High-throughput phenotyping to characterise range use behaviour in broiler chickens.

A key characteristic of free-range chicken farming is to enable chickens to spend time outdoors. However, each chicken may use the available areas for roaming in variable ways. To check if, and how, broilers use their outdoor range at an individual level, we need to reliably characterise range use behaviour. Traditional methods relying on visual scans require significant time investment and only provide discontinuous information. Passive RFID (Radio Frequency Identification) systems enable tracking individually tagged chickens' when they go through pop-holes; hence, they only provide partial information on the movements of individual chickens. Here, we describe a new method to measure chickens' range use and test its reliability on three ranges each containing a different breed. We used an active RFID system to localise chickens in their barn, or in one of nine zones of their range, every 30 seconds and assessed range-use behaviour in 600 chickens belonging to three breeds of slow- or medium-growing broilers used for outdoor production (all <40 g daily weight gain). From those real-time locations, we determined five measures to describe daily range use: time spent in the barn, number of outdoor accesses, number of zones visited in a day, gregariousness (an index that increases when birds spend time in zones where other birds are), and numbers of zone changes. Principal Component Analyses (PCAs) were performed on those measures, in each production system, to create two synthetic indicators of chickens' range use behaviour. The first two PCA axes represented over 90% of the variance of the five measures and were both consistent over time and correlated with independent visual scans. Contributions of the five measures to the PCAs were similar among breeds, except for the correlation between the number of outdoor accesses and the four other measures. PC1 correlated with time spent inside the barn and zone changes frequency, whilst PC2 was explained by exploration of the range. Taken together, PC1 and PC2 indicators showed that range use increased with age, outdoor temperature (in spring), and did not differ between males and females. Importantly, daily scores for both indicators were repeatable among individuals - particularly in PC1 - showing inter-individual variability on range-use. The characterisation of broiler behaviour around their range with these reliable and repeatable indicators provides novel tools to help understand individual variations of range-use in free-range farming.

Foraging Behavior Shows Individual-Consistency Over Time, and Predicts Range Use in Slow-Growing Free-Range Male Broiler Chickens.

Recent research on free-range chickens shows that individual behavioral differences may link to range use. However, most of these studies explored individual behavioral differences only at one time point or during a short time window, assessed differences when animals were out of their social group and home environment (barn and range), and in specific tests or situations. Therefore, it is yet unclear how different behaviors relate to range use and how consistent these behaviors are at the individual level. To fill this gap, we here aimed to describe the behavioral budget of slow-growing male broiler chickens (S757N) when in their social group and home environment during the whole rearing period (from the second week of life to the twelfth week, before slaughter), and to relate observed behavioral differences to range use. For this, we followed a sample of individuals in two flocks (n = 60 focal chickens out of 200 chickens per flock), over two seasons, during three periods: before range access (from 14 to 25 days old), during early range access (first weeks of range access, from 37 to 53 days old), and during late range access (last weeks of range access, from 63 to 87 days old). By the end of each period, individual tests of exploration and social motivation were also performed, measuring exploration/activity and sociability propensities. Our results show that foraging (i.e., pecking and scratching at the ground) was the only behavior that correlated to range use for all three rearing periods, independent of the season. Foraging was also the only behavior that showed within-individual consistency from an early age and across the three rearing periods. Foraging may, therefore, serve as a useful behavioral predictor of range use in free-range broiler chickens. Our study increases the knowledge of how behaviors develop and relate to each other in a domesticated and intensely selected species, and improves our understanding of the biology of free-range broiler chickens. These findings can, ultimately, serve as a foundation to increase range use and improve chicken welfare.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

Working for food is related to range use in free-range broiler chickens.

When animals prefer to make efforts to obtain food instead of acquiring it from freely available sources, they exhibit what is called contrafreeloading. Recently, individual differences in behavior, such as exploration, were shown to be linked to how prone an individual may be to contrafreeload. In this work, our main objective was to test whether and how individual differences in range use of free-range broiler chickens (Gallus gallus domesticus) were related to the individual motivation to contrafreeload. We also verified whether other behavioral variations could relate to range use. To that aim, over three different periods (before range access, first weeks of range access, and last weeks of range access), chickens with different ranging levels (low and high rangers) were submitted to a contrafreeloading test and had different behaviors recorded (such as foraging, resting, locomotion) in their home environment. During the contrafreeloading test, chickens were conditioned to one chamber presenting a foraging substrate and mealworms, while in the other chamber, mealworms were freely available on the floor. During testing trials, chickens had access to both empty chambers, and the time spent in each chamber was quantified. On average, low rangers preferred the chamber where mealworms were easily accessible (without the foraging substrate), while high rangers preferred the chamber where mealworms were accessible with difficulty, showing greater contrafreeloading. Out of ten behaviors recorded in chickens' home environment, foraging was the only one that differed significantly between our two ranging groups, with low rangers foraging, on average, significantly less than high rangers. These results corroborate previous experiences suggesting that range use is probably linked to chickens' exploratory trait and suggest that individual differences in free-range broiler chickens are present even before range access. Increasing our knowledge of individual particularities is a necessary step to improve free-range chicken welfare on the farm.

