Finding biomarkers of experience in animals.

At a time when there is a growing public interest in animal welfare, it is critical to have objective means to assess the way that an animal experiences a situation. Objectivity is critical to ensure appropriate animal welfare outcomes. Existing behavioural, physiological, and neurobiological indicators that are used to assess animal welfare can verify the absence of extremely negative outcomes. But welfare is more than an absence of negative outcomes and an appropriate indicator should reflect the full spectrum of experience of an animal, from negative to positive. In this review, we draw from the knowledge of human biomedical science to propose a list of candidate biological markers (biomarkers) that should reflect the experiential state of non-human animals. The proposed biomarkers can be classified on their main function as endocrine, oxidative stress, non-coding molecular, and thermobiological markers. We also discuss practical challenges that must be addressed before any of these biomarkers can become useful to assess the experience of an animal in real-life.

Positive Human Contact and Housing Systems Impact the Responses of Piglets to Various Stressors.

This experiment studied the effects of lactation housing systems and human interaction on piglets' responses to routine stressors. Forty litters of piglets were reared in either a standard farrowing crate (FC) or a loose farrowing and lactation pen (LP; PigSAFE pen) and received either routine contact with humans (C) or regular opportunities for positive human contact (+HC; 3 min of patting, stroking and scratching 5 times/week). Behavioural and physiological responses to routine husbandry procedures, weaning, novelty and humans were studied in addition to effects on piglet growth, injuries and survival. Compared to C piglets, +HC piglets vocalised for shorter durations (p = 0.018) during husbandry procedures and showed a lower intensity of escape behaviour during iron injection (p = 0.042) and oral vaccination (p = 0.026) at 3 d of age, capture at 2 wk of age (p < 0.001), and intramuscular vaccination (p = 0.005) at 3 wk of age. +HC piglets at 2 wk of age were faster than C piglets to approach (p = 0.048) and interact (p = 0.042) with a stationary unfamiliar human. Compared to LP piglets, FC piglets showed a lower intensity of escape behaviour during capture and iron administration by a stockperson at 3 d of age (p = 0.043). FC piglets at 2 wk of age were faster than LP piglets to approach (p = 0.005) and interact (p = 0.027) with a novel object and approach (p = 0.009) and interact (p = 0.008) with an unfamiliar human. FC piglets had fewer injuries than LP piglets at 2 wk of age (p = 0.004). +HC pigs had fewer injuries than C pigs after weaning (p = 0.003). After weaning there were more pigs from LP than FC observed to be upright (both stationary, p = 0.002 and walking, p = 0.024), vocalizing (p = 0.004), nosing another pig (p = 0.035) and nosing the pen floor (p = 0.038). There were no significant effects on neutrophil:lymphocyte ratios or plasma cortisol concentrations 1.5 h after weaning. However, 25 h after weaning +HC pigs had higher haptoglobin concentrations than C pigs (p = 0.002), and C/LP pigs had higher cortisol concentrations than +HC/LP and C/FC pigs (p = 0.012). There were no significant effects on piglet growth, the number of piglets born alive or the number stillborn, however there were more piglets weaned from FC than LP (p = 0.035). The results from this experiment raise questions that require further research on the ability of pigs reared in loose pens to cope with stressors such as exposure to humans, novelty, husbandry procedures and weaning. This experiment also provides evidence that regular positive human interaction reduces pigs' fear of humans and husbandry procedures imposed by stockpeople. More research is required to determine if any of these effects are sustained long-term.

An Extended Photoperiod Increases Milk Yield and Decreases Ovulatory Activity in Dairy Goats.

