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Conditions for assessing cortisol in sheep: the total form in blood v. the free form in saliva
- S. Andanson, A. Boissy, I. Veissier
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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
- P. R. Tamioso, A. Boissy, X. Boivin, H. Chandèze, S. Andanson, É. Delval, C. A. Taconeli, D. Hazard, C. F. M. Molento
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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
- A. Destrez, A. Boissy, L. Guilloteau, S. Andanson, A. Souriau, K. Laroucau, E. Chaillou, V. Deiss
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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.