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Physical exercise can act as a physiological and a mental stressor. Monitoring exercise-induced stress is therefore essential to understand racehorses ‘responses to effort and to ensure their welfare. Stress perceived by the horse during physical activity can be measured using various indicators, including stress-related hormones such as cortisol and adrenaline, and other neuromodulators such as serotonin, all involved in the stress response and its regulation. Another approach to assess physiological and emotional responses to stimuli such as exercise is through behaviours and facial movements. In this study, we aimed to 1) evaluate the changes in these three hormones following a trotting exercise, 2) determine the changes in behaviour and facial movements in response to the same exercise and 3) investigate potential relationships between hormonal variations and specific behavioural patterns that could serve as indicators of exercise-induced stress in horses. Fourteen French Standardbred horses from two stables were monitored over one day. In the morning, they performed an interval training trotting exercise. Behaviours and facial movements were recorded via video for 2 min 30 both before and just after exercise. Saliva and blood samples were collected at four time points: before exercise, just after exercise, 1 h post-exercise and 24 h post-exercise to assess salivary cortisol, and serum concentrations concentration of adrenaline and serotonin. Results showed significant post-exercise increases in all three hormones with peak concentrations observed immediately after exercise, and elevated cortisol and adrenaline levels persisting one hour later. These variations are consistent with normal physiological responses to physical effort, reflecting activation of regulatory systems rather than necessarily indicating negative stress. However, inter-individual variability in the magnitude of these responses suggests that horses did not all experience the exercise in the same way, highlighting potential differences exercise-induced stress. In terms of behaviour, horses exhibited higher frequencies of facial movements, particularly mouth movements, after exercise than before. Moreover, increases in serotonin and adrenaline concentrations were positively associated with agitation-related behaviours (pawing and head turning) and mouth movements. Overall, our findings suggest that a behavioural profile characterised by increased agitation and mouth movements may reflect a post-exercise arousal response in French Standardbreds. These behaviours, in association with hormonal changes, could provide a useful non-invasive tool to assess to assess horses’ response to exercise, and potentially exercise-induced stress. However, further studies are needed to confirm this interpretation, particularly by investigating the potential effects of post-exercise management practices such as cross-tying, which may induce frustration.

We simultaneously measured the fecal microbiota and multiple environmental and host-related variables in a cohort of 185 healthy horses reared in similar conditions during a period of eight months. The pattern of rare bacteria varied from host to host and was largely different between two time points. Among a suite of variables examined, equitation factors were highly associated with the gut microbiota variability, evoking a relationship between gut microbiota and high levels of physical and mental stressors. Behavioral indicators that pointed toward a compromised welfare state (e.g. stereotypies, hypervigilance and aggressiveness) were also associated with the gut microbiota, reinforcing the notion for the existence of the microbiota-gut-brain axis. These observations were consistent with the microbiability of behaviour traits (> 15%), illustrating the importance of gut microbial composition to animal behaviour. As more elite athletes suffer from stress, targeting the microbiota offers a new opportunity to investigate the bidirectional interactions within the brain gut microbiota axis.

Elite horse athletes that live in individual boxes and train and compete for hours experience long-term physical and mental stress that compromises animal welfare and alters the gut microbiota. We therefore assessed if a temporary period out to pasture with conspecifics could improve animal welfare and in turn, favorably affect intestinal microbiota composition. A total of 27 athletes were monitored before and after a period of 1.5 months out to pasture, and their fecal microbiota and behavior profiles were compared to those of 18 horses kept in individual boxes. The overall diversity and microbiota composition of pasture and control individuals were temporally similar, suggesting resilience to environmental challenges. However, pasture exposure induced an increase in Ruminococcus and Coprococcus that lasted 1-month after the return to individual boxes, which may have promoted beneficial effects on health and welfare. Associations between the gut microbiota composition and behavior indicating poor welfare were established. Furthermore, withdrawn behavior was associated with the relative abundances of Lachnospiraceae AC2044 group and Clostridiales family XIII. Both accommodate a large part of butyrate-producing bacterial genera. While we cannot infer causality within this study, arguably, these findings suggest that management practices maintained over a longer period of time may moderate the behavior link to the gut ecosystem beyond its resilience potential.

Horses are mainly housed in individual boxes. This housing system is reported to be highly detrimental with regard to welfare and could trigger the expression of four behavioural indicators of a compromised welfare state: stereotypies, aggressiveness toward humans, unresponsiveness to the environment, and stress-related behaviours. The aim of this study was to identify housing and management factors that could alleviate the detrimental effects of individual boxes on welfare. A total of 187 horses were observed over 50 days by scan sampling. The impact of 12 factors was investigated on the expression of the four behavioural indicators in three different analyses. The results show that the majority of factors tested did not influence the expression of the behavioural indicators. Only three (straw bedding, a window opening onto the external environment, and a reduced quantity of concentrated feed) would have beneficial, although limited, effects. Furthermore, the longer the horses spent in individual boxes, the more likely they were to express unresponsiveness to the environment. To preserve the welfare of horses, it seems necessary to allow free exercise, interactions with conspecifics, and fibre consumption as often as possible, to ensure the satisfaction of the species’ behavioural and physiological needs.