Short day length is associated with reduced milk production in dairy ruminants. Dairy ruminants have been kept in lit sheds during winter to extend the day length and stimulate milk production. However, there studies are few on the effect of an extended photoperiod on the ensuing reproductive performance of dairy goats. The aim of this study was to examine the effect of long day photoperiod (LDPP) and exposure to bucks on milk production and plasma progesterone and prolactin in dairy goats. The study was conducted in 122 non-pregnant lactating dairy goats over 18 weeks from April to August (late autumn and winter in the Southern Hemisphere). The goats were kept in open sided sheds in which the control treatment received ambient lighting while the LDPP treatment received 16 h of light, including artificial lighting. In June, July and August synchronised does were randomly assigned each month to the presence or absence of a buck and ovulatory activity determined from plasma progesterone. Plasma progesterone concentrations were reduced (0.73 vs. 0.46 pmol, p < 0.001) while prolactin concentrations were increased (0.095 vs. 1.33 ng/mL, p < 0.001) in LDPP goats. The former response was most marked in late winter (0.58 vs. 0.004 pmol, p < 0.001) indicating a lack of functional corpora lutea. While there was no overall effect of buck exposure on plasma progesterone concentrations there was a three-way interaction such that plasma progesterone concentrations were increased (p < 0.05) by exposure to bucks in LDPP goats in August (late winter) but not at other times. Milk production was increased in LDPP goats over the latter stages of the study (1. 55 vs. 1.82 L/d, p < 0.05). Also, persistency of lactation was greater in LDPP goats with fewer goats drying off (13 vs. 0%, p < 0.05). These findings suggest that LDPP can increase milk production and persistence while decreasing ovulatory activity in dairy goats.

Impact of the COVID-19 Pandemic on the Welfare of Animals in Australia.

We report on the various responses in Australia during 2020 to minimize negative impacts of the COVID-19 pandemic on the welfare of animals. Most organizations and individuals with animals under their care had emergency preparedness plans in place for various scenarios; however, the restrictions on human movement to contain the spread of COVID-19, coupled with the economic impact and the health effects of COVID-19 on the skilled workforce, constituted a new threat to animal welfare for which there was no blueprint. The spontaneous formation of a national, multisectoral response group on animal welfare, consisting of more than 34 organizations with animals under their care, facilitated information flow during the crisis, which helped to mitigate some of the shocks to different organizations and to ensure continuity of care for animals during the pandemic. We conclude that animal welfare is a shared responsibility, and accordingly, a multisectoral approach to animal welfare during a crisis is required. Our experience demonstrates that to safeguard animal welfare during crises, nations should consider the following: a national risk assessment, clear communication channels, contingency plans for animal welfare, a crisis response group, and support systems for animal care providers. Our findings and recommendations from the Australian context may inform other countries to ensure that animal welfare is not compromised during the course of unpredictable events.

Anogenital distance reflects the sex ratio of a gilt's birth litter and predicts her reproductive success1.

Anogenital distance (AGD) has been used to reflect masculinization in litter-bearing species. As masculinization affects behavior and reproduction, AGD could be measured to assist in selecting gilts with a temperament more suited to commercial production and greater reproductive potential. We hypothesized that gilts from a male-biased litter would have a longer AGD and poorer reproductive performance. In Exp. 1, AGD and weight were measured at day 1, day 21, and week 16 of age for gilts from male-biased litters (≥60% males; n = 51) and female-biased litters (≥60% females; n = 51). Sow AGD was measured 3 d after farrowing. In Exp. 2, AGD was measured at gilt selection at approximately 24 wk of age and gilts followed to second parity. Litter sex ratio affected AGD at 16 wk of age, with gilts from female-biased litters having longer AGD (mean ± SEM, 9.1 ± 0.7 mm vs. 11.0 ± 0.6 mm, P = 0.013). Anogenital distance was not different on day 1 or day 21. There was no effect of sex ratio on weight at any time, and sow AGD was not associated with the sex ratio of her litter. Gilts with an AGD longer than the mean of 11.55 mm were heavier (mean ± SEM, 118.8 ± 0.4 kg vs. 117.7 ± 0.4 kg, P = 0.023), were achieved puberty earlier (179.6 ± 0.6d vs. 182.2 ± 0.6 d, P = 0.001), were mated younger (200.6 ± 0.6 d vs. 203.2 ± 0.6 d, P = 0.001), and were more likely to be mated (91% vs. 83%, P = 0.005) than gilts with an AGD shorter than the mean. Gilts with an AGD greater than 11.55 mm had a greater born alive litter size (11.79 ± 0.20 vs. 11.20 ± 0.19, P = 0.018) compared with gilts with an AGD shorter than 11.55 mm. At 16 wk, AGD was associated with sex bias and could be used as a selection tool to predict reproductive success of the first parity, with a longer AGD being associated with gilts that had been born into a female-biased litter and that had better reproductive performance.

Sex bias of the birth litter affects surge but not tonic LH secretion in gilts.

