Maternal reactivity of ewes at lambing is genetically linked to their behavioural reactivity in an arena test.

In sheep, the bond between the dam and her lambs is established during the first hours of a lamb's life. Genetic variability for behavioural reactivity of ewes assessed in an arena test performed 24 hr after lambing has already been reported. However, there is no evidence that this reactivity represents the ewe's maternal reactivity at lambing in outdoor conditions. The objective of this study was to investigate whether or not the behavioural reactivity of ewes in the arena test is genetically related to their maternal reactivity measured at lambing. A total of 935 Romane ewes were studied. The maternal reactivity of ewes at the outdoor lambing site was recorded in response to a human approach and to the handling of the lambs. Their behavioural reactivity was also recorded 24 hr post-lambing in the arena test that involved a separation from the litter and a human presence. Flight distance, aggressive reaction, time to restore contact with the litter, maternal behaviour scores and vocalizations recorded at the lambing site were heritable (0.12-0.34). All of these behaviours were genetically correlated with the behavioural reactivity in the arena test. The highest genetic correlations (from 0.60 to 0.90) were found amongst maternal behavioural scores, flight distance and high-pitched bleats. In conclusion, behavioural reactivity in the arena test can be used to assess early maternal reactivity in standardized conditions. Phenotyping of ewes' behavioural reactivity with a simplified arena test can be performed for genetic improvement in maternal behaviour in sheep.

A Non-Invasive Millimetre-Wave Radar Sensor for Automated Behavioural Tracking in Precision Farming-Application to Sheep Husbandry.

The automated quantification of the behaviour of freely moving animals is increasingly needed in applied ethology. State-of-the-art approaches often require tags to identify animals, high computational power for data collection and processing, and are sensitive to environmental conditions, which limits their large-scale utilization, for instance in genetic selection programs of animal breeding. Here we introduce a new automated tracking system based on millimetre-wave radars for real time robust and high precision monitoring of untagged animals. In contrast to conventional video tracking systems, radar tracking requires low processing power, is independent on light variations and has more accurate estimations of animal positions due to a lower misdetection rate. To validate our approach, we monitored the movements of 58 sheep in a standard indoor behavioural test used for assessing social motivation. We derived new estimators from the radar data that can be used to improve the behavioural phenotyping of the sheep. We then showed how radars can be used for movement tracking at larger spatial scales, in the field, by adjusting operating frequency and radiated electromagnetic power. Millimetre-wave radars thus hold considerable promises precision farming through high-throughput recording of the behaviour of untagged animals in different types of environments.

Genetic parameters estimates for ewes' behavioural reactivity towards their litter after lambing.

In livestock, improving maternal reactivity towards the litter is an important issue in breeding strategies to promote production and animal welfare. As of yet, no studies have investigated the within-breed genetic variation of maternal reactivity in sheep. The objective of this study was to estimate the genetic parameters of maternal reactivity traits. A total of 1,095 primiparous and 1,441 multiparous Romane ewes were phenotyped 24 hr postlambing using a behavioural test (arena test, AT) over a 10-year experimental period. The test consisted of three successive phases evaluating the ewe's attraction to her litter, reactivity to separation from her litter, and reactivity to a conflict between attraction to her litter and avoidance of a motionless human. The ewes were reared exclusively on rangelands (South of France) and lambed outdoors in the spring. High-pitched bleating and low-pitched bleating in the AT were mostly highly heritable (0.39-0.46). Heritabilities were moderate for proximity to the litter in the presence of a human (0.27) and low for locomotion and vigilance in the AT (0.09-0.15). The measurements of a given behaviour in the three phases of the AT were highly genetically correlated. Few genetic correlations were found between the different behavioural traits in the AT, the highest correlations being between high-pitched bleating and low-pitched bleating (-0.43 to -0.77). In conclusion, our findings demonstrate moderate-to-high heritability for maternal reactivity traits. These traits could be included in genetic selection schemes to enhance maternal attachment provided there is no unfavourable link with other production traits.

Effects of prenatal stress and emotional reactivity of the mother on emotional and cognitive abilities in lambs.