Societal concerns for animal welfare extend to all domestic species, including high-level sport horses. The welfare of these horses, notably highlighted during the recent Olympics, has garnered significant public interest, prompting inquiries into their living conditions. Animal welfare studies have emphasised three key needs crucial to equine welfare: unlimited access to forage, freedom of movement, and social interactions with peers, commonly referred to as the “3Fs”—access to Forage, Freedom of movement, and interactions with Friend conspecifics. However, the feasibility and benefits of satisfying these needs specifically for sport horses remain unexplored. Indeed, they may face unique challenges such as high physical workload, extensive travel, limited time in their home stables, weight management, and high economic value necessitating careful handling. Consequently, restrictions on feeding, freedom of movement, and social contact are often deemed necessary. This field study aims to assess the actual level of implementation of welfare in high-level sport horses by evaluating body condition, injury risk, and behavioural welfare indicators in their home stable. To achieve this objective, the welfare of 56 high-level sport horses competing internationally was assessed using behavioural indicators of welfare through scan sampling (abnormal behaviours, i.e., stereotypies, aggression towards humans, withdrawn behaviour, and alert behaviours; positions of the ears in a backward position while foraging, watching behaviours, and through other Animal Welfare Indicators (AWIN) protocol measures). This study shows that there exists a large variability among horses regarding their access to the 3Fs, with some of them having a lot of restrictions and others not, meaning it is possible to respect them while competing at a high level. Second, we observed that the fewer restrictions the horses experience regarding the 3Fs while in their home stables, the better their welfare, as demonstrated by the indicators we assessed. These results undeniably support the fact that unrestricted access to forage, the ability to move freely outdoors, and the opportunity to interact socially with conspecifics are fundamental needs of horses that could be provided to horses, also to high-performance ones. It is therefore essential that solutions are put in place to ensure that these conditions are met.

Health and performance of vaulting horses cantering with reins might be affected by rein tensions. The primary aim of this present study was to measure rein and lunge line tensions in international-level vaulting horses with several types of reins adjusted in accordance with the requirements of the FEI Vaulting Rules and study the effect of reins types on it. The secondary aim was to evaluate behavioural signs of discomfort under the same conditions and study the effect of reins types on it. The relationship between tensions and behavioural signs of discomfort was then explored. Thirty-nine international-level vaulting horses took part in this study and tested side reins either with an elastic part, all elastic or without elastic, draw reins with an upper adjustment triangle and side reins fixed on the noseband. Tensions of the left and right reins and the lunge line were measured with sensors at a rate of 80 Hz for a canter of 1min30 on a 15-meter circle, without a vaulter, and behavioural analyses (on the locomotion, tail, head, mouth and ears movements) were performed using video filmed by a camera attached to the lunger’s chest. Mixed models revealed that the Root mean square (RMS) of the tensions of the left and right reins, of the total, as well as the lunge line, were significantly influenced by rein type (p<0.001) in all cases). Total RMS tension ranged from 6.2 kg to 11.7 kg with a right RMS tension superior to left RMS (p < 0.001). When tension was high, mouth opening increased (p = 0.007). It was with draw reins that total tension (p < 0.05) and mouth openings were lower (p = 0.005), even if a high proportion of variance of these discomfort behaviours could be explained by the individual horse.

We simultaneously measured the fecal microbiota and multiple environmental and host-related variables in a cohort of 185 healthy horses reared in similar conditions during a period of eight months. The pattern of rare bacteria varied from host to host and was largely different between two time points. Among a suite of variables examined, equitation factors were highly associated with the gut microbiota variability, evoking a relationship between gut microbiota and high levels of physical and mental stressors. Behavioral indicators that pointed toward a compromised welfare state (e.g. stereotypies, hypervigilance and aggressiveness) were also associated with the gut microbiota, reinforcing the notion for the existence of the microbiota-gut-brain axis. These observations were consistent with the microbiability of behaviour traits (> 15%), illustrating the importance of gut microbial composition to animal behaviour. As more elite athletes suffer from stress, targeting the microbiota offers a new opportunity to investigate the bidirectional interactions within the brain gut microbiota axis.

Stereotypies in animals are thought to arise from an interaction between genetic predisposition and sub-optimal housing conditions. In domestic horses, a well-studied stereotypy is crib-biting, an abnormal behaviour that appears to help individuals to cope with stressful situations. One prominent hypothesis states that animals affected by stereotypies are cognitively less flexible compared to healthy controls, due to sensitization of a specific brain area, the basal ganglia. The aim of this study was to test this hypothesis in crib-biting and healthy controls, using a cognitive task, reversal learning, which has been used as a diagnostic for basal ganglia dysfunction. The procedure consisted of exposing subjects to four learning tasks; first and second acquisition, and their reversals. For each task, we measured the number of trials to reach criterion and heart rate and heart-rate variability. Importantly, we did not try to prevent crib-biters from executing their stereotypic behaviour. We found that the first reversal learning task required the largest number of trials, confirming its challenging nature. Interestingly, the second reversal learning task required significantly fewer trials to reach criterion, suggesting generalisation learning. However, we did not find any performance differences across groups; both stereotypic and control animals required a similar numbers of trials and did not differ in their physiological responses. Our results thus challenge the widely held belief that crib-biting horses, and stereotypic animals more generally, are cognitively impaired. We conclude that cognitive underperformance may occur in stereotypic horses if they are prevented from crib-biting to cope with experienced stress.

