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Summary. The hypothesis that the secretion of gonadotrophins would be reduced by zinc deficiency was tested in five groups of four young Merino rams (initial liveweight 22 kg). Four groups were fed ad libitum with diets containing 4, 10, 17 or 27 μg Zn g⁻¹. The effects of loss of appetite on the deficient diet was controlled by feeding a fifth group (pair-fed control) at a rate of 27 μg Zn g⁻¹, but the amount of feed offered was restricted to that eaten voluntarily by the deficient (4 μg Zn g⁻¹) group. Blood was sampled every 20 min for 32 h on two occasions before the treatments were imposed and 96 days later, at the end of the experiment. The rams were injected with gonadotrophin-releasing hormone (GnRH; 10 ng kg⁻¹ i.v.) after each serial sampling, and with naloxone (1 mg kg⁻¹ i.v.) 24 h after the end of the final GnRH test.

In the group that were fed the diet with the lowest zinc content, the concentration of zinc in blood plasma was reduced to 18% of that in the pair-fed controls (P < 0·05) and was within the deficient range. The appetite of the deficient rams was half that of the controls fed 27 μg Zn g⁻¹ ad libitum and there was no increase in liveweight or testicular diameter during pubertal development. Similar, but smaller, effects were observed in the pair-fed controls. There were no significant differences between pair-fed and deficient groups in the frequency of the luteinizing hormone (LH) pulses or in the concentration of follicle-stimulating hormone (FSH), but the secretion of gonadotrophins was markedly lower in both groups than in the control rams fed ad libitum. The response to GnRH was not affected by treatment, but the increase in LH pulse frequency evoked by naloxone was lower in the deficient animals than in other groups. The animals fed zinc at intermediate rates (10–17 μg μ⁻¹) showed similar responses to the controls fed ad libitum.

It is concluded that the specific effects of zinc deficiency on testicular function were small. Most of the reduction in testicular growth in rams fed a deficient diet was not specifically related to the trace element, but was due to the fall in energy and protein intake caused by the loss of appetite. This leads to a reduction in the frequency of GnRH pulses secreted by the hypothalamus, and to low rates of gonadotrophin secretion by the pituitary gland.

Keywords: zinc; testis; gonadotrophin; nutrition; sheep

Corticotrophin-releasing hormone (CRH) has been proposed as a mediator of the antireproductive effects of stress through an action within the hypothalamus to inhibit GnRH secretion. This hypothesis was tested in sheep by studying the responses to central administration of CRH in both sexes and in both seasons. Sexually mature, Ile-de-France ewes and Romanov rams that had been gonadectomized and implanted with a permanent guide cannula into the third cerebral ventricle were used. Ewes were studied in the presence and absence of exogenous oestradiol plus progesterone, in both the breeding and anoestrous seasons. All rams were treated with testosterone and were studied only during the breeding season. Each observation involved serial samples (every 10 min) of jugular blood for 5 h before (control) and 5 h after an intracerebroventricular (icv) injection of either saline (vehicle) or 5 nmoles CRH in 20 μl vehicle. The saline injections did not affect any of the endocrine variables measured; however, CRH always increased cortisol concentrations in jugular plasma. In the absence of treatment with replacement sex steroids, icv injection of CRH had no effect on pulsatile LH secretion in females either during the breeding season or during anoestrus. However, LH pulse frequency and mean LH concentrations increased significantly on every occasion on which animals were treated with sex steroids. Treatment with CRH also increased LH secretion in the testosterone-treated rams. It is concluded that, contrary to the hypothesized role of CRH as an inhibitor of reproductive activity, this neuropeptide stimulates pulsatile LH (and thus GnRH) secretion, at least in this species. The fact that gonadal steroids seem to be obligatory for the expression of this effect suggests that the protocols used in past studies need to be reassessed.

Summary. Two experiments were conducted in Ile-de-France ewes to study changes in pulsatile LH secretion in ewes ovariectomized during anoestrus or during the midluteal phase of the oestrous cycle. In Exp. 1, blood samples were taken every 20 min for 12 h the day before ovariectomy (Day 0). After ovariectomy, samples were taken every 10 min for 6 h (10 ewes per group), on Days 1, 3, 7 and 15. In Exp. 2 samples were taken every 10 min for 6 h (10 ewes per group) on Days 7,15,30,60,90,120,150 and 180 after ovariectomy. Further samples were taken (5 ewes per group) at 9 and 12 months after ovariectomy.

