Arcuate nucleus kisspeptin response to increased nutrition in rams.

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

Twenty-four-hour profiles of metabolic and stress hormones in sheep selected for a calm or nervous temperament.

Even in the absence of stressors, temperament is associated with changes in the concentration of stress-responsive hormones and, possibly because of such changes, temperament can affect metabolism. We tested whether, in sheep bred for temperament for 14 generations, "nervous" females have greater concentrations of stress-responsive hormones in the absence of stressors than "calm" females, and whether these differences are associated with changes in the concentrations of metabolic hormones. In resting "calm" (n = 8) and "nervous" (n = 8) sheep, concentrations of cortisol, prolactin, leptin, and insulin were measured in blood plasma sampled via jugular catheter every 20 min for 24 h. The animals were individually penned, habituated to their housing and human handling over 7 wk, and fed before sampling began. Diurnal variation was evident for all hormones, but a 24-h cortisol pattern was detected in only 7 individuals. There was no effect of temperament on any aspect of concentrations of cortisol or prolactin, but "calm" animals had greater concentrations of insulin in the early afternoon than "nervous" animals (14.5 ± 1.1 vs 10.0 ± 1.6 µU/mL; P = 0.038), and a similar tendency was seen for leptin (P = 0.092). We conclude that selection for temperament affects the concentration of metabolic hormones in the absence of stressors, but this effect is independent of stress-responsive hormones.

Genetic selection for temperament affects behaviour and the secretion of adrenal and reproductive hormones in sheep subjected to stress.

We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.

Role of male novelty and familiarity in male-induced LH secretion in female sheep.

Ewes supposedly need to be separated from rams before male stimuli can increase gonadotrophin secretion and induce ovulation. In the present study, we investigated the LH response of ewes to 'novel' and 'familiar' rams after varying periods of separation. In Experiment 1, ewes (n = 8 per treatment) were separated from familiar rams for 15 min or 1 month and then exposed to either familiar rams, novel rams or novel wethers. After 15 min or 1 month of separation, exposure to novel rams increased pulsatile LH secretion (P < 0.05) and induced an LH surge in all ewes whereas exposure to familiar rams or novel wethers had no effect on LH secretion (P > 0.1). After 1 month of separation, re-exposure to the same familiar rams increased pulsatile LH secretion (P < 0.05) in six of eight ewes, but only induced an LH surge in two of eight ewes. In Experiment 2, familiar rams were removed and returned after 15 min, 1 day or 17 days (n = 5 per treatment). None of these treatments affected LH secretion. We conclude that separation of ewes from rams is a prerequisite for familiar rams to increase LH secretion, but is not necessary if the rams are novel.

Sociosexual stimuli and gonadotropin-releasing hormone/luteinizing hormone secretion in sheep and goats.

Sociosexual stimuli have a profound effect on the physiology of all species. Sheep and goats provide an ideal model to study the impact of sociosexual stimuli on the hypothalamic-pituitary-gonadal axis because we can use the robust changes in the pulsatile secretion of luteinizing hormone as a bioassay of gonadotropin-releasing hormone secretion. We can also correlate these changes with neural activity using the immediate early gene c-fos and in real time using changes in electrical activity in the mediobasal hypothalamus of female goats. In this review, we will update our current understanding of the proven and potential mechanisms and mode of action of the male effect in sheep and goats and then briefly compare our understanding of sociosexual stimuli in ungulate species with the "traditional" definition of a pheromone.

Genetic differences in temperament determine whether lavender oil alleviates or exacerbates anxiety in sheep.

Growing concerns about the risk of addiction to benzodiazepines have led to increasing interest in alternative therapies to treat anxiety and depression. Lavender oil (Lavendula augustifolia) is reportedly anxiolytic in a number of species but little is known about how it affects individuals that are more or less anxious when faced with a stressor. In this study, we used changes in locomotor activity and the plasma concentrations of cortisol to test whether lavender oil would reduce behavioral and endocrine correlates of anxiety in calm and nervous sheep exposed to an isolation stressor. During the non-breeding season, 'calm' or 'nervous' female sheep from the UWA temperament flock were exposed to a mask containing either 1 mL of 10% lavender oil (calm: n=8; nervous: n=8) or peanut oil (calm: n=8; nervous: n=8). After 30 min, each sheep was isolated for 5 min and then returned to the group. Blood was sampled prior to the mask, prior to isolation, 1 min and 30 min after isolation to profile changes in the plasma concentrations of cortisol. Agitation score, locomotor activity and vocalizations were recorded as correlates of anxiety associated with the isolation stressor. Irrespective of whether they were exposed to lavender oil, calm sheep had a lower agitation score (P<0.001), crossed the central lines of the isolation box less frequently (P<0.001), expressed fewer vocalizations (P<0.001) and had lower plasma concentrations of cortisol immediately after isolation (P<0.001) than nervous sheep. Exposure of calm sheep to lavender oil decreased the agitation score (P<0.001), frequency of vocalizations (P<0.05), decreased the number of crosses of the central lines of the isolation box (P<0.05), and the plasma concentrations of cortisol prior to isolation (P<0.05) (after mask application) compared to calm control sheep. Exposure of nervous sheep to lavender oil increased the frequency of vocalizations (P<0.05), the number of sheep attempting to escape (P<0.05) and the plasma concentrations of cortisol 30 min after isolation (P<0.05) compared to nervous control sheep. We conclude that genetic differences in temperament determine whether lavender oil alleviates or exacerbates the behavioral and/or endocrine correlates of anxiety in sheep.