Consequences of prenatal stress on emotional reactivity and cognitive abilities in offspring are under-documented in precocial mammals. Here, we investigated to what extent emotional reactivity, judgment bias and spatial learning abilities of lambs are affected by chronic stress during late pregnancy and by their dams' emotional reactivity. The 20 highest-responsive (HR) and 20 lowest-responsive (LR) ewes from a population of 120 Romane ewes were selected according to their pre-mating reactivity to social isolation in a new environment. Over the final third of pregnancy, 10 HR ewes and 10 LR ewes were exposed daily to various unpredictable aversive events such as restraint, mixing groups and transport while the other 20 selected ewes were not. In a human and an object test, prenatally-stressed lambs were more fearful than control lambs, but the prenatal stress effect was moderated by the reactivity of the mothers: prenatally-stressed lambs from ewes with high emotional reactivity were more affected. Prenatally-stressed lambs did not perform as well as control lambs in a maze test and showed pessimistic-like judgment in a cognitive bias test. Prenatally-stressed lambs were thus characterized by a negative affective state with increased fear reactions and impaired cognitive evaluation. The development of negative moods could have long-lasting consequences on the coping strategies of the lambs in response to their rearing conditions.

Identification of QTLs for behavioral reactivity to social separation and humans in sheep using the OvineSNP50 BeadChip.

BACKGROUND: Current trends in sheep farming practices rely on animals with a greater level of behavioral autonomy than before, a phenotype that actively contributes to the sustainability of animal production. Social reactivity and reactivity to humans are relevant behavioral traits in sheep, known for their strong gregariousness and weak tolerance to handling, which have previously been reported with moderate to high heritabilities. To identify loci underlying such behaviors, we performed a genome study in Romane lambs. RESULTS: The experiment was carried out on 934 male and female lambs allocated into 9 half-sib families (average of 103 lambs per family) and reared outside. After weaning, all the lambs were individually exposed to 4 standardized behavioral tests combining social isolation, exposure to humans or handling, confinement and novelty (i.e. arena test, corridor test, isolation box test, shearing test). A broad range of behaviors including vocalizations, locomotion, vigilance and flight distance, as well as the cortisol response to handling, were collected. All lambs were genotyped using the Illumina OvineSNP50 BeadChip. QTL detection was performed by linkage, association and joint linkage and association analyses using the QTLmap software. Five main QTL regions were identified on sheep chromosomes (Ovis Aries Region, OAR) 12, 16, 19, 21 and 23 among many other QTLs with small to moderate effects. The QTLs on OAR12, 16 and 21 showed significant associations with social reactivity. The QTLs on OAR19 and 23 were found to be associated with reactivity to humans. No overlapping QTLs were identified for the different traits measured in the behavioral tests, supporting the hypothesis that different genetic factors influence social reactivity and tolerance to humans. CONCLUSION: The results of this study using ovine SNP data suggest that in domestic sheep the behavioral responses to social separation and exposure to humans are under polygenic influence. The most relevant QTLs reported in the present study contain interesting candidate genes previously described to be associated with various emotional and social behaviors in mammals.

Mild effects of gestational stress and social reactivity on the onset of mother-young interactions and bonding in sheep.

Consequences of prenatal stress on mother-young relationships are well-documented in altricial mammals but less so in precocial mammals. In this study, we investigated the effects of unpredictable aversive events on maternal behavior and mutual mother-young recognition in pregnant ewes while accounting for modulatory effects of ewe reactivity. From a population of 120 Romane-breed ewes, we selected 20 high-responsive (HR) and 20 low-responsive (LR) ewes according to pre-mating reactivity assessed in isolation tests. Over the final third of pregnancy, 10 HR ewes and 10 LR ewes were exposed daily to various aversive events such as social isolation, mixing and transport (stressed ewes), while the other 20 ewes were not exposed to aversive events (control ewes). Although the treatment induced chronic stress, physiologically confirmed by an increase in salivary cortisol following transport and sham shearing, maternal behavior of stressed ewes observed during the first 30 min postpartum and in the selectivity test 1 h 30 min later did not differ from controls. However, in a maternal motivation test performed 48 h postpartum, stressed ewes vocalized less than controls when separated from their lambs, and walked less readily past an unknown object to reach their lambs. Lambs of stressed ewes spent more time near their dam in a preference test performed 15 h after birth compared to control-ewe lambs. HR ewes spent more time grooming their lambs than LR ewes. We posit that domestication could have selected animals displaying robust expression of maternal behavior related to social reactivity and producing offspring that are better adapted to challenging situations.