The physiology and behavior of gilts that develop in a male-biased litter can differ from gilts that develop in a female-biased litter. We hypothesized that gilts from male-biased litters will have a delayed and attenuated luteinizing hormone (LH) surge, and reduced LH pulse frequency and amplitude compared to gilts from female-biased litters. Gilts were selected at birth from male-biased (>60% males n = 10) or female-biased (>60% females n = 9) litters. From 18 wk of age, detection of puberty using daily boar contact began and their subsequent estrous periods were synchronized with oral progestogen (altrenogest). On day 3 after altrenogest withdrawal, blood samples were obtained from 6 gilts per sex bias group at 10 min intervals from 0900 to 2100 h to determine LH pulse amplitude and frequency. From 0900 on day 4, all 19 gilts were sampled every 4 h until the end of estrus to characterize LH surge dynamics. There were no differences between groups in LH pulse characteristics. Compared to gilts from female-biased litters, the LH surge in gilts from male-biased litters was delayed [56.00 ± 3.32 h vs. 43.11 ± 3.76 h (mean ± standard error of the mean (SEM)), P < 0.05], the duration was decreased [29.78 ± 2.12 h vs. 37.71 ± 1.19 h (mean ± SEM), P < 0.05] and the total secretion as measured by area under the curve was decreased (91.42 ± 9.52 ng/mL vs. 120.28 ± 9.48 ng/mL, P < 0.05). Our results indicate that a male-biased uterine environment has different effects on the tonic secretion of LH than the LH surge, with only some elements of the LH surge being affected.

Effects of selective serotonin reuptake inhibitor treatment on plasma oxytocin and cortisol in major depressive disorder.

BACKGROUND: Oxytocin is known for its capacity to facilitate social bonding, reduce anxiety and for its actions on the stress hypothalamopituitary adrenal (HPA) axis. Since oxytocin can physiologically suppress activity of the HPA axis, clinical applications of this neuropeptide have been proposed in conditions where the function of the HPA axis is dysregulated. One such condition is major depressive disorder (MDD). Dysregulation of the HPA system is the most prominent endocrine change seen with MDD, and normalizing the HPA axis is one of the major targets of recent treatments. The potential clinical application of oxytocin in MDD requires improved understanding of its relationship to the symptoms and underlying pathophysiology of MDD. Previous research has investigated potential correlations between oxytocin and symptoms of MDD, including a link between oxytocin and treatment related symptom reduction. The outcomes of studies investigating whether antidepressive treatment (pharmacological and non-pharmacological) influences oxytocin concentrations in MDD, have produced conflicting outcomes. These outcomes suggest the need for an investigation of the influence of a single treatment class on oxytocin concentrations, to determine whether there is a relationship between oxytocin, the HPA axis (e.g., oxytocin and cortisol) and MDD. Our objective was to measure oxytocin and cortisol in patients with MDD before and following treatment with selective serotonin reuptake inhibitors, SSRI. METHOD: We sampled blood from arterial plasma. Patients with MDD were studied at the same time twice; pre- and post- 12 weeks treatment, in an unblinded sequential design (clinicaltrials.govNCT00168493). RESULTS: Results did not reveal differences in oxytocin or cortisol concentrations before relative to following SSRI treatment, and there were no significant relationships between oxytocin and cortisol, or these two physiological variables and psychological symptom scores, before or after treatment. CONCLUSIONS: These outcomes demonstrate that symptoms of MDD were reduced following effective treatment with an SSRI, and further, stress physiology was unlikely to be a key factor in this outcome. Further research is required to discriminate potential differences in underlying stress physiology for individuals with MDD who respond to antidepressant treatment, relative to those who experience treatment resistance.

Neuroadrenergic dysfunction along the diabetes continuum: a comparative study in obese metabolic syndrome subjects.

