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.

Positive effects of melatonin treatment on the reproductive performance of young border leicester rams mated to merino ewes in spring: preliminary observations.

Poor reproductive performance of Merino ewe flocks when mated to Border Leicester rams during spring may be due to seasonality of the Border Leicester breed. Two approaches were taken to test this assumption. Six young (12 months old) or six mixed-age (12, 24 and ≥36 months old) Border Leicester rams were either treated or not treated with melatonin implants (2 × 2 design) 6 weeks before the four groups of rams were each put with approximately 300 Merino ewes for an 8-week mating period. Implants were inserted in early September (experiment 1). The second approach was to yard or not yard ewes and mixed-age rams on several occasions during the first 3 weeks of the mating period (experiment 2). Pregnancy rate and twinning percentage were assessed by ultrasonography. In experiment 1, melatonin treatment in young rams increased (p < 0.001) pregnancy rate from 5.0% to 92.6%, but mixed-age rams did not respond (90.7% vs 89.5% for melatonin and non-melatonin treatments, respectively). Twinning rate was similar (p > 0.05) for ewes mated to either melatonin or non-melatonin-treated young rams (36.8% vs 40.0%, respectively), whereas melatonin significantly improved (p < 0.05) twinning rate in those ewes mated to mixed-age rams (49.1% vs 36.1%). After 6 weeks of melatonin treatment, scrotal circumference was greater (p < 0.05) in both young and mixed-aged rams than in untreated counterparts. In experiment 2, yarding of ewes and rams overnight on several occasions early in the mating period reduced (p < 0.001) pregnancy rate compared with non-yarded counterparts (89.5% vs 65.5%). Twinning rate was not affected (37.7% vs 36.1%, respectively). In summary, melatonin treatment of Border Leicester rams significantly improved flock reproductive performance in spring due to improved pregnancy rates with young rams and improved litter size with mixed-age rams.

Early human contact and housing for pigs - part 1: responses to humans, novelty and isolation.

The development of fear and stress responses in animals can be influenced by early life experiences, including interactions with humans, maternal care, and the physical surroundings. This paper is the first of three reporting on a large experiment examining the effects of the early housing environment and early positive human contact on stress resilience in pigs. This first paper reports on the responses of pigs to humans, novelty, and social isolation. Using a 2 × 2 factorial design, 48 litters of pigs were reared in either a conventional farrowing crate (FC) where the sow was confined or a loose farrowing pen (LP; PigSAFE pen) which was larger, more physically complex and allowed the sow to move freely throughout the farrowing and lactation period. Piglets were provided with either routine contact from stockpeople (C), or routine contact plus regular opportunities for positive human contact (+HC) involving 5 min of scratching, patting and stroking imposed to the litter 5 days/week from 0-4 weeks of age. The positive handling treatment was highly effective in reducing piglets' fear of humans, based on +HC piglets showing greater approach and less avoidance of an unfamiliar person at 3 weeks of age. There was evidence that this reduction in fear of humans lasted well beyond when the treatment was applied (lactation), with +HC pigs showing greater approach and less avoidance of humans in tests at 6, 9 and 14 weeks of age. The +HC treatment also reduced piglets' fear of a novel object at 3 weeks of age, and for pigs in FC, the cortisol response after social isolation at 7 weeks of age. Rearing in FC compared to LP reduced piglets' fear of novelty at 3 weeks of age, as well as their vocalisations and cortisol response to isolation at 7 weeks of age. The FC pigs showed greater approach and less avoidance of humans compared to LP pigs at 3, 4 and 6 weeks of age, but not at 9 and 14 weeks of age. These results show that positive handling early in life can reduce pigs' fear of humans, fear of novelty and physiological stress response to social isolation. The LP pigs were reared in a more isolated environment with less overall contact with stockpeople and other pigs, which may have increased their fear responses to humans and novel situations, suggesting that different housing systems can modulate these pigs' responses.

Early human contact and housing for pigs - part 3: ability to cope with the environment.