Group size elicits specific physiological response in herbivores.

With increasing group size, individuals commonly spend less time standing head-up (scanning) and more time feeding. In small groups, a higher predation risk is likely to increase stress, which will be reflected by behavioural and endocrine responses. However, without any predator cues, we ask how the predation risk is actually processed by animals as group size decreases. We hypothesize that group size on its own acts as a stressor. We studied undisturbed groups of sheep under controlled pasture conditions, and measured in situ the cortisol and vigilance responses of identified individuals in groups ranging from 2 to 100 sheep. Both vigilance and average cortisol concentration decreased as group size increased. However, the cortisol response varied markedly among individuals in small groups, resulting in a lack of correlation between cortisol and vigilance responses. Further experiments are required to explore the mechanisms that underlie both the decay and the convergence of individual stress in larger groups, and whether these mechanisms promote adaptive anti-predator responses.

Classification of pig calls produced from birth to slaughter according to their emotional valence and context of production.

Vocal expression of emotions has been observed across species and could provide a non-invasive and reliable means to assess animal emotions. We investigated if pig vocal indicators of emotions revealed in previous studies are valid across call types and contexts, and could potentially be used to develop an automated emotion monitoring tool. We performed an analysis of an extensive and unique dataset of low (LF) and high frequency (HF) calls emitted by pigs across numerous commercial contexts from birth to slaughter (7414 calls from 411 pigs). Our results revealed that the valence attributed to the contexts of production (positive versus negative) affected all investigated parameters in both LF and HF. Similarly, the context category affected all parameters. We then tested two different automated methods for call classification; a neural network revealed much higher classification accuracy compared to a permuted discriminant function analysis (pDFA), both for the valence (neural network: 91.5%; pDFA analysis weighted average across LF and HF (cross-classified): 61.7% with a chance level at 50.5%) and context (neural network: 81.5%; pDFA analysis weighted average across LF and HF (cross-classified): 19.4% with a chance level at 14.3%). These results suggest that an automated recognition system can be developed to monitor pig welfare on-farm.

Changes in Heart Rate Variability during a tonic immobility test in quail.

Tonic immobility (TI) is an unlearned fear response induced by a brief physical restraint and characterized by a marked autonomic nervous system involvement. This experiment aimed at studying the relative involvement of both autonomic sub-systems, the sympathetic and parasympathetic nervous systems, during TI, by analyzing Heart Rate Variability. Quail selected genetically for long (LTI) or short (STI) TI duration and quail from a control line (CTI) were used. The animals were surgically fitted with a telemetric device to record electrocardiograms before and during a TI test. Heart rate did not differ between lines at rest. The induction of TI, whether effective or not, induced an increase in HR characterized by a shift of the sympathovagal balance towards a higher sympathetic dominance. Parasympathetic activity was lower during effective than during non-effective inductions in CTI quail. During TI, the increase in sympathetic dominance was initially maintained and then declined, while relative parasympathetic activity remained low, especially in CTI and STI lines. The end of tonic immobility was characterized by a rise in overall autonomic activity in all lines and an increase in parasympathetic influence in CTI and STI quail. To conclude, the susceptibility to TI cannot be explained only by autonomic reflex changes. It is probably strongly related to the perception of the test by the quail. During TI, the differences between lines in autonomic responses probably reflect behavioural differences in the fear response.

Perception of animal sentience by Brazilian and French citizens: The case of sheep welfare and sentience.

