EEG studies have suggested that cattle perceive pain when bled without stunning. The present study on bleeding without stunning, compared cows that had received a local anaesthetic on the site of the bleeding cut (Lurocaine; Luro cows) one hour before bleeding with a 35 cm knife, with cows that had not (saline: Placebo cows). Various physiological indicators potentially related to pain or stress were evaluated. Increases in heart rate (P < 0.02) and cortisol levels (P < 0.001) during slaughter, compared to control levels are indicative of slaughter stress in both groups. GSH/GSSG ratio, plasma PGE2, TNFα, and NO levels and blood haematocrit levels at slaughter were not influenced by slaughter or treatment. At bleeding, excluding two out of the 15 cows with non-missing data, Placebo cows presented a longer delay between the loss of the corneal reflex and respiratory arrest. Post-mortem, Longissimus muscle of Placebo cows had a faster pH decline and remained warmer. Overall, results suggest greater stress levels in this group, probably due to pain. Plasma or brain β-endorphin contents in relevant brain structures did not increase following bleeding in either group, thus not supporting the hypothesis that stress or pain-induced release of endorphins reduces pain perceived following the cut. Furthermore, according to existing knowledge, plasma β-endorphins do not reduce pain perceived. Thus, both our study and previous research do not provide evidence that slaughter without stunning does not cause pain or other forms of stress in at least part of the animals.

Cortisol is often used as a stress indicator in animal behaviour research. Cortisol is commonly measured in plasma and can also be measured in saliva. Saliva contains only the free form of cortisol, which is biologically active, and saliva sampling is not invasive and may therefore be less stressful. Our study aims to guide the choice between the measurements of cortisol in plasma v. saliva depending on experimental conditions. We analysed the effect of the level of cortisol in plasma on the concentration of cortisol in saliva compared to plasma and the effect of saliva sampling v. jugular venepuncture on the cortisol response. In Experiment 1, blood and saliva were collected simultaneously under conditions in which the expected cortisol release in blood varied: in an undisturbed situation or after the isolation of lambs from their pens or the administration of exogenous ACTH (six animals per treatment). In Experiment 2, we subjected lambs to saliva sampling, venepuncture or neither of these for 8 days to evaluate how stressful the sampling method was and whether the animals habituated to it by comparing the responses between the first and last days (four animals per treatment). All animals were equipped with jugular catheters to allow regular blood sampling without disturbance. Samples were collected 15 min before any treatment was applied, then at various time points up to 135 min in Experiment 1 and 45 min in Experiment 2. In Experiment 1, we observed a strong correlation between salivary and plasma cortisol concentrations (r = 0.81, P < 0.001). The ratio between salivary and plasma cortisol concentrations was 0.106 on average. This ratio was higher and more variable when the cortisol concentration in plasma was below 55 nmol/l. In Experiment 2, venepuncture induced a larger cortisol response than saliva sampling or no intervention on day 1 (P < 0.02); this difference was not observed on day 8, suggesting that sheep habituated to venepuncture. We recommend the measurement of cortisol in saliva to avoid stressing animals. However, when the expected concentration in plasma is below 55 nmol/l, the cortisol in saliva will reflect only the free fraction of the cortisol, which may be a limitation if the focus of the experiment is on total cortisol. In addition, if cortisol is measured in plasma and blood is collected by venepuncture, we recommend that sheep be habituated to venepuncture, at least to the handling required for a venepuncture.