Pasture access is widely recognized for its welfare, behavioural, and health benefits in horses compared to individual stabling. Our recent studies have shown that even short-term pasture exposure enhances welfare and induces lasting changes in gut microbiota, promoting health-associated bacterial populations. This study investigated the transcriptomic signature of 22 horses following a standardized pasture protocol, with peripheral blood gene expression analysed before pasture access (T0) and three months after returning to individual stabling (T1). First, using sPLS regression, we correlated gene expression profiles with behavioural welfare indicators measured at T0 and T1. Horses with pasture access exhibited significantly altered blood transcriptome compared to controls, with aggressiveness towards humans emerging as the strongest behavioural correlate of gene expression. Bioinformatic analyses revealed that aggressiveness-associated genes were linked to inflammation, apoptosis, and cell differentiation/growth. Then, horses with pasture access were divided in resilient and non-resilient according to the improvement of their behaviour after time on pasture. Differentially expressed genes post versus pre-pasture, compared between resilient and non-resilient horses for aggressiveness showed that inflammatory signals were downregulated in both subgroups, with a more pronounced effect in resilient horses. This suggests that resilient individuals are better equipped to modulate inflammatory responses in low-stress environments like pasture. Surprisingly, for unresponsiveness to the environment - a trait linked to depressive-like states - resilient horses displayed increased inflammatory signaling (e.g., IRF and CD40 activation) post-pasture, while non-resilient horses showed activation of anti-inflammatory PPAR signaling. Notably, non-resilient horses exhibited molecular signatures associated with organismal death, morbidity, and growth failure, indicating maladaptive physiological states. In contrast, resilient horses demonstrated activation of growth-related pathways (e.g., BMP2 and BMPR1A), suggesting a shift toward anabolic and developmental processes. This study underscores the behavioural and molecular benefits of pasture access for horses, particularly in reducing aggressiveness and inflammatory signaling in resilient individuals. The findings highlight the complex interplay between behaviour, inflammation, and resilience, with pasture access promoting adaptive physiological and molecular responses. Further research is needed to elucidate the long-term implications of these transcriptomic changes and their broader relevance to equine welfare.Competing Interest StatementThe authors have declared no competing interest.Footnotes* A minor error has been corrected: the sPLS analysis was performed on the data measured at T0 and T1, and not only at T1 as indicated in the previous version.* https://doi.org/10.5281/zenodo.18622341* https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE310717Funder Information DeclaredInstitut Français du Cheval et de l'Équitation, https://ror.org/05pf1p208

Physical exercise can act as a physiological and a mental stressor. Monitoring exercise-induced stress is therefore essential to understand racehorses’ responses to effort and ensuring their welfare. Stress perceived by the horse during physical activity can be measured using various indicators, including stress-related hormones such as cortisol and adrenaline, and other neuromodulators such as serotonin, all involved in the stress response and its regulation. Another approach to assess physiological and emotional responses to stimuli such as exercise is through behaviours and facial movements. In this study, we aimed to 1) evaluate the changes in these three hormones following a trotting exercise, 2) determine the changes in behaviour and facial movements in response to the same exercise and 3) investigate a potential relationship between hormonal variations and specific behavioural patterns that could serve as indicators of exercise-induced stress in horses. Fourteen French Standardbred horses from two stables were monitored over one day. On the morning they performed a trotting exercise. Behaviours and facial movements were recorded via video for 2min30 both before and just after exercise. Saliva and blood samples were collected at four time points: before exercise, just after exercise, 1h post-exercise and 24h post-exercise to assess the concentration of salivary cortisol, serum concentration of adrenaline and serotonin. Results showed significant post-exercise increases in all three hormones with peak concentrations observed immediately after exercise (general mixed models, p<0.05), and elevated cortisol and adrenaline levels persisting one hour later (general mixed models, p<0.05). In terms of behaviours, horses exhibited higher frequencies of facial movements and especially mouth movements after exercise than before (MANOVA, F=5.85, p<0.01). Moreover, increases in serotonin and adrenaline concentrations were positively associated with agitation-related behaviours (pawing and head turning) and mouth movements (general linear models, p<0.01). This association may reflect a rebound effect of bit pressure on the mouth, which may be greater with greater perceived stress during exercise, and the increased arousal effects of adrenaline, and to a lesser extent, serotonin. Overall, our findings suggest that a behavioural profile characterised by increased agitation and mouth movements may serve as a useful non-invasive indicator of exercise-induced stress in French Standardbreds.