The study compared the perception of ordinary citizens from Curitiba, Brazil (OB) and Clermont-Ferrand, France (OF), as well as OB, Brazilian veterinarians (VB), biologists (BB) and animal scientists (AB), concerning animal welfare and sentience. An online survey containing 18 open-ended, multiple choices and Likert scale questions was conducted from November 2014 to May 2016. The survey covered questions on demographics, perception of animal welfare, sheep welfare, sentience and animals' emotional capacities. In total, 1103 respondents participated in the survey (388 OB, 350 OF, 248 VB, 92 BB and 25 AB); data were compared using non-parametric tests. Brazilian citizens (46.9%) believed more than OF (3.7%) that welfare is not considered for farm animals and OB attributed higher scores of suffering to sheep during management procedures (median 4, severe suffering) than OF (3, moderate suffering). Additionally, OB gave higher scores of emotions to animals (5) than OF (4). In general, OB and BB had similar perceptions; OB and BB differed from VB and AB who were similar to each other. Citizens (46.9%) and BB (29.3%) believed more than VB (18.5%) and AB (12.0%) that welfare is not considered for farm animals; OB and BB also attributed higher scores of suffering to sheep during management procedures than VB and AB. Women and older respondents showed higher perception of animal welfare issues. There was no clear correlation between perception of animal welfare or sentience and education. Overall, ordinary citizens differed on their perceptions of welfare and sentience in livestock and specifically in sheep, and sheep suffering during management procedures. Ordinary citizens from Curitiba showed higher perception of animal welfare issues as compared to respondents from Clermont-Ferrand and to veterinarians and animal scientists. Ensuring a better consideration of welfare at farm level and in educational programs seems warranted according to the results of this study.

Are there facial indicators of positive emotions in birds? A first exploration in Japanese quail.

The positive aspect of emotions, like pleasure, remains overlooked in birds. Our aim was to contribute to the exploration of facial indicators of positive emotions. To observe contrasting emotional expressions, we used two lines of Japanese quail divergently selected on their inherent fearfulness: a fearful line (long tonic immobility duration: LTI) and a less fearful line (short tonic immobility duration: STI). To induce positive emotions, we gave individual quail the opportunity to perform a rewarding behaviour, dustbathing, in an unfamiliar cage. More STI than LTI quail expressed dustbathing and latencies to dustbathe were significantly shorter in STI than LTI quail. This result indicated that the lines of quail differed in their fearfulness of the situation. We observed crown feather height, throat feather angle and pupil surface before (control) and during dustbathing. We found significant increases in crown feather height, pupil area and angle of throat feathers between the control and the dustbathing phases in STI quail, and pupil area correlated positively with crown feather height. In LTI quail, the angle of throat feathers increased during dustbathing, but the other parameters did not differ. We argue that variation in crown feather height and pupil area may provide indications of positive emotions in Japanese quail.

Emotional experience in sheep: predictability of a sudden event lowers subsequent emotional responses.

The study of emotions in animals can be approached thanks to a framework derived from appraisal theories developed in cognitive psychology, according to which emotions are triggered when the individual evaluates challenging events. This evaluation is based on a limited number of criteria such as the familiarity and the predictability of an event. If animals are able to experience emotions rather than simply displaying reflex responses to their environment, then their appraisal of events should, as in humans, modulate their emotional responses. We tested this hypothesis by comparing vocalisations, feeding behaviour, and the startle and cardiac responses of lambs submitted to a sudden event that could or could not be predicted. Lambs able to predict the sudden event thanks to a light cue (associative predictability) showed weaker suddenness-induced startle and cardiac responses and spent more time feeding than their counterparts, thus supporting the existence of an emotional experience in these animals. Furthermore, lambs submitted to the regular appearance of the sudden event (temporal regularity) vocalised less and left less unconsumed food deliveries than lambs submitted to random appearances of the sudden event (controls). These results underline that the cognitive abilities of animals should be taken into account when assessing their emotional experiences and more generally their mood states, which are underlying factors of animal welfare.

Assessment of positive emotions in animals to improve their welfare.

It is now widely accepted that good welfare is not simply the absence of negative experiences, but rather is primarily the presence of positive experiences such as pleasure. However scientific investigation of positive emotions has long been neglected. This paper addresses two main issues: first, it reviews the current state of scientific knowledge that supports the existence of positive affective states in animals and, second, it suggests possible applications of this knowledge that may enhance quality of life under animal management conditions. In the first part of the paper, recent advances in psychology and neuroscience are reviewed to provide pragmatic frameworks based on cognitive processes (such as positive anticipation, contrast and controllability) for further investigations of positive emotions in animals. Thereafter, the neurobiological bases of positive emotions are highlighted in order to identify behavioral and physiological expressions of positive experiences in animals. Monitoring both the autonomic nervous system (via heart rate and its variability) and the immune system could offer relevant tools to better assess emotional states in animals, complementary to classical adrenocortical measures. In the second part of the paper, useful strategies for enhancing positive experiences (such as physical, social and cognitive enrichment or putative genetic selection) are outlined. Then this paper emphasizes practical applications for assessing and promoting positive emotions that may help in providing animals with a better quality of life. Play, affiliative behaviors and some vocalizations appear to be the most promising convenient indicators for assessing positive experiences in laboratory and farm animals under commercial conditions.