Gentle handling seems to elicit positive states in sheep. The study investigated whether spatial distance alters sheep responses to brushing and whether spatial distance is influenced by reactivity. Twenty Romane ewes were assessed in three sessions: in Sessions 1 and 3, one grid separated the test animal from pen mates, with no distance between them, and in Session 2 two grids separated the test animal from pen mates by a distance of about 1.7 m. Ewes had been genetically selected for low (R-) or high (R+) behavioural reactivity to social isolation. Body postures, head orientation, ear postures, closed and half-closed eyes, tail wagging and feeding behaviour, in addition to heart rate (HR) and HR variability, as the root mean square of successive differences (RMSSD), standard deviation of all normal-to-normal (NN) intervals (SDNN), RMSSD/SDNN ratio and ratio between low-frequency (LF) and high-frequency (HF) powers (LF/HF) were assessed. Data were analysed using generalized linear models and linear mixed models. Session, genetic line and phase (pre-, brushing and post-brushing) were considered fixed effects. Increased distance in Session 2 might not have influenced ewes' responses. Fewer changes in ear postures were noted in Session 3 than 1 (P<0.01), suggesting that ewes were more relaxed in Session 3. The RMSSD/SDNN ratio was higher mainly during brushing in Sessions 1 and 3 (P<0.05), indicating that ewes were more relaxed during brushing, and at no distance between pen mates. However, spatial distance influenced R- and R+ ewes' responses; R+ ewes performed more asymmetric ear postures in Session 2 than 1 and 3 (P<0.01), and in Session 3 than 1 (P<0.01), indicating that spatial distance had a negative effect on R+ ewes. Low reactive ewes spent less time on horizontal ear postures in Session 2 than 1 and 3 (P<0.01), and R+ ewes spent more time on horizontal postures in Session 1 than 3 (P<0.01). Curiously, R- ewes spent more time eating and ruminating in Session 3 than 1 (P<0.01), and in Session 2 than 1 and 3 (P<0.01), whereas R+ ewes ate and ruminated more in Session 1 than 3 (P<0.05). Higher HR was found among R- ewes in Session 2 than 1 and 3, and in Session 3 than 1 (P<0.01). High reactive ewes showed higher HR in Session 1 than 3 (P<0.01). The findings suggest that the social context might influence sheep responses to gentle handling, and the effects depend on their reactivity traits.

L’objectif était de suivre la douleur induite par la castration chez des ovins et d’évaluer l'efficacité de différentes stratégies analgésiques. Vingt-quatre moutons Texel mâles âgés de 12 mois ont été castrés par pince burdizzo sans anesthésie (Cast), après une anesthésie locale (Cast + AL) ou après une anesthésie locale associée des AINS (Cast +AL +AINS). Les réponses comportementales et physiologiques des moutons ont été enregistrées durant quatre périodes : P0 (-24h à -1 h), P1 (0 à +2 h), P2 (+3 h à +32 h) et P3 (+36 h à +78 h). Les périodes en fonction des traitements ont pu être parfaitement différenciées grâce aux 23 indicateurs de l’approche multiparamétrique. Les lots Cast en P0, Cast + AL en P0 et Cast + AL + AINS en P0, P1 et P2 ont présenté les valeurs les plus faibles pour tous les indicateurs, reflétant probablement l'absence de douleur et d’inconfort. Les moutons des lots Cast en P1 et P2, et Cast + AL en P2 ont montré des signes cliniques et une attention réduite, un rapport LF/HF élevé et des taux de cortisol élevés, reflétant une douleur aiguë. Les lots Cast et Cast + AL en P3 ont une température élevée et des concentrations en haptoglobine, glucose et SAA élevées. Ainsi, les signes comportementaux de douleur ont été présents pendant les 32 premières heures suivant la castration ; l'anesthésie locale a été efficace, mais seulement pendant son temps d'action (2 h) ; l'analgésie multimodale (AL+AINS) a été efficace jusqu'à 3 jours après la castration. Ces résultats, notamment ceux liés au comportement des moutons, peuvent aider les vétérinaires et les éleveurs à mieux détecter la douleur et ainsi, à mieux prendre en charge le soulagement de la douleur.

Many biological studies seek to confirm the effect of a treatment on the levels of chemical markers in biological tissues. This requires choosing the analytical method best able to detect the difference between basal levels and those found after treatment. We propose a new approach to calculate a criterion we call the 'discrimination threshold' (DT), and we applied it to an analytical method that we developed to determine cortisol in cattle plasma to detect stress. DT is derived from the measurement uncertainty (MU), and combines the variabilities of both the analytical method and the marker. The uncertainty of the analytical method comes from the method validation study. The marker variability (here cortisol) is modeled from a literature review. The graphical representation of DT allows estimating the applicability of the method. Our analytical method is shown to perform well when the difference in blood cortisol exceeds 18 ng.mL(-1). (C) 2018 Elsevier B.V. All rights reserved.

