Conditions for assessing cortisol in sheep: the total form in blood v. the free form in saliva.

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.

Spatial distance and reactivity traits alter the positive perception of brushing by ewes.

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.

Effects of a chronic stress treatment on vaccinal response in lambs.

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.

Effects of feed deprivation on behavioral reactivity and physiological status in Holstein cattle.

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 ß-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.

The motivation of cows to walk as thwarted by tethering.

Cows are often housed in tie-sheds for some part of the year (from 20 to 80% of adult cows in European countries). By contrast, regulations aiming at improving animal welfare generally provide for more opportunity to express behavior and so limit tethering (the European directive for sows). To inform current debate on whether cows should be tethered from an animal welfare point of view, 2 experiments were conducted to determine whether cows were motivated to walk (if their walking motivation was thwarted by tethering) and whether this frustration resulted in acute or chronic stress. In Exp. 1, fifteen cows were housed in tie-stalls for 1, 3, 9, or 27 d or loose-housed in a pen equipped with free-stalls for 27 d according to a Latin square design. On d 28, cows were observed in an 80-m(2) arena where they could walk or run for 10 min. In Exp. 2, fifteen cows were either loose-housed or tie-housed with or without 1 h of daily access to an exercise area according to a Latin square design (treatment duration of 27 d). On d 28, cows were observed in the test arena for 10 min as in Exp. 1. In addition, in Exp. 2, milk was sampled twice weekly for cortisol concentrations and cows underwent a chronic stress test (challenge with ACTH, followed by blood samples for cortisol concentrations). When they were tied with no access to exercise, cows displayed a greater locomotor activity in the test arena (time spent walking x 1.4 in Exp. 1, P < 0.05; time spent trotting x 5 in Exp. 2, P < 0.05), whatever the duration of tie-housing. Regular exercise caused locomotor activity in the arena test to revert to levels observed when cows were loose-housed. Basal cortisol concentrations in milk decreased with time whatever the housing condition (P < 0.001). This decrease was more marked when cows were tied than when they were loose-housed (P < 0.05). Cortisol responses to ACTH were similar between treatments. Adult cows are motivated to walk, and tethering thwarts this motivation. However, the frustration produced by tethering does not result in either acute or chronic physiological stress responses. We recommend that cows housed in tie-sheds be given regular access to an exercise area.

Variations in meat pH of beef bulls in relation to conditions of transfer to slaughter and previous history of the animals.

Animals are subjected to various events that cause physical exhaustion and psychological stress during transfer to slaughter. This can lead to defective meat quality. Some animals may be better able to withstand the stress of transfer, depending on their previous experience of transport and on their finishing conditions (mixing, farmers' attitudes). The objective of this study was to assess the impact of 1) the conditions of transfer to slaughter (including duration of the journey, waiting time at lairage, etc.); and 2) the bulls' previous history (including experience in transport, mixing during finishing, and the farmers' attitudes) on the reactions of bulls to transfer and on their meat quality. We conducted a survey in commercial conditions. The history of the bulls and the facilities on the farms were noted; farmers were questioned on their attitudes; the bulls' reactions to loading into and unloading from the truck were observed; journey-related data were collected; and cortisol concentration at slaughter and the pH of the LM and the rectus abdominis were measured. Our study confirmed that certain physical factors associated with transport can increase stress and limit the decline of meat pH. These factors include the absence of loading facilities on the farm, transport on a warm day, or a short waiting time at lairage. Social aspects also played a role; the presence of bulls from the same finishing group limited stress and improved the pH decline. Events and management before transfer were also of importance; the farmer awareness of the sensitivity of bulls to humans or to feeding schedules but the absence of a positive attitude toward close contacts with bulls were all likely to limit stress or its consequences on meat pH. Although these results need to be confirmed in controlled experiments, they suggest that good management of beef bulls before and during transfer is essential to meat quality.

Calves' responses to repeated social regrouping and relocation.

Because of welfare concerns and increased labor efficiency, calves are increasingly housed in groups. To reduce variability in live weight within groups, farmers frequently regroup calves according to growth rate. We assessed the consequences of repeated regrouping and relocation on the welfare of 32 male Holstein calves housed in pairs. Animals of half of the pairs (regrouped calves) were placed in a new pen with a new partner once a week for 14 wk. Animals of the other half of the pairs (control calves) stayed in the same pen with the same partner. Behavior was observed for the 3 h following four mixings and for 24 h after all relocations were finished. The functioning of the hypothalamic-pituitary-adrenal axis and of the sympathetic nervous system were assessed. Calves were weighed once a week, their health was assessed daily, and abomasa were inspected when the calves were slaughtered. Calves reacted to the first mixing by interacting with the new partner and increasing their general activity (sniffing the partner in regrouped calves vs controls: 5.5 vs 2.9, P < 0.01; percentage time stepping: 3.2 vs 1.3, P < 0.001). This effect disappeared by the ninth mixing. After all relocations were completed, regrouped calves were more active at the end of the day and less active at night (P < 0.05). Cortisol responses to exogenous ACTH were higher in regrouped calves (integrated response: 6,688 vs 5,508 ng x min/mL, P < 0.01). Basal cortisol levels, ACTH responses to corticotropin-releasing hormone, activities of catecholamine-synthesizing enzymes (tyrosine hydroxylase and phenylethanolamine N-methyl transferase), and the incidence of health problems and growth rates did not differ between the two groups. Regrouped calves had fewer abomasal ulcers. Apart from the increased sensitivity of the adrenal cortex of regrouped calves to ACTH and the modification in the daily rhythm of activity, there was no clear evidence that repeated regrouping and relocation stresses calves. Aggression between calves was rare, and calves seemed to habituate to repeated mixing.

Adrenocorticotropic hormone and cortisol in calves after corticotropin-releasing hormone.

The aim for this study was to analyze responsiveness of the hypothalamo-pituitary-adrenocortical axis to exogenous bovine corticotropin-releasing hormone (bCRH) in calves. Two dose-response studies were carried out, using either bCRH alone (dose rates of 0, .01, .03, and .1 microg bCRH/kg live weight) or in combination with arginine-vasopressin (bCRH:AVP, 0:0, .1:.05, .5:.25, and 1:.5 microg kg live weight). The bCRH was administered i.v. to calves (n = 5 to 7 per dose) housed individually or in groups. Serial blood samples were obtained from before to 300 min after injection and analyzed for plasma ACTH and cortisol concentrations. The lowest bCRH dose that produced a response in all calves was .1 microg/kg. In the experiment using bCRH with AVP, increasing the bCRH dose from .1 to 1 microg/kg resulted in an increase in peak ACTH concentration (321 vs. 2,003 pg/mL) but did not significantly affect the peak cortisol concentration (37 vs. 40 ng/mL). The time to reach the peak cortisol concentration increased with the dose of bCRH with AVP (from 38 to 111 min). The ACTH and cortisol concentrations determined at any time between 20 and 90 min after bCRH injection were correlated to the integrated responses calculated as areas under the ACTH and the cortisol curves (r between .61 and .99, P<.05). In comparison with results from studies in humans, pigs, and sheep, our data showed that the pituitary of calves seems less sensitive to CRH than that of other mammals, despite a greater capacity to produce ACTH. Moreover, the calf's adrenals seem to have a lower capacity to produce cortisol than adrenals of other mammals. As in other species, it seems that AVP enhances the release of ACTH and cortisol. For CRH challenge to be used in calves, we suggest injecting at least .1 microg of bCRH/kg live weight either with or without AVP and taking several blood samples before injection and between 20 and 90 min after injection.