Sexual experience and temperament affect the response of Merino ewes to the ram effect during the anoestrous season.

In seasonally anoestrous ewes of many breeds, the introduction of rams triggers an increase in gonadotrophin secretion that induces ovulation, a phenomenon known as the 'ram effect'. The ram effect is a practical method for mating ewes outside the natural breeding season, and also can provide synchronised lambing, but the variability of the response, especially in young animals, reduces its potential for widespread application. The aim of our study was to assess two factors that are thought to contribute to the variability in young ewes: temperament and sexual experience. We used anovulatory ewes from a flock that had been genetically selected for 'calm' or 'nervous' temperament and compared the endocrine and ovarian responses to the ram effect in four groups (each n=15): 'calm' and parous (3-6 years old); 'calm' and nulliparous (2 years old); 'nervous' and parous; and 'nervous' and nulliparous. Parous ewes, independently of their temperament, exhibited a faster endocrine response and a higher proportion of females cycling after ram introduction than nulliparous ewes. 'Nervous' ewes exhibited a higher proportion of females cycling after ram introduction than calm ewes, but only in the nulliparous group. We conclude that temperament exerts little influence on the response to the ram effect in sexually experienced ewes, and that females of 'nervous' temperament appear to respond better when sexually 'naive'. Both sexual experience and temperament need to be taken into consideration when flock management involves the ram effect. Finally, some ewes were cyclic at ram introduction, yet exhibited an increase in LH secretion even in the presence of high concentrations of progesterone. The mechanism by which the inhibitory effect of progesterone on LH secretion was bypassed needs to be clarified.

Do cyclic female goats respond to males with an increase in LH secretion during the breeding season?

The male effect is currently only used during seasonal or lactational anoestrus because the response is thought to be blocked in cyclic females by periods of elevated progesterone. In this study, we tested whether cyclic, female goats would respond to male exposure with an increase in pulsatile LH secretion. During May (breeding season; Southern Hemisphere) the cycles of 16 Australian Cashmere goats were synchronised using intravaginal progesterone pessaries. Pessary insertion was staggered to produce groups in their early luteal (EL; n=8) and late luteal phases (LL; n=8). The LL group was retrospectively subdivided into mid-luteal (ML; n=4) and late luteal (LL; n=4) groups due to differences in oestrous cycle length that emerged during the study. Male exposure stimulated an increase in LH pulse frequency in the EL and LL groups (P<0.01) but not in the ML group (P>0.1). This increase was accompanied by an increase in basal and mean concentrations of LH in the LL group (P<0.05) but not in EL (P<0.1) or ML (P>0.1) group. There was no effect of male exposure on LH pulse amplitude (P>0.1). Progesterone concentrations differed among all groups on the day of male exposure (P<0.05) and declined significantly over the 12-h sampling period in the LL group (P<0.05). Prolactin concentrations declined in the EL group but did not change significantly in the ML or LL group. In conclusion, male exposure induced an increase in pulsatile LH in goats in the early and late luteal phases of the oestrous cycle. The high concentrations of progesterone in females in the mid-luteal phase appeared to block the male effect.

Ram novelty and the duration of ram exposure affects the distribution of mating in ewes exposed to rams during the transition into the breeding season.