Welfare concerns not only the absence of negative emotional experiences but also the presence of positive experiences. However, scientific investigation on positive emotions in farm animals has been long neglected. This study aimed to investigate the perception of sheep to positive stimuli and whether emotional reactivity influences such perception, through behavioural and cardiac indicators. Therefore, 38 Romane ewes belonging to two divergent lines, high (R+) or low (R-) reactive, were submitted to the presence of a familiar experimenter (HUM) or brushing by a familiar experimenter (BRU). The procedure was divided in three phases: before (2.5 min), during (3.0 min) and after (2.5 min) HUM or BRU. Body postures, head orientation, ear changes and postures, closed and half-closed eyes, tail wagging and feeding behaviour were analyzed, and heart rate variability parameters (RMSSD, RMSSD/SDNN and LF/HF ratios) were estimated. Data were analyzed using linear models with different correlation structures. The models considered treatment, genetic line and phase as fixed effects, including their interactions. Brushed ewes showed fewer body posture, head orientation and ear posture changes and a higher proportion of closed and half-closed eyes, tail wagging and rumination during the procedure, when compared to ewes submitted to the human presence (P < 0.05). Both BRU and HUM ewes had lower HR during and after the procedures and higher RMSSD/SDNN rate during the procedures, reflecting the activation of the parasympathetic nervous system in response to both treatments (P < 0.05). Behavioural and cardiac results suggest that both treatments induced a relaxing state in sheep, more pronounced for the brushing treatment. Interestingly, an anticipatory agitation before the treatment was observed mainly in BRU sheep. Highly reactive ewes expressed less head orientation changes and a higher proportion of horizontal ear postures and feeding behaviour in response to brushing than when submitted to human presence (P < 0.05), indicating that brushing might have elicited a more relaxing effect on R+ animals. Differences between R+ and R- ewes suggest that reactivity might modulate sheep responses to assumed positive stimuli.

Under temperate climates, cattle are often at pasture in summer and are not necessarily provided with shade. We aimed at evaluating in a temperate region (Belgium) to what extent cattle may suffer from heat stress (measured through body temperature, respiration rate and panting score, cortisol or its metabolites in milk, and feces on hot days) and at assessing the potential benefits of shade. During the summer of 2012, 20 cows were kept on pasture without access to shade. During the summer of 2011, ten cows had access to shade (young trees with shade cloth hung between them), whereas ten cows had no access. Climatic conditions were quantified by the Heat Load Index (HLI). In animals without access to shade respiration rates, panting scores, rectal temperatures, and milk cortisol concentrations increased as HLI increased in both 2011 and 2012. Fecal cortisol metabolites varied with HLI in 2011 only. When cattle had access to shade, their use of shade increased as the HLI increased. This effect was more pronounced during the last part of the summer, possibly due to better acquaintance with the shade construction. In this case, shade use increased to 65% at the highest HLI (79). Shade tempered the effects on respiration, rectal temperature, and fecal cortisol metabolites. Milk cortisol was not influenced by HLI for cows using shade for > 10% of the day. Therefore, even in temperate areas, cattle may suffer from heat when they are at pasture in summer and providing shade can reduce such stress.

Farming systems can expose animals to chronic mild stress which is known to induce negative affective state. Affective state in animals, as in humans, can be assessed through behavioral cues. This study aimed to describe the effect of a chronic mild stress, known to induce a negative affective state, on sheep health through their response to vaccination. The study used 15 lambs subjected to a model of chronic mild stress for 15 weeks and 15 lambs reared under conventional farming as a control group. After 7 weeks of stressful treatment, the lambs were individually exposed to a judgment bias test to assess a putative stress-induced 'pessimism.' After 15 weeks of stressful treatment, antibody immune response was measured after an injection of a live vaccine challenge (Chlamydia abortus attenuated vaccine strain 1B). Stressed lambs displayed a pessimistic-like perception in the judgment bias test, revealing a negative affective state. Stressed and control animals showed different immunological reactions to vaccine challenge: stressed sheep had lower hemoglobin concentrations and higher platelet, granulocyte and acute-phase protein concentrations. Antibody response induced by the vaccine strain was not different between stressed and control sheep. Our results suggest that negative affective state induced by chronic stress treatment may induce a stronger inflammatory response to vaccine challenge in sheep. Improvement of animal health may be achieved through consideration of stressors that may affect the emotional and immunological state of sheep.

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.