Early experiences can have long-term impacts on stress adaptability. This paper is the last of three in a series on early experiences and stress in pigs, and reports on the effects of early human contact and housing on the ability of pigs to cope with their general environment. Using a 2 × 2 factorial design, 48 litters of pigs were reared in either a farrowing crate (FC) or a loose farrowing pen (LP; PigSAFE pen) which was larger, more physically complex and allowed the sow to move freely. Piglets were provided with either routine contact from stockpeople (C), or routine contact plus regular opportunities for positive human contact (+HC) involving 5 min of scratching, patting and stroking imposed to the litter 5 days/week from 0 to 4 weeks of age. At 4 weeks of age (preweaning), C piglets that were reared in FC had considerably lower concentrations of serum brain-derived neurotrophic factor (BDNF) than piglets from the other treatment combinations. Compared to C pigs, +HC pigs had fewer injuries at 4 weeks of age. There were no clear effects of human contact on BDNF concentrations or injuries after weaning, or on basal cortisol or immunoglobulin-A concentrations, behavioural time budgets, tear staining, growth, or piglet survival. Compared to FC piglets, LP piglets showed more play behaviour and interactions with the dam and less repetitive nosing towards pen mates during lactation. There was no evidence that early housing affected pigs' behavioural time budgets or physiology after weaning. Tear staining severity was greater in LP piglets at 4 weeks of age, but this may have been associated with the higher growth rates of LP piglets preweaning. There was no effect of lactation housing on growth after weaning. Preweaning piglet mortality was higher in the loose system. The findings on BDNF concentrations, injuries and play behaviour suggest improved welfare during the treatment period in +HC and LP piglets compared to C and FC piglets, respectively. These results together with those from the other papers in this series indicate that positive human interaction early in life promotes stress adaptability in pigs. Furthermore, while the farrowing crate environment deprives piglets of opportunities for play behaviour and sow interaction, there was no evidence that rearing in crates negatively affected pig welfare or stress resilience after weaning. Whether these findings are specific to the two housing systems studied here, or can be generalised to other housing designs, warrants further research.

Early human contact and housing for pigs - part 2: resilience to routine husbandry practices.

The ability of pigs to cope with routine farming practices can affect their welfare. This paper is part of a series on early experiences and stress, and reports on the effects of early human contact and housing on the responses of pigs to routine husbandry practices. Using a 2 × 2 factorial design, 48 litters of pigs were raised in either a conventional farrowing crate (FC) or a loose farrowing pen (LP; PigSAFE pen) which was larger, more physically complex and allowed the sow to move freely. Piglets were provided with either routine contact from stockpeople (C), or routine contact plus regular opportunities for positive human contact (+HC) involving 5 min of scratching, patting and stroking imposed to the litter 5 days/week from 0 to 4 weeks of age. At 4 weeks of age, piglets were weaned and re-housed with controlled mixing of litters within treatment. At 4 days of age, after only 3 bouts of the handling treatment, +HC pigs showed less escape behaviour than C pigs after capture by a stockperson for vaccinations and tail docking, and shorter durations of vocalisations throughout the procedures. The  +HC pigs also showed less escape behaviour when captured by a stockperson at 3 weeks of age. The FC pigs showed less escape behaviour than LP pigs after capture by a stockperson at 4 days of age but not at 3 weeks of age. Serum cortisol concentrations were lower in FC pigs than LP pigs 2 h after weaning but not at 49 h after weaning, whereas serum cortisol concentrations were lower in  +HC pigs than C pigs at 49 h after weaning but not at 2 h after weaning. In the period from 0 to 1 h after weaning, C pigs from LP performed the most escape attempts, although escape attempts were rare overall. When being moved out of the home pen by a stockperson at 21 weeks of age, FC pigs showed less baulking than LP pigs, but there were no detected effects of human contact treatment. In conclusion, both housing system and human contact during lactation affected the stress responses of pigs to routine husbandry practices. The +HC and FC pigs appeared to cope better than C and LP pigs, based on lower responses indicative of stress including escape behaviour, vocalisations and cortisol concentrations. These findings are consistent with corresponding reductions in fear that were reported in Part 1 of this series of papers.

The effects of dietary weight loss with or without exercise training on liver enzymes in obese metabolic syndrome subjects.