This study compared the affect of short-term and continuous exposure to rams during the transition between anoestrus and the breeding season on the distribution of mating and subsequent lambing. Further, within ewes continuously exposed to rams we investigated the effect of replacing these rams every 17 days with 'novel' rams. During August (late anoestrus, Northern Hemisphere), multiparous, North of England mule ewes were allocated to one of four groups: SVR ewes were exposed to vasectomised rams for 24h on Day 0 (short term; n=109), RVR ewes were exposed to vasectomised rams for 24h on Days 0, 17 and 34 (short term; n=113); PVR ewes were exposed to vasectomised rams on Day 0 and remained with the same rams for the duration of the pre-mating period (continuous; n=104); NVR ewes were continuously exposed to vasectomised rams from Day 0 with the rams replaced with 'novel' rams every 17 days (continuous; n=113). Blood samples were collected from a subset of ewes (n=22 per group) to monitor progesterone. On Day 50, harnessed, entire rams were introduced for mating and raddle marks recorded daily for the first 17 days. The median date of mating occurred 1 day earlier in NVR ewes than PVR ewes (P<0.05). A synchrony score calculated from the blood sampled ewes showed that the distribution of mating was more synchronised in PVR and NVR ewes than SVR and RVR ewes (P<0.001). PVR and NVR ewes had an earlier onset of cyclic activity than RVR ewes (P<0.01). However, only NVR ewes differed from SVR ewes in this variable (P<0.05). Within ewes lambing to first service, the median date of lambing of PVR, NVR and SVR ewes occurred at least 2 days earlier than RVR ewes (at least P<0.05). Further, PVR and NVR ewes had a more compact distribution of lambing than SVR and RVR ewes (P<0.05) and lambing was more compact in NVR ewes than PVR ewes (P<0.05). In conclusion, ewes in continuous contact with rams prior to mating had a more synchronised distribution of mating and lambing than ewes given only short-term exposure to rams. This distribution of mating in continuous ram exposed ewes can be further enhanced by periodic exposure to novel rams.

Can audio-visual or visual stimuli from a prospective mate stimulate a reproductive neuroendocrine response in sheep?

Stimuli from a prospective mate increase the secretion of luteinising hormone (LH) in sheep. This 'male effect' in ewes and 'female effect' effect in rams is predominantly mediated by olfactory signals, though it is thought that non-olfactory signals play synergistic or substitutive roles. In this study, we tested whether exposure to visual or audio-visual stimuli from a prospective mate would stimulate an increase in LH secretion in ewes (Experiment 1) and rams (Experiment 2). In Experiment 1, groups of eight Merino ewes were exposed to one of three stimuli midway through a frequent blood-sampling regimen: full ram contact, still images of rams, a video of ewes and rams mating. Control ewes (n = 8) were completely isolated from rams. Exposure to still images of rams appeared to stimulate an increase in mean LH concentrations (P < 0.05) and tended to increase LH pulse frequency (P < 0.1), but the response was significantly smaller than that observed in ewes exposed to rams (P < 0.01). Audio-visual stimuli had no effect on any parameters of LH secretion (P > 0.1). In Experiment 2, Merino rams were allocated to either an Exposure (n = 7) or a Control (n = 7) group. Exposure rams underwent two exposure periods midway through a frequent blood-sampling regimen; exposure to still images of ewes and audio recorded during mating of ewes and rams (audio-visual exposure); exposure to oestrous ewes (ewe exposure). Control rams were sampled at the same frequency but remained isolated from ewe stimuli. Exposure of rams to the audio-visual stimuli did not affect any parameters of LH secretion (P > 0.1). In contrast, exposure to oestrous ewes increased LH pulse frequency (P < 0.05) and advanced the onset of the next LH pulse (P < 0.05). In conclusion, visual signals appear to be involved in eliciting the neuroendocrine response of ewes to rams and are of greater importance to this phenomenon in ewes (male effect) than rams (female effect). However, overall the visual and audio-visual signals used in this study were far less effective than stimulus animals, suggesting that these stimuli are less important than olfactory signals, or a combination of olfactory and audio-visual signals.

Prior exposure of maiden ewes to rams enhances their behavioural interactions with rams but is not a pre-requisite to their endocrine response to the ram effect.

In this study, we tested whether prior experience with rams would modify the behavioural and endocrine responses of maiden ewes to rams. During mid-anoestrus, sexually naïve, maiden ewes were exposed to rams for 7 days (ram experienced, RE; n=61) or isolated from rams (ram naïve, RN; n=63). All ewes were subsequently isolated from rams. In Experiment 1, RE (n=55) and RN (n=57) ewes were introduced to rams during late anoestrus. RE ewes had more total and positive interactions with rams than RN ewes (P<0.001). RE ewes showed more ram seeking behaviour and spent more time in proximity of rams than RN ewes (at least; P<0.05). In Experiment 2, RE (n=6) and RN (n=6) ewes were introduced to rams midway through a frequent blood sampling regime in late anoestrus. Ram introduction stimulated an increase in LH pulse frequency and basal LH in both RE and RN ewes (at least P<0.05). RE ewes had an increase in mean LH concentrations (P<0.01) that failed to reach significance in RN ewes (P<0.1). There was no significant effect of prior experience with rams on LH pulse frequency, amplitude or whether ewes had an LH surge. In conclusion, prior experience with rams is important in developing appropriate ewe-ram interactions but is not a pre-requisite to the endocrine response to the ram effect.

