Chronic elevation of plasma cortisol causes differential expression of predominating glucocorticoid in plasma, saliva, fecal, and wool matrices in sheep.

There is increasing interest in using nonblood measures of glucocorticoids to assess the physiological response to chronic stress conditions. In sheep, cortisol has been measured in various matrices including saliva, feces, and wool, but comprehensive studies of the relationship between plasma concentrations of cortisol and concentrations in these nonblood matrices are lacking. Therefore, we tested the hypothesis that administration of cortisol to sheep would result in elevated concentrations of cortisol in blood, saliva, feces, and wool. Merino ewes were administered with saline or 2 mg/kg BW/d hydrocortisone acetate (HCA) by intramuscular (i.m.) injection for 28 d. This treatment was imposed to mimic circulating cortisol concentrations experienced during periods of chronic stress. Cortisol and cortisone were directly measured in plasma, saliva, and wool before, during, and after treatment with saline or HCA. A 14-d pre-treatment and a 14-d post-treatment period were used to measure time taken for glucocorticoid concentrations in each of the matrices to return to baseline levels. Cortisol was also measured in feces before, during, and after treatment. Wool growth was also measured. Before treatment, there was no difference in the concentration of cortisol or cortisone in plasma, saliva, feces, or wool in animals treated with saline or HCA. In contrast, treatment with HCA increased (P < 0.05) concentrations of both cortisol and cortisone in plasma, saliva, and wool and of cortisol in feces. In plasma, cortisol concentrations were higher than cortisone (P < 0.05), whereas saliva cortisol and cortisone concentrations did not differ significantly. In wool, the concentration of cortisone was about 19-fold higher than that of cortisol during treatment and post-treatment periods. Treatment with HCA inhibited wool growth. These results demonstrate that an increase in glucocorticoids in the blood of sheep is reflected in increases in saliva (after 7 d of treatment), feces (21 d), and wool (14 d). Therefore, measures of glucocorticoids in these matrices may provide a measure of activation of the adrenal glands over time in sheep, thereby providing a retrospective indicator of chronic stress. With respect to wool, it appears that cortisol is predominantly metabolized to cortisone in the skin or wool follicle and is stored as cortisone. Therefore wool cortisone may also provide an important measure in quantifiying chronic stress in sheep.

Brain-derived neurotrophic factor in serum as an animal welfare indicator of environmental enrichment in pigs.

Environment enrichment is a rising topic for animal welfare but measures to identify effective enrichment interventions are lacking. In humans and rodent species, environmental enrichment increases brain-derived neurotrophic factor (BDNF), the most abundant neurotrophin in the brain. Higher BDNF concentration is ultimately linked to higher stress resilience, and BDNF in the hippocampus enhances learning and memory. In addition, BDNF concentrations in the brain and blood are correlated, offering the opportunity to use peripheral BDNF as a minimally invasive measure of effective enrichment reflecting neural changes. This study investigated changes in serum BDNF following the provision of environmental enrichment to pigs. Pigs were housed in different environments during lactation (enriched vs barren) and after weaning (enriched vs barren), using a 2 × 2 factorial design and the provision of a foraging block as enrichment. Pigs provided with foraging enrichment during lactation or after weaning tended to have higher serum BDNF concentrations than pigs housed in a barren environment, and this effect was significant for pigs enriched during lactation when sampled 5 wk after weaning. Brain-derived neurotrophic factor concentration reduced as the pigs aged from 3 to 11 wk. The measurement of BDNF in serum brings a practical approach to study the effects of environmental enrichment on neurobiological changes in domestic animals. A better understanding of the factors modulating BDNF and its link to welfare states could bring insight into the benefits of stimulating an animal's life.

Response to gonadotrophins differs for gilts from female- and male-biased litters.

In several species, females masculinised by abnormal androgen exposure in utero have poor reproductive performance and gilts born into litters with a male bias are likely exposed to greater androgen concentrations prenatally than gilts born into female-biased litters. At 24h of age, piglet plasma testosterone concentrations in gilts from male-biased litters (>60% male; n=22) or female-biased litters (>60% female; n=27) were not different. At 18 wks of age, all gilts received an injection of 400IU equine chorionic gonadotrophin plus 200IU human chorionic gonadotrophin to stimulate oestrus. Two weeks after the injection gilts were slaughtered and ovaries collected for determination of numbers of corpora lutea (CL). Compared to gilts from female-biased litters, gilts from male-biased litters were more likely to ovulate (86.0% vs 59.5%, P=0.047) and had more CL (13.1±1.5 vs 7.2±1.7, P=0.015). The present data indicate an effect of birth litter sex-bias on pre-pubertal physiological development, possibly involving organisational effects at the ovarian cellular level impacting on future ovarian function. Potential impacts on subsequent fertility remain to be determined.

INVITED REVIEW: The usefulness of measuring glucocorticoids for assessing animal welfare.