Neuroadrenergic function in type 2 diabetic (T2D) patients without neuropathy is poorly characterized. We therefore compared sympathetic nervous system activity at rest and during an oral glucose tolerance test in obese metabolic syndrome (MetS) subjects classified as glucose intolerant (impaired glucose tolerance [IGT]; n = 17) or treatment-naive T2D (n = 17). Untreated subjects, matched for age (mean 59 ± 1 year), sex, BMI (32.4 ± 0.6 kg/m(2)), and family history of diabetes were studied. We measured resting muscle sympathetic nerve activity (MSNA) by microneurography, whole-body norepinephrine kinetics by isotope dilution, insulin sensitivity by euglycemic-hyperinsulinemic clamp (steady-state glucose utilization adjusted for fat-free mass and steady-state insulin concentration [M/I]), and MetS components. T2D subjects had higher resting MSNA burst incidence (67 ± 4 versus 55 ± 3 bursts per 100 heartbeats; P = 0.05) and arterial norepinephrine levels (264 ± 33 versus 167 ± 16 pg/mL; P = 0.02), lower plasma norepinephrine clearance (by 17%; P = 0.03), and reduced neuronal reuptake compared with IGT subjects (by 46%; P = 0.04). Moreover, norepinephrine spillover responses to glucose ingestion were blunted in T2D subjects. The M/I value independently predicted whole-body norepinephrine spillover (r = -0.47; P = 0.008), whereas fasting insulin level related to neuronal norepinephrine reuptake (r = -0.35, P = 0.047). These findings demonstrate that progression to T2D is associated with increased central sympathetic drive, blunted sympathetic responsiveness, and altered norepinephrine disposition.

Reward processing in anorexia nervosa.

Individuals with anorexia nervosa (AN) demonstrate a relentless engagement in behaviors aimed to reduce their weight, which leads to severe underweight status, and occasionally death. Neurobiological abnormalities, as a consequence of starvation are controversial: evidence, however, demonstrates abnormalities in the reward system of patients, and recovered individuals. Despite this, a unifying explanation for reward abnormalities observed in AN and their relevance to symptoms of the illness, remains incompletely understood. Theories explaining reward dysfunction have conventionally focused on anhedonia, describing that patients have an impaired ability to experience reward or pleasure. We review taste reward literature and propose that patients' reduced responses to conventional taste-reward tasks may reflect a fear of weight gain associated with the caloric nature of the tasks, rather than an impaired ability to experience reward. Consistent with this, we propose that patients are capable of 'liking' hedonic taste stimuli (e.g., identifying them), however, they do not 'want' or feel motivated for the stimuli in the same way that healthy controls report. Recent brain imaging data on more complex reward processing tasks provide insights into fronto-striatal neural circuit dysfunction related to altered reward processing in AN that challenges the relevance of anhedonia in explaining reward dysfunction in AN. In this way, altered activity of the anterior cingulate cortex and striatum could explain patients' pathological engagement in behaviors they consider rewarding (e.g., self-starvation) that are otherwise aversive or punishing, to those without the eating disorder. Such evidence for altered patterns of brain activity associated with reward processing tasks in patients and recovered individuals may provide important information about mechanisms underlying symptoms of AN, their future investigation, and the development of treatment approaches.

Baseline sympathetic nervous system activity predicts dietary weight loss in obese metabolic syndrome subjects.

CONTEXT: The sympathetic nervous system is an important physiological modulator of basal and postprandial energy expenditure. OBJECTIVE: Our objective was to investigate whether the variability of weight loss attained during hypocaloric dietary intervention is related to individual differences in baseline sympathetic drive and nutritional sympathetic nervous system responsiveness. PARTICIPANTS AND METHODS: Untreated obese subjects (n = 42; body mass index = 32.1 ± 0.5 kg/m(2)), aged 57 ± 1 yr, who fulfilled Adult Treatment Panel III metabolic syndrome criteria participated in a 12-wk weight loss program using a modified Dietary Approaches to Treat Hypertension (DASH) diet. Muscle sympathetic nerve activity (MSNA) was measured by microneurography at rest and in a subset of subjects during a standard 75-g oral glucose tolerance test. RESULTS: Weight loss (6.7 ± 0.5 kg) was independently predicted by baseline resting MSNA burst incidence (r = 0.38; P = 0.019), which accounted for 14.3% of the variance after adjustment for age and baseline body weight. Weight loss-resistant subjects in the lower tertile of weight loss (4.4 ± 0.3%) had significantly blunted MSNA responses to oral glucose at baseline compared with successful weight losers (9.6 ± 0.8%). Absolute Δ MSNA averaged -7 ± 2, -6 ± 5, and -3 ± 3 bursts per 100 heartbeats at 30, 60, and 90 min after glucose in the weight loss-resistant group. Corresponding values in the successful weight loss group were 9 ± 3, 12 ± 3, and 15 ± 4 bursts per 100 heartbeats (time × group interaction, P = 0.004). CONCLUSIONS: These findings indicate that baseline sympathetic drive and nutritional sympathetic responsiveness may be important prognostic biological markers for weight loss outcome.