Behavioral and physiological reactions in dogs to a veterinary examination: Owner-dog interactions improve canine well-being.

In order to improve well-being of dogs during veterinary visits, we aimed to investigate the effect of human social interactions on behavior and physiology during routine examination. Firstly, we assessed the impact of a standardized veterinary examination on behavioral and physiological indicators of stress in dogs. Secondly, we examined whether the owner's tactile and verbal interactions with the dog influenced behavioral and physiological stress-associated parameters. A randomized within-subjects crossover design was used to examine behavior (n=33), rectal temperature (n=33), heart rate (HR) (n=18), maximal ocular surface temperature (max OST) (n=13) and salivary cortisol concentrations (n=10) in healthy privately owned pet dogs. The study consisted of two experimental conditions: a) "contact" - owner petting and talking to the dog during the examination; b) "non-contact" - owner present during the examination but not allowed to interact with the dog. Our findings showed that the veterinary examinations produced acute stress responses in dogs during both "contact" and "non-contact" conditions, with significant increases in lip licking, HR, and max OST. A significant decrease in attempts to jump off the examination table (p=0.002) was observed during the examination in the "contact" compared to the "non-contact" condition. In addition, interactions of owners showed an attenuating effect on HR (p=0.018) and max OST (p=0.011) in their dogs. The testing order (first vs. second visit) had no impact on behavioral and physiological parameters, suggesting that dogs did not habituate or sensitize to the examination procedure. Moreover, the duration of the owner-dog interactions had no significant impact on the behavioral and physiological responses of their dogs. This study demonstrates that owner-dog interactions improve the well-being of dogs during a veterinary examination. Future research may assist in further understanding the mechanisms associated with reducing stress in dogs in similar settings.

Valence of Facial Cues Influences Sheep Learning in a Visual Discrimination Task.

Sheep are one of the most studied farm species in terms of their ability to process information from faces, but little is known about their face-based emotion recognition abilities. We investigated (a) whether sheep could use images of sheep faces taken in situation of varying valence as cues in a simultaneous discrimination task and (b) whether the valence of the situation affects their learning performance. To accomplish this, we photographed faces of sheep in three situations inducing emotional states of neutral (ruminating in the home pen) or negative valence (social isolation or aggressive interaction). Sheep (n = 35) first had to learn a discrimination task with colored cards. Animals that reached the learning criterion (n = 16) were then presented with pairs of images of the face of a single individual taken in the neutral situation and in one of the negative situations. Finally, sheep had to generalize what they had learned to new pairs of images of faces taken in the same situation, but of a different conspecific. All sheep that learned the discrimination task with colored cards reached the learning criterion with images of faces. Sheep that had to associate a negative image with a food reward learned faster than sheep that had to associate a neutral image with a reward. With the exception of sheep from the aggression-rewarded group, sheep generalized this discrimination to images of faces of different individuals. Our results suggest that sheep can perceive the emotional valence displayed on faces of conspecifics and that this valence affects learning processes.

Appraisal process in sheep (Ovis aries): interactive effect of suddenness and unfamiliarity on cardiac and behavioral responses.

Appraisal theories state that an emotion results from an individual's evaluation of a stimulating event, which is based on a set of criteria. Each criterion triggers specific expressions that can be modulated by the outcome of other criteria. The authors analyzed the effects of the criteria of suddenness, unfamiliarity, and their combination on behavioral and cardiac responses in lambs (Ovis aries). Lambs were exposed to a visual stimulus that appeared either rapidly or slowly and was either unfamiliar or familiar. Suddenness induced a startle response and an increase in heart rate. Unfamiliarity induced an orientation response. The combination of suddenness and unfamiliarity enhanced both the heart rate increase and the behavioral orientation response. These results support the hypotheses of specificity and modulation of emotional expressions.

Behavioural expression of positive anticipation for food or opportunity to play in lambs.