Social motivation and the use of distal, but not local, featural cues are related to ranging behavior in free-range chickens (Gallus gallus domesticus).

Animals can navigate an environment relying on different sources of information, such as geometrical or featural cues. The favoring of one type of information over another depends on multiple factors, such as inter-individual differences in behavior and cognition. Free-range chickens present different range use patterns, which may be explained by behavioral and cognitive differences. However, how behavior, cognition, and range use intercorrelate is still poorly understood. In this work, we aimed to further understand possible differences in behavior and cognition between two groups of free-range broiler chickens: those who frequently explore their range ('high rangers') and those who prefer to stay in or near the barn ('low rangers'). Prior to range access, individual behavior was measured in open field-, emergence-, and social motivation tests. To investigate cognitive differences, we analyzed whether exploratory behavior was linked to different performances in the use of distal and local spatial cues during an orientation task. During the social motivation test, low rangers showed a higher inclination to be near conspecifics than did high rangers. Our orientation tests show that chickens preferred to orientate themselves using the local cues over the distal cues. Individual differences were only found for distal, but not for local, cue use suggesting that demanding tasks are more efficient in revealing individual cognitive differences. Our results suggest that considering variation in social motivation may allow a more comprehensive understanding of chicken range use. Our results also support the importance of incorporating multiple aspects of individual differences to understand individual reactions to its environment.

Uninhibited chickens: ranging behaviour impacts motor self-regulation in free-range broiler chickens (Gallus gallus domesticus).

Inhibiting impulsive, less flexible behaviours is of utmost importance for individual adaptation in an ever-changing environment. However, problem-solving tasks may be greatly impacted by individual differences in behaviour, since animals with distinct behavioural types perceive and interact with their environment differently, resulting in variable responses to the same stimuli. Here, we tested whether and how differences in ranging behaviour of free-range chickens affect motor self-regulation performance during a cylinder task. For this task, subjects must refrain from trying to reach a food reward through the walls of a transparent cylinder and detour to its open sides, as a sign of inhibition. Free-range chickens exhibited an overall low performance in the motor self-regulation task (31.33 ± 13.55% of correct responses), however, high rangers showed significantly poorer performance than the low rangers (23.75 ± 9.16% versus 40 ± 12.90%, respectively). These results give further support to the impacts of individual behavioural differences on cognitive performances. This is the first demonstration to our knowledge of a relationship between exploratory tendencies and motor self-regulation for an avian species.

Relationship between ranging behavior and spatial memory of free-range chickens.

Different personalities may lead to different ways of processing environmental information; however, the relationship between personality and cognition is not fully understood as studies on diverse species present contrasting results. As there is great within-flock variability of outdoor ranging behavior in free-range broiler chickens, we tested whether and how ranging behavior impacts on individual spatial memory abilities. The experiment was conducted on one flock (n = 200) reared in the same conditions throughout the study, to simulate on-farm situations. As the ranging behavior was stable over time, we compared two distinct groups of male chickens: one that visited the range more (High rangers) and one that was more prone to staying in the poultry house (Low rangers). To test the spatial memory, individuals (n = 30) went through two main phases in an arena with 8 cups. For the familiarization phase, individuals were submitted to one trial per day, for seven days, to a situation where all eight cups were baited with mealworms. Animals had to reach a criterion of 5 cups visited out of 8 to advance to the next phase. For the spatial test, only four cups were baited and systematically placed at the same location. This last phase comprised two trials per individual per day, for nine days. During these two phases, latency to visit cups and the number of visits and revisits of all cups were recorded. Low ranger chickens took less time to attain the pre-established threshold of visiting 5 cups out of 8, over the familiarization phase. During the spatial test, the latency to visit four cups decreased between the within-day trials for low ranger chickens and increased for high ranger chickens. Moreover, in the within-day trial analysis, low ranger chickens exhibited an improvement on spatial memory and better spatial memory compared to high ranger chickens. Different speed-accuracy trade-offs may explain these differences between low and high ranger chickens and the way individuals interact and solve the task. Our study strengthens the scientific evidence relating consistent individual differences in behavior, with the ranging behavior of free-range chickens, and cognitive performance during a spatial memory task.

Effects of gut microbiota transfer on emotional reactivity in Japanese quails (Coturnix japonica).