The estrous cycle of the ewe is resistant to disruption by repeated, acute psychosocial stress.

Five experiments were conducted to test the hypothesis that psychosocial stress interferes with the estrous cycle of sheep. In experiment 1, ewes were repeatedly isolated during the follicular phase. Timing, amplitude, and duration of the preovulatory luteinizing hormone (LH) surge were not affected. In experiment 2, follicular-phase ewes were subjected twice to a "layered stress" paradigm consisting of sequential, hourly application of isolation, restraint, blindfold, and predator cues. This reduced the LH pulse amplitude but did not affect the LH surge. In experiment 3, different acute stressors were given sequentially within the follicular phase: food denial plus unfamiliar noises and forced exercise, layered stress, exercise around midnight, and transportation. This, too, did not affect the LH surge. In experiment 4, variable acute psychosocial stress was given every 1-2 days for two entire estrous cycles; this did not disrupt any parameter of the cycle monitored. Lastly, experiment 5 examined whether the psychosocial stress paradigms of experiment 4 would disrupt the cycle and estrous behavior if sheep were metabolically stressed by chronic food restriction. Thirty percent of the food-restricted ewes exhibited deterioration of estrous cycle parameters followed by cessation of cycles and failure to express estrous behavior. However, disruption was not more evident in ewes that also encountered psychosocial stress. Collectively, these findings indicate the estrous cycle of sheep is remarkably resistant to disruption by acute bouts of psychosocial stress applied intermittently during either a single follicular phase or repeatedly over two estrous cycles.

Effect of RF-amide-related peptide-3 on luteinizing hormone and follicle-stimulating hormone synthesis and secretion in ovine pituitary gonadotropes.

GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for beta-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHbeta and FSHbeta subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.

Role of estradiol in cortisol-induced reduction of luteinizing hormone pulse frequency.

Precise control of pulsatile GnRH and LH release is imperative to ovarian cyclicity but is vulnerable to environmental perturbations, like stress. In sheep, a sustained (29 h) increase in plasma cortisol to a level observed during stress profoundly reduces GnRH pulse frequency in ovariectomized ewes treated with ovarian steroids, whereas shorter infusion (6 h) is ineffective in the absence of ovarian hormones. This study first determined whether the ovarian steroid milieu or duration of exposure is the relevant factor in determining whether cortisol reduces LH pulse frequency. Prolonged (29 h) cortisol infusion did not lower LH pulse frequency in ovariectomized ewes deprived of ovarian hormones, but it did so in ovariectomized ewes treated with estradiol and progesterone to create an artificial estrous cycle, implicating ovarian steroids as the critical factor. Importantly, this effect of cortisol was more pronounced after the simulated preovulatory estradiol rise of the artificial follicular phase. The second experiment examined which component of the ovarian steroid milieu enables cortisol to reduce LH pulse frequency in the artificial follicular phase: prior exposure to progesterone in the luteal phase, low early follicular phase estradiol levels, or the preovulatory estradiol rise. Basal estradiol enabled cortisol to decrease LH pulse frequency, but the response was potentiated by the estradiol rise. These findings lead to the conclusion that ovarian steroids, particularly estradiol, enable cortisol to inhibit LH pulse frequency. Moreover, the results provide new insight into the means by which gonadal steroids, and possibly reproductive status, modulate neuroendocrine responses to stress.

Cortisol reduces gonadotropin-releasing hormone pulse frequency in follicular phase ewes: influence of ovarian steroids.