AIM: Insulin resistance and visceral adiposity are predisposing factors for fatty liver disease. The main objectives of this study were (i) to compare the effects of caloric restriction (CR) alone or together with moderate-intensity aerobic exercise training (CR+EX) on liver enzymes, a surrogate marker of liver injury, in obese metabolic syndrome (MetS) subjects and (ii) to identify anthropometric, metabolic, cardiovascular and dietary predictors of changes in liver enzymes. METHODS: Sedentary men and women (n = 63), aged 55 ± 6 (s.d.) years with body mass index 32.7 ± 4.1 kg/m(2) and confirmed MetS, were randomized to 12-week CR, CR+EX or no treatment (Control). RESULTS: Weight loss averaged 7.6% in the CR and 9.1% in the CR+EX group (time effect, p < 0.001; group effect, p = 0.11); insulin sensitivity improved by 49 and 45%, respectively (both p < 0.001). Fitness (maximal oxygen consumption) increased by 19% in the CR+EX group only (p < 0.001). Alanine aminotransferase (ALT) levels decreased by 20% in the CR and 24% in the CR+EX group (time effect, both p < 0.001; group effect, p = 0.68); corresponding values for γ-glutamyltransferase (GGT) were -28 and -33%, respectively (time effect, both p < 0.001; group effect, p = 0.28). Reduction in abdominal fat mass (measured by DXA from L1 to L4) independently predicted ΔALT (r = 0.42, p = 0.005) and ΔGGT (r = 0.55, p < 0.001), whereas change in dietary saturated fat intake was independently associated with ΔALT (r = 0.35, p = 0.03). CONCLUSIONS: Reductions in central adiposity and saturated fat intake are key drivers of improvement in liver enzymes during lifestyle interventions. Exercise training did not confer significant incremental benefits in this study.

Acute and chronic stress-like levels of cortisol inhibit the oestradiol stimulus to induce sexual receptivity but have no effect on sexual attractivity or proceptivity in female sheep.

Stress-like levels of cortisol inhibit sexual receptivity in ewes but the mechanism of this action is not understood. One possibility is that cortisol interferes with the actions of oestradiol to induce sexual receptivity. We tested this hypothesis in 2 experiments with ovariectomised ewes that were artificially induced into oestrus by 12 days of i.m. injections of progesterone followed by an i.m. injection of oestradiol benzoate (ODB) 48 h later. In Experiment 1, ewes were randomly allocated to the following groups: saline infusion+25 µg ODB, saline infusion+50 µg ODB, cortisol infusion+25 µg ODB or cortisol infusion+50 µg ODB (n=5 per group). Saline or cortisol was infused i.v. for 40 h beginning at the ODB injection. In Experiment 2, ewes were infused with saline or cortisol (n=5 per group) for 5h beginning 1h before ODB injection. In both experiments, ewe sexual behaviour (attractivity, proceptivity and receptivity) was quantified every 6h. Blood samples were also collected. The cortisol infusion yielded plasma concentrations of cortisol similar to those seen during psychosocial stress. In both experiments, cortisol suppressed receptivity index (number of immobilisations by ewe/courtship displays by ram) and the number of times ewes were mounted but had no effect on attractivity or proceptivity, irrespective of the dose of ODB (Experiment 1). Cortisol also suppressed LH pulse amplitude. These results suggest that both an acute (5h) and chronic (40 h) infusion of cortisol inhibit oestradiol-induced sexual receptivity in ewes and that increasing the dose of ODB does not overcome the inhibitory effects of cortisol.

Sexual behaviour, semen quality and fertility of young Border Leicester rams administered melatonin during spring.

This study was conducted to test the hypothesis that between year variability in reproductive response to melatonin treatment of young Border Leicester (BL) rams in spring was related to sexual maturity at the time of treatment. Two variables of sexual maturity (mating behaviour and semen quality) were examined. In Experiment 1, mating activity of melatonin-treated and untreated rams was examined at two sites before a flock mating at one site. In Experiment 2, testosterone concentrations, semen quality and variables of in vitro fertilising capacity were examined. In Experiment 1, melatonin treatment did not alter sexual behaviour (latency to nose, total nosings, courtings including Flehmen expression, attempted mounts, mounts, ejaculations) with the exception of a lesser (P < 0.05) Flehmen expression at Site 1 whereas values for most variables were greater (P < 0.05) at Site 2. Treatment resulted in a greater pregnancy rate (89 % and 82 %, respectively; P < 0.05) and advanced distribution of pregnancies (P < 0.001) at Site 1. Testicular size and testosterone concentrations were also greater (P < 0.05) as a result of melatonin treatment in spring whilst the reverse occurred in autumn. In Experiment 2, sperm motility and in vitro fertilising capacity were greater (P < 0.05) and acrosome damage less (P < 0.05) as a result of melatonin treatment. In conclusion, variability in age at sexual maturity of young BL rams at spring mating can be reduced with melatonin treatment and is associated with differences in sexual behaviour, testicular growth, testosterone concentrations, and sperm quality.