There were significant interactions between season and day of sampling for the interval between LH pulses in both experiments. LH pulse frequency increased within 1 day of ovariectomy and the increase was more rapid during the breeding season. There were clear seasonal differences in pulse frequency in Exp. 2. Compared with ewes ovariectomized in anoestrus, pulse frequency was significantly higher for ewes ovariectomized in the breeding season, from Day 7 until Day 120. Once pulse frequency had increased in ewes about the time of the normal breeding season, pulse frequency remained high and subsequent seasonal changes were greatly reduced.

Pulse amplitude increased immediately after ovariectomy to reach a maximum on Day 7 and there were no differences between season of ovariectomy in the initial changes in amplitude. In Exp. 2, changes in amplitude followed changes in pulse interval and there was a significant interaction between season and day of sampling. There were no significant effects of season on nadir LH concentrations which increased throughout the duration of the experiments.

These results show that, in ovariectomized ewes, LH pulse frequency observed on a given day depends on time after ovariectomy, season at the time of sampling and on previous exposure of ewes to stimulatory effects of season. The direct effects of season on LH pulse frequency and seasonal changes in sensitivity to steroid feedback may contribute to control of the breeding season and their relative contributions to the beginning and end of the breeding season may differ.

Summary. Three experiments were conducted to study changes in pulsatile secretion of LH and FSH during the breeding season or anoestrus in ovariectomized Ile-de-France ewes fed different amounts of the phyto-oestrogen coumestrol. In Exp. 1, conducted during the breeding season, ewes (3–4 per group) were fed lucerne supplying 4, 18 or 30 mg coumestrol per ewe per day for 15 days. Experiments 2 and 3 were conducted during seasonal anoestrus. In Exp. 2, ewes (4 per group) were fed lucerne supplying coumestrol concentrations ranging from 4 to 38 mg/ewe/day for 15 days. In Exp. 3, ewes (10 per group) were fed lucerne supplying 14 or 125 mg coumestrol/ewe/day for 15 days. During the breeding season, an increased concentration of coumestrol in the diet significantly decreased the amplitude of LH pulses. There were no effects on LH pulse frequency or on FSH concentrations. During seasonal anoestrus, there were no significant effects on LH pulse frequency, or amplitude and no significant effect on FSH concentration. These results show that high concentrations of coumestrol in lucerne diets would not explain seasonal variation in LH pulse frequency in ovariectomized ewes. However, lucerne diets with increased coumestrol concentrations can influence LH release during the breeding season.

Summary. The effects of season and of oestradiol and progesterone on the tonic secretion of LH were studied in ovariectomized Merino and Suffolk ewes, two breeds which differ markedly in the seasonal pattern of their reproductive activity. In the absence of exogenous steroids, the frequency of LH pulses was lower and the amplitude of the pulses was higher in anoestrus than in the breeding season for Merino and Suffolk ewes 30 days after ovariectomy. In long-term (190 days) ovariectomized ewes, this seasonal change in LH secretion was observed in Suffolk ewes only. During seasonal anoestrus, treatment of ewes with subcutaneous oestradiol-17β implants (3, 6 or 12 mm in length) decreased the frequency of LH pulses in a dose-dependent manner, with Suffolk ewes being far more sensitive to the inhibitory effects of oestradiol than Merino ewes. The lowest dose of oestradiol (3 mm) had no effect on the secretion of LH in Merino ewes, but reduced secretion in Suffolk ewes. Treatment of ewes with the highest dose of oestradiol (12 mm) completely abolished LH pulses in Suffolk ewes, whereas infrequent pulses remained evident in Merino ewes. During the breeding season, oestradiol alone had no effect on the pulsatile release of LH in either breed, but in combination with progesterone there was a significant reduction in LH pulse frequency. Progesterone effectively decreased LH secretion in both breeds in both seasons. It was concluded that differences between breeds in the 'depth' of anoestrus could be related to differences in the sensitivity of the hypothalamus to both negative feedback by oestradiol and the direct effects of photoperiod.

