The discrimination threshold: A selection criterion for analytical methods based on measurement uncertainty - Application to animal stress studies.

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.

Heat stress in cows at pasture and benefit of shade in a temperate climate region.

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.

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.

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.

Human-lamb bonding: oxytocin, cortisol and behavioural responses of lambs to human contacts and social separation.

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.

An assessment of the impact of rumenocentesis on pain and stress in cattle and the effect of local anaesthesia.

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 4h 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.

Exploration of the hypothalamic-pituitary-adrenal function as a tool to evaluate animal welfare.

Measuring HPA axis activity is the standard approach to the study of stress and welfare in farm animals. Although the reference technique is the use of blood plasma to measure glucocorticoid hormones (cortisol or corticosterone), several alternative methods such as the measurement of corticosteroids in saliva, urine or faeces have been developed to overcome the stress induced by blood sampling itself. In chronic stress situations, as is frequently the case in studies about farm animal welfare, hormonal secretions are usually unchanged but dynamic testing allows the demonstration of functional changes at several levels of the system, including the sensitization of the adrenal cortex to ACTH and the resistance of the axis to feedback inhibition by corticosteroids (dexamethasone suppression test). Beyond these procedural aspects, the main pitfall in the use of HPA axis activity is in the interpretation of experimental data. The large variability of the system has to be taken into consideration, since corticosteroid hormone secretion is usually pulsatile, follows diurnal and seasonal rhythms, is influenced by feed intake and environmental factors such as temperature and humidity, age and physiological state, just to cite the main sources of variation. The corresponding changes reflect the important role of glucocorticoid hormones in a number of basic physiological processes such as energy metabolism and central nervous system functioning. Furthermore, large differences have been found across species, breeds and individuals, which reflect the contribution of genetic factors and environmental influences, especially during development, in HPA axis functioning. Usually, these results will be integrated with data from behavioral observation, production and pathology records in a comprehensive approach of farm animal welfare.