Stress-like elevations in plasma glucocorticoids suppress gonadotropin secretion and can disrupt ovarian cyclicity. In sheep, cortisol acts at the pituitary to reduce responsiveness to GnRH but does not affect GnRH pulse frequency in the absence of ovarian hormones. However, in ewes during the follicular phase of the estrous cycle, cortisol reduces LH pulse frequency. To test the hypothesis that cortisol reduces GnRH pulse frequency in the presence of ovarian steroids, the effect of cortisol on GnRH secretion was monitored directly in pituitary portal blood of follicular phase sheep in the presence and absence of a cortisol treatment that elevated plasma cortisol to a level observed during stress. An acute (6 h) cortisol increase in the midfollicular phase did not lower GnRH pulse frequency. However, a more prolonged (27 h) increase in cortisol beginning just before the decrease in progesterone reduced GnRH pulse frequency by 45% and delayed the preovulatory LH surge by 10 h. To determine whether the gonadal steroid milieu of the follicular phase enables cortisol to reduce GnRH pulse frequency, GnRH was monitored in ovariectomized ewes treated with estradiol and progesterone to create an artificial follicular phase. A sustained increment in plasma cortisol reduced GnRH pulse frequency by 70% in this artificial follicular phase, in contrast to the lack of an effect in untreated ovariectomized ewes as seen previously. Thus, a sustained stress-like level of cortisol suppresses GnRH pulse frequency in follicular phase ewes, and this appears to be dependent upon the presence of ovarian steroids.

Psychosocial stress inhibits amplitude of gonadotropin-releasing hormone pulses independent of cortisol action on the type II glucocorticoid receptor.

Our laboratory has developed a paradigm of psychosocial stress (sequential layering of isolation, blindfold, and predator cues) that robustly elevates cortisol secretion and decreases LH pulse amplitude in ovariectomized ewes. This decrease in LH pulse amplitude is due, at least in part, to a reduction in pituitary responsiveness to GnRH, caused by cortisol acting via the type II glucocorticoid receptor (GR). The first experiment of the current study aimed to determine whether this layered psychosocial stress also inhibits pulsatile GnRH release into pituitary portal blood. The stress paradigm significantly reduced GnRH pulse amplitude compared with nonstressed ovariectomized ewes. The second experiment tested if this stress-induced decrease in GnRH pulse amplitude is mediated by cortisol action on the type II GR. Ovariectomized ewes were allocated to three groups: nonstress control, stress, and stress plus the type II GR antagonist RU486. The layered psychosocial stress paradigm decreased GnRH and LH pulse amplitude compared with nonstress controls. Importantly, the stress also lowered GnRH pulse amplitude to a comparable extent in ewes in which cortisol action via the type II GR was antagonized. Therefore, we conclude that psychosocial stress reduces the amplitude of GnRH pulses independent of cortisol action on the type II GR. The present findings, combined with our recent observations, suggest that the mechanisms by which psychosocial stress inhibits reproductive neuroendocrine activity at the hypothalamic and pituitary levels are fundamentally different.

Cortisol interferes with the estradiol-induced surge of luteinizing hormone in the ewe.

Two experiments were conducted to test the hypothesis that cortisol interferes with the positive feedback action of estradiol that induces the luteinizing hormone (LH) surge. Ovariectomized sheep were treated sequentially with progesterone and estradiol to create artificial estrous cycles. Cortisol or vehicle (saline) was infused from 2 h before the estradiol stimulus through the time of the anticipated LH surge in the artificial follicular phase of two successive cycles. The plasma cortisol increment produced by infusion was approximately 1.5 times greater than maximal concentrations seen during infusion of endotoxin, which is a model of immune/inflammatory stress. In experiment 1, half of the ewes received vehicle in the first cycle and cortisol in the second; the others were treated in reverse order. All ewes responded with an LH surge. Cortisol delayed the LH surge and reduced its amplitude, but both effects were observed only in the second cycle. Experiment 2 was modified to provide better control for a cycle effect. Four treatment sequences were tested (cycle 1-cycle 2): vehicle-vehicle, cortisol-cortisol, vehicle-cortisol, cortisol-vehicle. Again, cortisol delayed but did not block the LH surge, and this delay occurred in both cycles. Thus, an elevation in plasma cortisol can interfere with the positive feedback action of estradiol by delaying and attenuating the LH surge.

Variation in kisspeptin and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropin-releasing hormone neurons in the brain: a novel medium for seasonal breeding in the sheep.

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

The effect of testosterone and season on prodynorphin messenger RNA expression in the preoptic area-hypothalamus of the ram.