Keywords: oestradiol; negative feedback; progesterone; sheep

Nutrition-induced changes in testicular size in Merino rams appear to involve both GnRH-dependent and -independent pathways. This hypothesis was tested by feeding mature Merino rams that had been actively immunized against BSA or GnRH conjugated to BSA a diet that maintained initial body weight or the same diet supplemented daily with 1.5 kg of lupin grain. Blood was sampled every 20 min for 24 h on days – 1, 19 and 70 relative to the change in diet. The plasma was used to assess the effects of treatments on changes in LH, FSH and testosterone concentrations. In the group immunized against BSA, FSH increased in lupin-supplemented rams compared with maintenance-fed rams, while LH and testosterone were not affected by diet. In comparison, the concentrations of LH, FSH and testosterone were significantly lower in the group immunized against GnRH than in rams immunized against BSA, but none of these endocrine variables was affected by nutrition. With both immunization treatments, the testes were significantly larger in lupin-supplemented than in maintenance-fed rams. In the group immunized against BSA, this difference was caused by testicular growth in lupin-supplemented rams, whereas in the group immunized against GnRH, lupin supplementation effectively maintained testicular mass, rather than allowed the regression observed in maintenance-fed rams. In conclusion, differences in testicular growth that were induced by dietary treatments in rams immunized against GnRH were not associated with changes in gonadotrophin or testosterone secretion. This supports the hypothesis that part of the effect of nutrition on testicular growth is independent of changes in GnRH secretion. The differences in testicular size observed in control rams were of similar magnitude to those observed in treated rams, but associated with large differences in plasma FSH concentrations, suggesting that this hormone plays an important role in this effect.

The effects of nutrition on the hypothalamo–pituitary–gonadal axis were studied in three groups of six mature Merino rams that were fed for 56 days with a ration that maintained their initial live mass (intermediate diet: 675 g chaff plus 175 g lupins), the same ration with a lupin supplement (high diet: 675 g chaff plus 825 g lupins), or about half of the intermediate ration (low diet: 475 g chaff plus 125 g lupins). Lupin seed provides a highly (95%) digestible source of energy and protein. Plasma concentrations of LH, FSH, testosterone and inhibin were measured in blood samples collected over 24 h on the day before dietary treatments began (day −1), then on days 0, 1, 5, 14, 28 and 56. Compared with the intermediate diet, the high diet significantly increased live mass within 14 days and testicular size within 28 days, and these differences increased steadily throughout the experiment. Plasma FSH concentrations and LH pulse frequency increased within 5 days, but these effects were maintained for only 14 days. Decreasing the nutritional status reduced live mass and testicular size within 7 days, led to a low LH pulse frequency that persisted throughout the experiment, but did not affect FSH concentrations. Significantly less testosterone was secreted over 24 h in the low dietary group than in the intermediate or high group until day 28. The high group tended to secrete more than the intermediate group, but only at the beginning of the experiment when LH pulse frequencies differed between these groups. The testosterone response to each endogenous LH pulse, or following an injection of ovine LH i.v. (200 ng kg⁻¹ live mass), was not related to testicular size or dietary treatment at any stage of the experiment. Similarly, plasma inhibin concentrations were not related to change of diet, despite large differences in testicular size. We concluded that the effects of nutritional status on testicular size in mature rams are at least partly mediated through changes in gonadotrophin secretion. Both increases and decreases in food supply affected LH pulse frequency, suggesting the involvement of hypothalamic mechanisms. However, the lack of an effect of a decrease in nutritional status on the secretion of FSH and inhibin and the inconsistent long-term relationship between LH pulse frequency and testicular size suggest that the effects of diet on testicular growth also involve mechanisms that are independent of changes in gonadotrophin secretion.