Glucocorticoids (corticosterone in birds and rodents and cortisol in all other mammals) are glucoregulatory hormones that are synthesized in response to a range of stimuli including stress and are regularly measured in the assessment of animal welfare. Glucocorticoids have many normal or non-stress-related functions, and glucocorticoid synthesis can increase in response to pleasure, excitement, and arousal as well as fear, anxiety, and pain. Often, when assessing animal welfare, little consideration is given to normal non-stress-related glucocorticoid functions or the complex mechanisms that regulate the effects of glucocorticoids on physiology. In addition, it is rarely acknowledged that increased glucocorticoid synthesis can indicate positive welfare states or that a stress response can increase fitness and improve the welfare of an animal. In this paper, we review how and when glucocorticoid synthesis increases, the actions mediated through type I and type II glucocorticoid receptors, the importance of corticosteroid-binding globulin, the role of 11 ß-hydroxysteroid dehydrogenase, and the key aspects of neurophysiology relevant to activating the hypothalamo-pituitary-adrenal axis. This is discussed in the context of animal welfare assessment, particularly under the biological functioning and affective states frameworks. We contend that extending the assessment of animal welfare to key brain regions afferent to the hypothalamus and incorporating the aspects of glucocorticoid physiology that affect change in target tissue will advance animal welfare science and inspire more comprehensive assessment of the welfare of animals.

Impact of psychosocial stress on gonadotrophins and sexual behaviour in females: role for cortisol?

This review focuses on the importance of cortisol in mediating the inhibitory effects of psychosocial stress on reproduction in females. In particular, we have summarized our research in sheep where we have systematically established whether cortisol is both sufficient and necessary to suppress reproductive hormone secretion and inhibit sexual behaviour. Our findings are put into context with previous work and are used to develop important concepts as well as to identify productive further lines of investigation. It is clear that cortisol is necessary to inhibit some, but not all, aspects of reproduction in female sheep. These actions vary with reproductive state, and there are important interactions with gonadal steroids. The impact of cortisol on the tonic secretion of gonadotrophin-releasing hormone and luteinizing hormone has been investigated extensively, but less is known about the surge secretion of these hormones and their effects on sexual behaviour. Furthermore, there are separate effects of cortisol in the brain (hypothalamus) and at the anterior pituitary, illustrating that there are different mechanisms of action. Thus, although cortisol is important in mediating some of the effects of stress on reproduction, we need to look beyond cortisol and investigate some of the other mechanisms and mediators that relay the effects of stress on reproduction. In this regard, we propose that a group of neurons in the hypothalamus that co-synthesize kisspeptin, neurokinin B and dynorphin, termed KNDy cells, play important roles in mediating the effects of cortisol on reproduction. This hypothesis needs to be rigorously tested.

The hypothalamo-pituitary-adrenal (HPA) axis in sheep is attenuated during lactation in response to psychosocial and predator stress.

Activation of the hypothalamo-pituitary-adrenal (HPA) axis by psychosocial stress is attenuated during lactation. We tested the hypothesis that lactating ewes will have attenuated HPA axis responses to isolation and restraint but will have greater responses to predator stress in the form of barking dogs. We imposed two 4 h stressors: psychosocial stress (isolation and restraint of ewes) and predator stress (barking dogs). Blood was collected intravenous every 10 min from nonlactating ewes (n = 6), lactating ewes with lambs present but not able to be suckled (n = 6), and lactating ewes with lambs present and able to be suckled (n = 6). Plasma cortisol and oxytocin were measured. For nonlactating ewes, cortisol increased (P < 0.01) in response to both stressors, and these increases were greater (P < 0.01) than that in the lactating animals. For lactating ewes with lambs present but unable to be suckled, cortisol increased (P < 0.05) in response to both stressors with a greater response to barking dogs (P < 0.05). For lactating ewes with lambs present and able to be suckled, cortisol increased (P < 0.01) in response to barking dogs only. Plasma oxytocin was greater (P < 0.01) in lactating ewes than in nonlactating ewes and did not change in response to the stressors. In conclusion, lactating ewes are likely to have a greater HPA axis response to a stressor that may be perceived to threaten the welfare of themselves and/or their offspring. The role of oxytocin in attenuation of the HPA axis to stress in sheep is unclear from the current research and requires further investigation.

Relationship between plasma and tissue corticosterone in laying hens (Gallus gallus domesticus): implications for stress physiology and animal welfare.

This study directly compared the dynamics of change in plasma corticosterone concentration with the dynamics of change in tissue corticosterone concentration in laying hens. In concert, we measured the rate of gluconeogenesis, glycogenesis, and glycolysis in the liver, kidney, skeletal muscle, and heart. We evaluated these changes acutely, over 3 h in response to an adrenocorticotropic hormone (ACTH) injection, and chronically, over 24 h in response to food and water deprivation. In response to ACTH injection, there was a significant (P < 0.05) increase in plasma corticosterone concentration and a parallel significant increase in corticosterone concentration in the skeletal muscle, kidney, and heart. However, the change in corticosterone concentration in the liver did not parallel the plasma, at times it was greater than the plasma, and there was a second significant increase (P < 0.05) in corticosterone concentration in the liver after 180 min. Under these conditions, the rate of gluconeogenesis in the liver decreased and the rate of glycogenesis increased. In contrast, after 12 h and 24 h of food and water deprivation plasma corticosterone concentration was increased, and this was paralleled by increased corticosterone concentration in the liver, an increase in the rate of gluconeogenesis and a decrease in the rate of glycogenesis. After ACTH injection, glucose concentration in the liver was not significantly depleted but after 12 h or 24 h of food and water deprivation it was significantly depleted (P < 0.05). Plasma corticosterone concentration provided different insight into the effect of the stressor on hen physiology under acute and chronic conditions. Our data suggest that extending our evaluation of stress to the site of corticosterone action, that is, the target tissue, may enhance our ability to evaluate stress and the welfare of laying hens.