Cattle are often disbudded in a procedure that is painful to them. In an effort to find practical ways to reduce poor welfare due to hot-iron disbudding, we tested a combination of sedation and nonsteroidal anti-inflammatory analgesia (NSAID) that farmers themselves can apply to calves at two different postnatal ages. We compared Prim’Holstein and Charolais calves, male or female, aged 1 or 4 weeks, subjected to hot-iron disbudding without (vigil calves), or with sedation (xylazine calves, 0.2mg/kg xylazine 2%), and to sham-disbudded calves without sedation or analgesia (control calves). Both groups of disbudded calves received NSAID (3mg/kg Ketoprofen 10 %®). Calf behavior was observed during disbudding and the following 15min, and then between 2h and 7h post-disbudding. Salivary cortisol and heart rate were measured from 20min before to 240min after disbudding. Compared to control and xylazine calves, vigil calves reacted strongly to disbudding by vocalizing (mean vocalizations during the 15min post-disbudding: vigil calves, 1.95; control calves, 0.2; and xylazine calves, 1.30, a value that was not significantly different from the other two), and higher salivary cortisol concentrations 30min post-disbudding (vigil calves, 4.08ng/mL; control calves, 1.59ng/mL; and xylazine calves, 1.18ng/mL). An increase in heart rate of 17bpm above baseline was observed in vigil calves during the 10min post-disbudding, whereas the heart rate of control calves did not vary and that of xylazine calves decreased by 39bpm. Significant differences in salivary cortisol concentration and heart rate were observed at 45min and 30min post-disbudding, respectively. Vocalizations were observed between 2 and 7h after disbudding in both vigil and xylazine calves. Responses to disbudding were the same whatever the age, sex, or breed of the calves. The results suggest that sedation with xylazine can reduce the stress calves experience right after disbudding but not after 2h, and that the pain produced by disbudding is the same at 1 or 4 weeks of age.

L’ecornage est une procedure douloureuse pour les veaux. Le but de cette etude etait de determiner si la combinaison d’un sedatif et d’un anti-inflammatoire non steroidien (AINS) est un moyen pour reduire la douleur a l’ecornage, et si l’expression de la douleur varie selon l’âge, la race ou le sexe du veau.Des veaux Holstein et Charolais des 2 sexes, âges de 7 ou 28 jours, ebourgeonnes et ayant recu un AINS (ketoprofene) seul ou couple a un sedatif (xylazine) ont ete compares a des veaux sans traitement etpour lesquels l’ebourgeonnage a ete simule. Leur comportement, leur taux de cortisol salivaire et leur frequence cardiaque ont ete suivis.Les veaux non sedates ont reagi plus fortement et avaient une frequence cardiaque et un taux de cortisol salivaire plus eleves. Ces effets n’ontpas ete observes chez les veaux sedates. Les reponses ont ete les memesquels que soient l’âge, le sexe ou la race.Les resultats sont en faveur d’un traitement antidouleur lors de l’ecornage, combinant sedation et anti-inflammatoire.

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.

Friendly interactions between humans and animals such as gentling or petting have been shown to have positive behavioural and physiological consequences in many species. In primates, rodents and dogs, oxytocin has been associated with tactile contact and anti-stress effects that may influence bonding and responses to stress situations. However the activation of the oxytocinergic system in other human–animal interactions such as with herbivores, had not yet been studied. Sixteen female lambs were reared by artificial feeding reinforced with 3× 30 s daily stroking sessions. At 6 weeks of age, the test consisted in measuring first plasma oxytocin and cortisol responses in lambs during a first 6-min phase in the home pen where the familiar caregiver gently stroked the lamb, and then physiological and behavioural responses in a test pen during a 20-min – phase of social isolation followed by a 20-min – phase of reunion with its familiar caregiver. The lambs expressed behavioural agitation during the whole period of isolation. A strong affiliative response towards the human and a sustained reduction of the agitation behaviour were observed during reunion. Lambs’ behaviours when isolated and when in contact with the human were correlated suggesting a response to social separation from the familiar caregiver more than to social isolation from congeners. No significant changes in cortisol levels were observed during the test. Oxytocin levels did not vary during human contact, but increased when the familiar caregiver left the lamb alone in the test pen. In conclusion, lambs displayed affiliative responses towards their caregiver, and the lack of cortisol response during isolation while oxytocin was released suggest an anti-stress effect of oxytocin.