Summary. Introduction of rams to ovariectomized ewes treated with oestradiol implants (N = 10) increased the frequency of LH pulses from 4·8 to 10·6 pulses per 12 h. This effect was reflected by increases in mean levels of LH and the basal levels upon which the pulses were superimposed. In ewes that had not been treated with oestradiol (N = 5), there was no significant increase in pulse frequency but mean and basal levels of LH increased slightly after the introduction of rams. In a second experiment, similar effects of the introduction of rams were seen in ovariectomized ewes treated with oestradiol or oestradiol + androstenedione (N = 16), but no significant effects of the rams were observed in untreated ewes (N = 8) or ewes treated only with androstenedione (N = 7). No preovulatory surges of LH were observed in the 30-h period after the introduction of rams.

It was concluded that the ram stimulus probably evokes the increase in pulse frequency by inhibiting the negative feedback action of oestradiol, and that the surge normally observed in entire ewes is dependent on the ovarian response to these pulses. However, the observation of responses in some ewes not treated with oestradiol also raises the possibility that the ram stimulus can act directly on the hypothalamic neurones that control the secretion of LH, and that this effect is enhanced in the presence of oestrogen.

Summary. Seasonally anovulatory Merino ewes isolated from rams were allocated to three treatments before the re-introduction of rams. Ten ewes received a single injection of progesterone (20 mg), 18 ewes received the injection of progesterone but had the ram-induced preovulatory surge of LH replaced by a series of injections of GnRH 24 h after the introduction of the rams, and 20 control ewes had no hormone treatment. Of the 48 ewes, 44 ovulated within 5 days of the introduction of rams and the treatments had no significant effect on the incidence of ovulation. The frequency of corpora lutea with a short life span (the interval between successive preovulatory surges of LH being 5·1 ± 0·9 days) was 72% for control ewes and 58% for ewes treated with progesterone and GnRH, but such CL were prevented completely after the injection of progesterone alone (P < 0·001). The injection of progesterone also delayed the preovulatory surge of LH (P < 0·001). These results suggest that progesterone assures normality of corpora lutea by lengthening the period of gonadotrophin priming of follicles before ovulation.

Summary. In Exp. 1, the changes in pulsatile LH secretion at the onset of the breeding season were observed in 20 intact, mature Saanen does. Blood was sampled every 20 min for 6 h each week from the beginning of August until the onset of ovulatory activity, as evidenced by cycles in plasma progesterone. The first doe ovulated at the end of August and all were cycling by the end of September. As the first ovulation approached, LH pulse frequency increased by 67% and mean levels of LH increased by 47%. These changes were progressive rather than abrupt. In Exp. 2, seasonal changes in the inhibition of pulsatile LH secretion by ovarian steroids were studied in ovariectomized Saanen does. The animals were untreated (N = 4) or given subcutaneous oestradiol implants (N = 4) and blood was sampled every 10 min for 6 h, twice during the breeding season and twice during the anoestrous season. In each season, the second series of samples was taken after the animals had been treated with progesterone, administered by intravaginal implants. Season did not significantly affect LH secretion in goats not treated with oestradiol, but LH pulse frequency was 54% lower during the anoestrous season than during the breeding season in oestradiol-treated goats. Mean LH concentrations were affected in the same manner as pulse frequency, but pulse amplitude was increased by oestradiol treatment in both seasons. Progesterone had no detectable effect on LH secretion in either season. In Exp. 3, the response to repeated melatonin injections at a set time after dawn was investigated in 11 oestradiol-treated, ovariectomized goats. They were placed under a regimen of long days (16 h light:8 h dark) and 1 month later 6 of them were injected daily (10 h after 'dawn') with 2 mg melatonin. The other 5 animals served as controls. Blood samples (every 10 min for 6 h) were taken just before and 38 and 72 days after the start of melatonin treatment. As the experiment progressed, LH pulse frequency increased by 20% in melatonin-treated goats but decreased by 43% in controls. Mean LH values were maintained in melatonin-treated females but decreased in the control group. Melatonin did not affect pulse amplitude.

The results of the experiments with ovariectomized does suggest that seasonal reproductive cycles in the goat are caused by changes in the duration of melatonin secretion at night, which induce alterations in the intensity with which oestradiol inhibits LH secretion. In turn, this may be responsibile for the gradual changes in the frequency of LH pulses observed in entire does. The frequency of LH pulses is one of the most important endocrine signals controlling ovarian activity so would form the final link in the chain connecting environmental and reproductive cycles.

Keywords: LH; goat; melatonin; steroids; season