Testosterone and season influence mRNA expression for the opioid, enkephalin, in the preoptic area and hypothalamus of rams. Dynorphin is another opioid which has been shown to play a role in the control of reproductive function in females. We now report effects of season and testosterone on the expression of prodynorphin mRNA in the hypothalamus of the ram. Castrated adult Romney Marsh rams (5/group) received vehicle or testosterone propionate (i.m.) during either the 'breeding' season or 'non-breeding' season. Prodynorphin mRNA expression was quantified in the hypothalami by in situ hybridisation. Testosterone treatment increased prodynorphin mRNA expression in the supraoptic nucleus and the bed nucleus of the stria terminalis in the breeding season but not during the non-breeding season. Prodynorphin mRNA expression was also higher in the breeding season than in the non-breeding season in the caudal preoptic area, paraventricular nucleus and accessory supraoptic nucleus, irrespective of treatment. No effects of treatment were observed in any other regions of the hypothalamus. We conclude that testosterone and season regulate prodynorphin mRNA expression in a region-specific manner, which may influence seasonal changes in reproductive function.

Responses of the hypothalamopituitary adrenal axis and the sympathoadrenal system to isolation/restraint stress in sheep of different adiposity.

There is evidence that levels of adipose tissue can influence responses of the hypothalamopituitary-adrenal (HPA) axis to stress in humans and rats but this has not been explored in sheep. Also, little is known about the sympathoadrenal responses to stress in individuals with relatively different levels of adipose tissue. We tested the hypothesis that the stress-induced activation of the HPA axis and sympathoadrenal system is lower in ovariectomized ewes with low levels of body fat (lean) than ovariectomized ewes with high levels of body fat (fat). Ewes underwent dietary manipulation for 3 months to yield a group of lean ewes (n = 7) with a mean (+/-SEM) live weight of 39.1 +/- 0.9 kg and body fat of 8.9 +/- 0.6% and fat ewes (n = 7) with a mean (+/-SEM) live weight of 69.0 +/- 1.8 kg and body fat of 31.7 +/- 3.4%. Fat ewes also had higher circulating concentrations of leptin than lean ewes. Blood samples were collected every 15 min over 8 h when no stress was imposed (control day) and on a separate day when 4 h of isolation/restraint was imposed after 4 h of pretreatment sampling (stress day). Plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, epinephrine and norepinephrine did not change significantly over the control day and did not differ between lean and fat ewes. Stress did not affect plasma leptin levels. All stress hormones increased significantly during isolation/restraint stress. The ACTH, cortisol and epinephrine responses were greater in fat ewes than lean ewes but norepinephrine responses were similar. Our results suggest that relative levels of adipose tissue influence the stress-induced activity of the hypothalamopituitary-adrenal axis and some aspects of the sympathoadrenal system with fat animals having higher responses than lean animals.

Does cortisol acting via the type II glucocorticoid receptor mediate suppression of pulsatile luteinizing hormone secretion in response to psychosocial stress?

This study assessed the importance of cortisol in mediating inhibition of pulsatile LH secretion in sheep exposed to a psychosocial stress. First, we developed an acute psychosocial stress model that involves sequential layering of novel stressors over 3-4 h. This layered-stress paradigm robustly activated the hypothalamic-pituitary-adrenal axis and unambiguously inhibited pulsatile LH secretion. We next used this paradigm to test the hypothesis that cortisol, acting via the type II glucocorticoid receptor (GR), mediates stress-induced suppression of pulsatile LH secretion. Our approach was to determine whether an antagonist of the type II GR (RU486) reverses inhibition of LH pulsatility in response to the layered stress. We used two animal models to assess different aspects of LH pulse regulation. With the first model (ovariectomized ewe), LH pulse characteristics could vary as a function of both altered GnRH pulses and pituitary responsiveness to GnRH. In this case, antagonism of the type II GR did not prevent stress-induced inhibition of pulsatile LH secretion. With the second model (pituitary-clamped ovariectomized ewe), pulsatile GnRH input to the pituitary was fixed to enable assessment of stress effects specifically at the pituitary level. In this case, the layered stress inhibited pituitary responsiveness to GnRH and antagonism of the type II GR reversed the effect. Collectively, these findings indicate acute psychosocial stress inhibits pulsatile LH secretion, at least in part, by reducing pituitary responsiveness to GnRH. Cortisol, acting via the type II GR, is an obligatory mediator of this effect. However, under conditions in which GnRH input to the pituitary is not clamped, antagonism of the type II GR does not prevent stress-induced inhibition of LH pulsatility, implicating an additional pathway of suppression that is independent of cortisol acting via this receptor.