this study shows that the social environment within a group can shape and enhance variation in general movement patterns of individual animals.

Groups and Individuals: Optical Flow Patterns of Broiler Chicken Flocks Are Correlated with the Behavior of Individual Birds.

Group level measures of welfare flocks have been criticized on the grounds that they give only average measures and overlook the welfare of individual animals. However, we here show that the group-level optical flow patterns made by broiler flocks can be used to deliver information not just about the flock averages but also about the proportion of individuals in different movement categories. Mean optical flow provides information about the average movement of the whole flock while the variance, skew and kurtosis quantify the variation between individuals. We correlated flock optical flow patterns with the behavior and welfare of a sample of 16 birds per flock in two runway tests and a water (latency-to-lie) test. In the runway tests, there was a positive correlation between the average time taken to complete the runway and the skew and kurtosis of optical flow on day 28 of flock life (on average slow individuals came from flocks with a high skew and kurtosis). In the water test, there was a positive correlation between the average length of time the birds remained standing and the mean and variance of flock optical flow (on average, the most mobile individuals came from flocks with the highest mean). Patterns at the flock level thus contain valuable information about the activity of different proportions of the individuals within a flock.

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.

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.

Improving intra- and inter-observer repeatability and accuracy of keel bone assessment by training with radiographs.

Assessing keel bone damage reliably and accurately is a requirement for all research on this topic. Most commonly, assessment is done on live birds by palpation and is therefore prone to bias. A 2-day Training School of the COST Action "Identifying causes and solutions of keel bone damage in laying hens" with 16 participants of variable experience was held where palpation of live hens was followed by consulting corresponding radiographic images of keel bones. We hypothesized that the inter-observer and intra-observer repeatabilities as well as the agreement between palpation and assessment from the radiograph (considered as the accuracy) would increase from day 1 to 2. Repeatability estimates were calculated using the R-package rptR and the change in level of accuracy on day 1 and 2 was analyzed with generalized linear models. As predicted, the inter-observer repeatabilities of the assessments of the fractures and deviations were improved by training, but this improvement differed for fractures and deviations between the cranial, middle, and caudal parts of the keel bone. Intra-observer repeatabilities before training also differed between the different parts of the keel bone and were highest for fractures at the caudal part of the keel bone. The training affected the accuracy of palpation to different degrees for the different parts of the keel bone. A training effect was found for the caudal part of the keel bone in regard to fractures and deviations, but for fractures the training effect was missing for the cranial part and for deviations it was missing for the middle part of the keel bone. In conclusion, the training school involving radiographs improved inter-observer repeatabilities in the diagnosis of fractures and deviations of keel bones and thus had the potential to lead to more comparable results among research groups.

Improving intra- and inter-observer repeatability and accuracy of keel bone assessment by training with radiographs.

Assessing keel bone damage reliably and accurately is a requirement for all research on this topic. Most commonly, assessment is done on live birds by palpation and is therefore prone to bias. A 2-day Training School of the COST Action "Identifying causes and solutions of keel bone damage in laying hens" with 16 participants of variable experience was held where palpation of live hens was followed by consulting corresponding radiographic images of keel bones. We hypothesized that the inter-observer and intra-observer repeatabilities as well as the agreement between palpation and assessment from the radiograph (considered as the accuracy) would increase from day 1 to 2. Repeatability estimates were calculated using the R-package rptR and the change in level of accuracy on day 1 and 2 was analyzed with generalized linear models. As predicted, the inter-observer repeatabilities of the assessments of the fractures and deviations were improved by training, but this improvement differed for fractures and deviations between the cranial, middle, and caudal parts of the keel bone. Intra-observer repeatabilities before training also differed between the different parts of the keel bone and were highest for fractures at the caudal part of the keel bone. The training affected the accuracy of palpation to different degrees for the different parts of the keel bone. A training effect was found for the caudal part of the keel bone in regard to fractures and deviations, but for fractures the training effect was missing for the cranial part and for deviations it was missing for the middle part of the keel bone. In conclusion, the training school involving radiographs improved inter-observer repeatabilities in the diagnosis of fractures and deviations of keel bones and thus had the potential to lead to more comparable results among research groups.

Keel bone fractures affect egg laying performance but not egg quality in laying hens housed in a commercial aviary system.

The aim of this study was to investigate the effect of keel bone fracture (KBF) severity and healing activity on individual productivity of laying hens. Focal hens (75 Lohmann Selected Leghorn (LSL), 75 Lohmann Brown (LB)) were housed alongside non-focal hens in 10 identical pens containing a commercial aviary system (15 focal hens per pen). Eggs of focal hens were identified by orally administering a dye on 3 consecutive days, resulting in a hen-specific color pattern in the yolk. Eggs were collected at 7 time points (37 to 61 weeks of age; WOA) for 5 d to determine individual laying performance and to assess egg quality. Radiographs were performed to score KBF severity on a continuous scale. Healing activity was scored as inactive, healing, or fresh. Linear mixed effects models were used for statistical analyses. We found an association between KBF severity and reduced egg laying performance with increasing age (P = 0.005). At 37 WOA, egg laying performance was similar across KBF severities, whereas at 61 WOA, performance in hens with the highest observed KBF severity was 16.2% lower than in hens without fractures. Hens with fresh fractures had a lower performance than hens with healing and inactive fractures at 37 WOA but higher performance at 61 WOA (P = 0.02). Egg quality parameters were not affected by fractures but were associated with an age × hybrid interaction (egg mass: P = 0.039, shell breaking strength: P = 0.03, shell width: P = 0.001). In conclusion, hens could maintain high performance irrespective of fracture severity until shortly after peak of lay, but seemed to redirect available resources towards fracture healing if a fresh fracture was present. At the end of lay, the negative effect of KBF fracture severity on individual production amplified indicating that hens were no longer able to cope with the physiological challenge of a fracture.