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.

Estradiol enables cortisol to act directly upon the pituitary to suppress pituitary responsiveness to GnRH in sheep.

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Reduced protein diet and amino acid concentration alter intestinal barrier function and performance of broiler chickens with or without synthetic glucocorticoid.

Little is known about the effect of low-protein (LP) diets on intestinal barrier function and permeability. In the first part of this study, starting on day 9 of age, the growth performance of the birds fed 3 experimental diets in each phase of feeding (G/F: grower/finisher) was investigated. Three experimental diets were as follows: LP (170/150 g/kg CP) fortified with essential amino acids (EAA), standard protein (SP), (202/190 g/kg), and high protein (HP) (220/210 g/kg). LP and SP diets contained a similar level of EAA concentration, while the HP diet contained 10% above the Ross 308 specifications. Each diet was replicated 6 times (10 male birds per replicate). The second part investigated intestinal permeability (IP) and function on additional 72 birds. On days 14, 16, and 20, a total of 36 birds (12 birds per diet) were injected with dexamethasone (DEX) to induce leaky gut. Birds fed LP diets had lower body weight gain (BWG) and higher feed conversion ratio (FCR) compared with SP and HP diets in both grower and finisher phases of feeding. For the challenge part, DEX increased the FCR independent of diets. Diet and DEX interacted for BWG, whereby the effect of diets was only evident in sham-injected birds. Birds fed LP had a higher fluorescein isothiocyanate dextran (FITC-d) concentration indicating a more permeable intestine compared to HP, but similar to SP. DEX increased FITC-d concentration in all dietary treatments. Birds fed LP had less ileal zonula occludens-2 (ZO-2) expression in comparison with SP, but not HP. DEX increased the expression of Claudin3 and ZO-2 and reduced Claudin1 (P < 0.05) and junctional adhesion molecule 2 in the ileum. Expression of ileal Na+-dependent glucose transporter 1 (SGLT1) was upregulated in LP fed group. It is concluded that compared with SP, IP can be maintained in LP by supplementing EAA. However, when compared with HP, feeding birds with LP may lead to a higher IP. DEX had a profound independent effect on intestinal barrier function.

The effects of floor space and nest box access on the physiology and behavior of caged laying hens.

Confinement housing appears to be at the forefront of concern about laying hen welfare. This experiment examined the effects of floor space during rearing (315 or 945 cm2/bird) and adulthood (542 or 1648 cm2/bird) and access to a nest box on the welfare of caged laying hens. Measurements of the normality of biological functioning, such as plasma, egg albumen and yolk and fecal corticosterone concentrations, and heterophil to lymphocyte ratios, behavioral time budgets, mortality and efficiency of productivity, and measurement of hen preferences, such as choice behavior in Y maze tests, were used to assess hen welfare. There were no effects of treatment on physiological measurements. Hens given less space during adulthood spent less time mobile, inedible pecking, drinking, and preening and spent more time resting and feed pecking and sitting (P < 0.05). Hens with access to a nest box spent more time resting (P = 0.046) and less time sham dust bathing (P = 0.044) than hens without access to a nest box. There were no effects of space allowance on choice behavior for space or a nest box over food; however, hens with access to a nest box chose the nest box over food more than hens without access to a nest box (P = 0.0053). The present experiment provides no convincing evidence that either reducing space allowance in adulthood from 1648 to 542 cm2/bird or eliminating access to a nest box results in disruption of biological function. Less space and no access to a nest box did not increase the choice for more space or a nest box, respectively, over food in the preference tests. However, reduced floor space reduced behavioral freedom and denying access to a nest box eliminated the opportunity for the motivated behavior of laying their eggs in a discrete enclosed nest box, both of which presumably provide hens with the opportunity for positive affective experiences.

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe.

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.

Artificial lighting during winter increases milk yield in dairy ewes.