Short term, repeated exposure to rams during the transition into the breeding season improves the synchrony of mating in the breeding season.

The ram effect is widely used in Mediterranean breeds of sheep but its use in temperate genotypes is restricted by breed seasonality. However, ewes from these highly seasonal genotypes are sensitive to stimulation by rams close to the onset of the natural breeding season. In this study we developed a pre-mating protocol of repeated, short-term exposure to rams (fence-line contact or vasectomised rams) beginning during late anoestrus and continuing into the breeding season. We hypothesised that this pre-mating protocol would synchronise the distribution of mating of North of England Mule ewes during the breeding season above that observed in ewes isolated from rams prior to mating. Ram-exposed ewes were given contact with rams (Experiment 1: fence-line; FR, n=94 and Experiment 2: vasectomised rams; VR; n=103) for 24h on Days 0 (10 September), 17 and 34 of the experiment. Control ewes (Experiment 1; FC, n=98 and Experiment 2; VC; n=106) remained isolated from rams prior to mating. In Experiment 2, a subset of VR (n=35) and VC ewes (n=35) were blood sampled twice weekly to monitor their pre-mating progesterone profiles. At mating, harnessed entire rams were introduced, 17 or 16 days after the last ram exposure (Experiments 1 and 2) and raddle marks were recorded daily. The median time from ram introduction to mating was reduced in ewes given both fence-line and vasectomised ram contact (P<0.001), leading to a more compact distribution of mating and lambing (At least P<0.01). In the blood sampled VR ewes, there was a progressive decline in the number of days from ram exposure to the onset of dioestrus (at least P<0.05). This observation indicates that the cycles in VR ewes became increasingly synchronised over the pre-mating period, a pattern not evident in VC ewes. In conclusion, repeated, short-term exposure of ewes to rams during the transition into the breeding season is an effective method of synchronising the distribution of mating during the breeding season.

The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season.

Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n=3) [corrected] and late luteal (LL; n=6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentration [corrected] of LH (at least P<0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P<0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P<0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle.

The effects of ram exposure during progestagen oestrus synchronisation and time of ram introduction post progestagen withdrawal on fertility in ewes.

Three experiments were undertaken to investigate the effect of a pre-mating ram exposure during progestagen synchronisation treatment on time of breeding, ovulation rate, embryo quality and fertility and any interaction with time of ram introduction for breeding post sponge withdrawal. Crossbred ewes in experiment 1a (n = 348), 1b (mule; n = 133) and 2 (n = 58) underwent a 12-14 days synchronisation protocol. Three days prior to sponge withdrawal ewes were divided into Control (ewes in continued isolation from rams) or +Ram (ram-exposed) groups. Rams were introduced to +Ram ewes and remained with ewes until sponge withdrawal. Ewes in experiments 1a and 2 received eCG at sponge withdrawal and were reintroduced to rams at either 36 or 48 h post sponge removal (PSR). In experiment 1b, ewes did not receive eCG and were reintroduced to rams at 24 h PSR. In experiments 1a and 1b time of breeding, date of lambing and litter size were recorded. In experiment 2, ewes were slaughtered 5 days post breeding, reproductive tracts flushed and corpora lutea, ova and embryos assessed. Fewer +Ram ewes were mated by 96 h PSR (P < 0.001) than Control ewes in experiment 1a but not when rams were introduced earlier in experiment 1b. In experiment 1a, ram introduction at 36 h PSR improved conception to first service compared to introduction at 48 h PSR (P < 0.01) in both +Ram and Control groups. In experiments 1a and 1b, +Ram ewes had reduced litter size caused by more single births (1a; P < 0.001, 1b; P < 0.01). In experiment 2, +Ram ewes had fewer corpora lutea than Control ewes (P < 0.001) but embryo quality was similar. However, more good embryos were produced when rams were introduced for breeding at 36 h compared to 48 h PSR (P < 0.001). We conclude that a pre-mating ram exposure during the synchronisation treatment reduced the number of ewes mated at and conceiving to the first service. This was partially overcome by introducing rams for breeding earlier (24 or 36 h compared to 48 h PSR) but the most dramatic decrease in fertility was due to a reduction in ovulation rate in the ram-exposed ewes.

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes.

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.