A Reliable Method to Assess Keel Bone Fractures in Laying Hens From Radiographs Using a Tagged Visual Analogue Scale.

Up to 97% of laying hens housed in aviary systems are affected by keel bone fractures. Due to the scope of the problem, multiple efforts investigating causes and consequences of fractures have been conducted. The most frequently used techniques to detect fractures lack accuracy and provide only vague information (palpation) or cannot be conducted longitudinally (dissection). Radiographic imaging overcomes these weaknesses as it allows longitudinal observations and provides detailed information for individual fractures of which a single keel may have several at different locations and of different origins. However, no standardized system exists to assess fracture severity from radiographs if multiple fractures are present. The aim of this study was therefore to test the reliability of a scoring system assessing the aggregate severity of multiple fractures, taking into account the characteristics of all present fractures (e.g., locations, callus formation, width of fracture gaps). We developed a scoring system based on a tagged visual analogue scale, ranging from score 0 (no fracture) to score 5 (extremely severe) with intermediate tags for scores 1, 2, 3, and 4. A catalog of example scores was provided to describe the range of each score visually. An online tutorial with an introduction, training and scoring session was completed by 14 participants with varying experience involving laying hens and keel bone damage. For inter-observer reliability, we found an Intraclass correlation coefficient (ICC) of 0.985 with a 95% confidence interval of 0.974 < ICC < 0.993 (average-rating, absolute-agreement, two-way random-effects model). Intraclass correlation coefficient for intra-observer reliability was 0.923 with a 95% confidence interval of 0.879 < ICC < 0.951 (single-rating, absolute-agreement, two-way mixed-effects model). Intra-observer reliability ranged from 0.704 to 1.0 indicating excellent agreement and similar ratings across and within participants. Further, high ICCs suggest that the introduction and the training sessions provided were adequate tools to prepare observers for the assessment task despite various backgrounds of the participants. Nonetheless, the validity of this scoring system needs to be investigated further in order to link responses of interest and biological relevance with the specific severity values resulting from our scoring system.

Susceptibility to keel bone fractures in laying hens and the role of genetic variation.

Keel bone fractures are a well-known welfare problem in modern commercial laying hen systems. The present study sought to identify genetic variation in relation to keel bone fracture susceptibility of 4 distinct crossbred and one pure line, and by extension, possible breeding traits. Susceptibility to fractures were assessed using an ex vivo impact testing protocol in combination with a study design that minimized environmental variation to focus on genetic differences. The 5 crossbred/pure lines differed in their susceptibility to keel bone fractures with the greatest likelihood of fracture in one of the 3 commercial lines and the lowest susceptibility to fractures in one of the experimental lines. Egg production at the hen-level did not differ between the crossbred/pure lines (P > 0.05), though an increased susceptibility to keel bone fractures was associated with thinner eggshells and reduced egg breaking strength, a pattern consistent among all tested crossbred/pure lines. Our findings suggest an association between egg quality and bone strength which appeared to be independent of crossbred/pure line. The findings indicate the benefit of the impact methodology to identify potential breeding characteristics to reduce incidence of keel fracture as well as the potential relationship with eggshell quality.

Soft perches in an aviary system reduce incidence of keel bone damage in laying hens.

Keel bone fractures and deviations are one of the major welfare and health issues in commercial laying hens. In non-cage housing systems like aviaries, falls and collisions with perches and other parts of the housing system are assumed to be one of the main causes for the high incidence of keel bone damage. The objectives of this study were to investigate the effectiveness of a soft perch material to reduce keel bone fractures and deviations in white (Dekalb White) and brown laying hens (ISA Brown) kept in an aviary system under commercial conditions. In half of 20 pens, all hard, metal perches were covered with a soft polyurethane material. Palpation of 20 hens per pen was conducted at 18, 21, 23, 30, 38, 44 and 64 weeks of age. Production data including egg laying rate, floor eggs, mortality and feed consumption were collected over the whole laying period. Feather condition and body mass was assessed twice per laying period. The results revealed that pens with soft perches had a reduced number of keel bone fractures and deviations. Also, an interaction between hybrid and age indicated that the ISA hybrid had more fractured keel bones and fewer non-damaged keel bones compared with the DW hybrid at 18 weeks of age, a response that was reversed at the end of the experiment. This is the first study providing evidence for the effectiveness of a soft perch material within a commercial setting. Due to its compressible material soft perches are likely to absorb kinetic energy occurring during collisions and increase the spread of pressure on the keel bone during perching, providing a mechanism to reduce keel bone fractures and deviations, respectively. In combination with genetic selection for more resilient bones and new housing design, perch material is a promising tool to reduce keel bone damage in commercial systems.