In Australia, the supply of sheep milk is reduced during the winter. Housing dairy animals under lights during winter is a simple technique to increase milk yield; however, it is difficult to predict the magnitude of this increase in dairy ewes, because there are few corroborating data. We studied 220 East Friesian crossbred ewes (50 primiparous and 170 multiparous ewes, respectively) that lambed in April to May 2007 (late autumn, southern hemisphere) and were weaned from their lambs within 24 h of parturition and milked exclusively by machine. These ewes were ranked according to their milk production, and ewes producing > or =1,000 mL/d of milk were allocated to 1 of 2 groups. One group of ewes was kept indoors under a long-day photoperiod (16 h of light), whereas the other group was kept indoors under a naturally declining day length. Ewes were maintained under these conditions for 8 wk. Milk yield was measured twice weekly, and ewe weight and condition were measured at weekly intervals. From a subset of ewes (n = 20 per group), milk samples were collected twice weekly at the morning milking to measure milk lipid, protein, and lactose, and blood samples were collected once a week to measure plasma prolactin concentrations. Mean daily milk yield was analyzed as a percentage of preexperimental milk yield because the milk yield of ewes housed under the long photoperiod was lower than that of ewes under a declining day length when the treatments began. Thus, the ewes under a long photoperiod yielded 91.7% of their starting yield by wk 8 of treatment, whereas ewes under a declining day length yielded 76.25% of their initial value (LSD = 5.1), and this divergence in milk yield was apparent by wk 2 of treatment. Mean plasma prolactin levels were greater in ewes housed under the long-day photoperiod (n = 20) compared with control ewes (n = 20) at wk 6 (168 +/- 27 vs. 72 +/- 19 ng/mL, respectively), wk 7 (125 +/- 28 vs. 37 +/- 7 ng/mL, respectively), and wk 8 of the experiment (132 +/- 35 vs. 31 +/- 7 ng/mL, respectively). The composition of the milk was similar between the groups at each time point, and milk from these ewes (n = 20 per group) contained, on average, 6.1 +/- 0.05% lipid, 4.8 +/- 0.02% protein, and 5.4 +/- 0.01% lactose (n = 309 samples). We concluded that ewes increase milk production in response to being housed under a long-day photoperiod during winter.

Isolation and restraint stress results in differential activation of corticotrophin-releasing hormone and arginine vasopressin neurons in sheep.

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Predicting milk yield in sheep used for dairying in Australia.

It is necessary to identify traits that are simple to measure and correlated with milk yield to select ewes for dairying from existing populations of sheep in Australia. We studied 217 primiparous and 113 multiparous (second parity, n = 51; third parity, n = 40; and fourth parity, n = 22) East Friesian crossbred ewes, for 2 consecutive lactations, that were milked by machine following a period of suckling (24 to 28 d). We measured lamb growth, milk production, milk yield, and residual milk during early lactation (

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.

Activation of the hypothalamo-pituitary-adrenal axis by isolation and restraint stress during lactation in ewes: effect of the presence of the lamb and suckling.

We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary-adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In experiment 1, blood samples were collected every 10 min from nonlactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In experiment 2, ewes (n = 6) were allocated to 1) nonlactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle, and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (control day) and for 4 h before and 4 h during stress (stress day). In experiment 1, the mean (+/-SEM) cortisol concentrations increased significantly (P < 0.05) in nonlactating ewes during stress but did not change in lactating ewes. In experiment 2, cortisol did not vary on the control day or pretreatment of the stress day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in nonlactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in nonlactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.

A sex difference in the cortisol response to tail docking and ACTH develops between 1 and 8 weeks of age in lambs.

It is important to understand factors that may influence responses to stress, as these factors may also influence vulnerability to pathologies that can develop when stress responses are excessive or prolonged. It is clear that, in adults, the sex of an individual can influence the cortisol response to stress in a stressor specific manner. Nevertheless, the stage of development at which these sex differences emerge is unknown. We tested the hypothesis that there are sex differences in the cortisol response to tail docking and ACTH in lambs of 1 and 8 weeks of age. We also established cortisol responses in males when tail docking was imposed alone and in combination with castration at these ages. In experiment 1, 1 and 8 week old male and female lambs were subjected to sham handling, tail docking or, in males, a combination of tail docking and castration. In experiment 2, we administered ACTH (1.0 IU/kg) to male and female lambs at 1 and 8 weeks of age. There were significant cortisol responses to all treatments at both ages. Sex differences in the cortisol responses to tail docking and ACTH developed between 1 and 8 weeks of age, with females having greater responses than males. The data suggest that the mechanism for the sex difference in response to tail docking may involve the adrenal glands. At both ages, in males, the cortisol response to the combined treatment of tail docking and castration was significantly greater than that for tail docking alone.

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.