Rumenocentesis is commonly used to collect rumen fluid to screen for acidosis. This study was designed to investigate whether rumenocentesis induced pain and stress and, if so, whether local anaesthesia could limit this. Twenty-four dairy cows were assigned to one of three treatments: (1) rumenocentesis with local anaesthesia (AR); (2) rumenocentesis without local anaesthesia (R); and (3) local anaesthesia only (A). Treatments were performed in a restraining cage. The cows were placed in the cage on three consecutive days and anaesthesia and/or rumenocentesis was performed on the second day. Blood samples for cortisol determination and heart rate were recorded from 0.25 h before treatment until 4 h after. Behaviour was noted while the cows were caged. Feed intake and milk production were measured the week before treatment, on the day of treatment, and the day after. With all three treatments, cortisol concentrations and heart rate were increased while cows were in the cage. Cortisol, cardiac and behavioural responses were not significantly higher in the R and AR treatments than the A group. Cortisol concentrations and heart rate did not change between days. Feed intake and milk production were unaffected by the treatments. It was concluded that rumenocentesis does not appear more stressful than local anaesthesia or handling.

Needs for objective and automatic assessment of animal welfare are increasing in livestock production. The strong dam-offspring relationship is a good model to investigate the vocal expression of emotions in animals since removing the lamb induces behavioural distress in ewes (e.g. agitation and vocalisations). We analysed acoustic characteristics in bleats of ewes following an unpredictable separation from their lambs, in addition to their behavioural and physiological responses. Twenty four ewes of INRA 401 breed were individually exposed to 3 successive 3 min phases: 1) in contact with their lamb placed behind a grid, 2) at 6m away from the lamb and 3) again in contact. Behaviour, vocalisations and cardiac activity (via adhesive external electrodes and a telemetric remote system) were recorded and blood was collected just after the test by venipuncture for cortisol assay. After the lamb was moved away, the ewes were more active and more vigilant, they also bleated more, and their heart rate and cortisol levels dramatically increased. These results confirm that the separation induced behavioural and physiological responses of distress in ewes which reflect negative emotional states. Beside, acoustic analyses show that lamb withdrawal induced changes in the voice characteristics of mothers in correlation with their physiological and behavioural responses. Temporal parameters (e.g. total duration) increased, as did amplitude parameters (e.g. energy, RMS). Concerning the frequency parameters, fundamental frequency increased but frequency Bandwidth, Quartiles (Q25%), (Q50%), (Q75%) decreased. Therefore some bleat characteristics can be used as acoustic markers of emotional distress. Acoustic sensory modality could thus provide an objective basis of negative emotions in sheep, which measure could be easier to automate than other behavioural and physiological correlates of emotional reactions.

The present study evaluated whether feed deprivation can increase reactivity to stressful events, such as those that can occur at slaughter. Therefore, effects of 30 h of feed deprivation on behavior, including reactions to psychological stressors, and physiological status in cattle were determined. Sixteen Holstein cows (Exp. 1) and 32 Holstein heifers (Exp. 2) were either fed (FE) or 30-h feed deprived (FD). Throughout the first day of feed deprivation and during evening feed distribution to control animals, FD heifers and cows were more active than controls (P < 0.05). In Exp. 1, during a feeding test, in response to a sudden air blast arising from the bucket from which the cow was feeding, FD cows showed a longer latency to return to feed (P = 0.0002), spent less time in the bucket air blast zone (P = 0.008) and less time motionless (P = 0.03), and tended to withdraw over a longer distance (P = 0.07) than FE cows. In Exp. 2, during a reactivity test, FD heifers spent more (P = 0.0001) time motionless in response to social isolation than FE heifers. In Exp. 2, one-half of the FE and FD heifers were subjected to an additional physical and psychological stressor just before the reactivity test by driving them for 5 min through a labyrinth. Within heifers subjected to the additional stressor, FD heifers were less accepting of being detained (P = 0.05) and stroked (P = 0.003) by a familiar stockperson in a corner of the test arena. Compared with FE animals, FD heifers and FD cows had greater plasma cortisol concentrations (P < 0.05). Feed-deprived cows also had reduced β-hydroxybutyrate concentrations (P = 0.02) compared with FE cows. Thus, in cattle, FD influenced some of the classical indicators of energy metabolism and exacerbated reactivity to sudden events. In addition, when additional stressors were applied, FD cattle were more reluctant to accept handling. Results indicate that a multifactorial origin of stressors during the slaughter period may synergistically increase psychological stress of cattle.