Anticipatory behaviours may serve a useful tool in studying positive emotional states in animals. This study aimed to investigate if lambs express anticipatory behaviours for opportunities to play or a food reward and if these behaviours would differ depending on the anticipated event. Forty-two male lambs were allocated into three treatment groups (control, play, food). Play and food lambs were conditioned to anticipate play or food in a holding pen for three minutes prior to accessing a reward arena containing toy objects or concentrate, respectively. Control lambs returned to their home pen following three minutes in the holding pen. Compared to the control lambs, both play and food lambs differed in several behaviours frequencies and durations, e.g. by an increased frequency of behavioural transitions and duration of walking. Following these observations, food lambs received the toy objects when entering the reward arena. The subsequent session in the holding pen resulted in a decrease in number of behavioural transitions, time spent walking and an increase in time standing still. In conclusion, anticipating a positive event resulted in differences in behaviour compared to a control group, and these behaviours are affected when the anticipated event does not fit with the lambs' expectations.

Stress during pregnancy alters dendritic spine density and gene expression in the brain of new-born lambs.

Rodent studies show how prenatal stress (PS) can alter morphology in the cortico-limbic structures that support emotional and cognitive functions. PS-induced alteration is less well described in species with a gyrencephalic brain and complex earlier fetal development, and never in sheep at birth to rule out postnatal environment effects or influences of maternal behavior. This study aimed to assess the consequences of a mild chronic stress in pregnant ewes on the neurobiological development of their lambs at birth. During the last third of gestation, 7 ewes were exposed daily to various unpredictable and negative routine management-based challenges (stressed group), while 7 other ewes were housed without any additional perturbation (control group). For each group, a newborn from each litter was sacrificed at birth to collect its brain and analyze its expression levels of genes involved in neuronal dendritic morphology (Dlg4, Rac1, RhoA, Doc2b), synaptic transmission (Nr1, Grin2A, Grin2B) and glucocorticoid receptor (Nr3C1) in hippocampus (HPC), prefrontal cortex (PFC) and amygdala (AMYG). Results revealed that lambs from stressed dam (PS lambs) showed under-expression of Rac1 and Nr1 in PFC and overexpression of Dlg4 in AMYG compared to controls. To assess the morphological consequences of gene dysregulations, the dendritic morphology of pyramidal neurons was explored by Golgi-Cox staining in HPC and PFC. PS lambs had higher dendritic spine density in both structures and more stubby-type spines in the CA1 area of HPC than controls. This is the first demonstration in sheep that PS alters fetal brain, possibly reflecting functional changes in synaptic transmission to cope with adversity experienced in fetal life.

Do lambs perceive regular human stroking as pleasant? Behavior and heart rate variability analyses.

Stroking by humans is beneficial to the human-animal relationship and improves welfare in many species that express intraspecific allogrooming, but very few studies have looked at species like sheep that do not express such contact except around parturition. This study investigated the way lambs perceive regular human tactile contact using behavioral and physiological responses. Twenty-four lambs were reared and bucket-fed in groups of four. All were stroked daily by their familiar caregiver. At 8 weeks of age, the lambs were individually tested in their home pen but in a 1×1m open-barred pen after a 15 h period of habituation to physical separation from peers while remaining in visual and auditory contact. Half of the lambs received stroking by their caregiver for 8 min and half were exposed to their caregiver's immobile presence. Heart rate and heart rate variability were recorded and analyzed by 2-min slots over the same interval based on three measures: mean heart rate value (HR), root mean square of successive differences (RMSSD) and standard deviation of all intervals measured between consecutive sinus beats (SDNN). Behavioral responses (ear postures of the lamb and time spent in contact with the familiar caregiver, on the knees of the familiar caregiver, and moving) were recorded throughout the test. Lamb HR decreased continuously while in the presence of their caregiver. Lambs being stroked showed slower HR and higher RMSSD which reflected positive emotional states compared to lambs left unstroked. All behavioral variables were highly correlated with the main component axis of the PCA analyses: the more the animals stayed in contact with their caregiver, the less they moved and the more their ears were hanging. This first component clearly differentiates lambs being stroked or not. Behavioral and physiological observations support the hypothesis that gentle physical contact with the caregiver is perceived positively by lambs.