The interaction between the gut microbiota (GM) and the brain has led to the concept of the microbiota-gut-brain axis but data for birds remain scarce. We tested the hypothesis that colonization of germ-free chicks from a quail line selected for a high emotional reactivity (E+) with GM from a line with low emotional reactivity (E-) would reduce their emotional behaviour in comparison with germ-free chicks from an E+ line colonized with GM from the same E+ line. The GM composition analysis of both groups revealed a shift in terms of microbial diversity and richness between day 21 and day 35 and the GM of the two groups of quails were closer to each other at day 35 than at day 21 at a phylum level. Quails that received GM from the E- line expressed a lower emotional reactivity than quails colonized by GM from the E+ line in tonic immobility and novel environment tests carried out during the second week of age. This result was reversed in a second tonic immobility test and an open-field run 2 weeks later. These behavioural and GM modifications over time could be the consequence of the resilience of the GM to recover the equilibrium present in the E+ host, which is in part driven by the host genotype. This study shows for the first time that a GM transfer can influence emotional reactivity in Japanese quails, supporting the existence of a microbiota-gut-brain axis in this species of bird.

Does lipidomic serum analysis support the assessment of digestive efficiency in chickens?

The increasing cost of conventional feedstuffs used in poultry diets has bolstered interest in genetic selection for digestive efficiency (DE) to improve the adaptation of the birds to various alternative feedstuffs. However, DE measurement through AMEn is time-consuming and constraining. To simplify selection for DE, the potential of serum composition to predict AMEn was evaluated based on 40 birds from two broiler lines (D+ and D-) divergently selected on the fecal AMEn of a difficult-to-digest wheat-based diet. Differences in serum coloration were suspected between the two lines, and thus a spectrophotometric analysis was carried out, revealing a significant difference in absorption between 430 nm and 516 nm, corresponding to the signature of orange-red lipophilic pigments such as xanthophylls. To go further, the liposoluble fraction of the serum was explored for its lipidome by mass spectrometry. Discriminant analysis revealed that a pattern of 10 metabolites, including zeaxanthin/lutein, can explain 82% of the lipidomic differences between the two lines. Colorimetry combined with lipidomics studies confirmed the relationship between digestive efficiency and serum composition, which opens up new possibilities for using it as a quick and easy proxy of digestive efficiency.

Field evaluation of a gag/env heteroduplex mobility assay for genetic subtyping of small-ruminant lentiviruses.

Small-ruminant lentiviruses (SRLVs) display a high genetic diversity and are currently classified into five genotypes and an increasing number of subtypes. The co-circulation of subtypes in restricted geographical regions, combined with the occurrence of cross-species infection, suggests the need for development of a large-scale screening methodology for rapid monitoring of the prevalence of the various genetic subtypes and their genetic evolution. Here, a heteroduplex mobility assay (HMA) was developed for the rapid identification of group B subtypes. The assay was validated for both the p14 nucleocapsid-coding region of the gag gene and the V1-V2 region of the env gene using a panel of reference standards and was applied to the genetic subtyping of SRLV field isolates from five mixed flocks in France. Subtyping of 75 blood samples using the env HMA revealed a preferential distribution of subtypes B1 and B2 in sheep and goats, despite direct evidence for interspecies transmission of both subtypes. Adding the gag HMA to the env HMA provided evidence for dual infection and putative recombination between subtypes B1 and B2 in five goats, and between groups A and B in one sheep. Phylogenetic analysis revealed that 100 % (23/23) and 96.7 % (30/31) of samples were correctly classified using the gag and env HMAs, respectively. These results indicate that dual infection and recombination may be a significant source of new variation in SRLV and provide a useful tool for the rapid genetic subtyping of SRLV isolates, which could be relevant for the development of more accurate diagnosis of prevalent SRLV strains in different countries.

Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep.

Small ruminants lentiviruses (SRLV) nucleotide sequences spanning the V1V2 variable regions of the env gene were amplified by nested-PCR from 38 blood samples collected from 16 naturally infected sheep flocks in France. For the rapid SRLV group determination of field isolates, the PCR-amplified fragments were subjected to a SRLV-adapted heteroduplex mobility assay (HMA). All viral sequences were clearly assignable to the SRLV group B by HMA analysis. Twenty-seven SRLV isolates were selected for DNA sequence analysis. In each case, nucleotide comparison and phylogenetic analyses confirmed the genetic relationships inferred by HMA. Six SRLV isolates belonged to subtype B1, and 21 pertained to subtype B2, one flock being infected with both subtypes. Subtypes B1 and B2 were found with different frequencies and geographic spread, but exhibited similar genetic diversities. These results give a more complete picture of the distribution and heterogeneity of SRLV env subtypes in sheep and confirmed that multiple interspecies transmission occurred in the past. Furthermore, HMA appeared to be a rapid and reliable method to differentiate caprine arthritis encephalitis virus from maedi-visna virus.

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III.

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.