Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season.

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 +/- 0.9 h versus 48.3 +/- 0.8 h, P < 0.02; ovariectomized model: 40.8 +/- 0.6 h versus 42.6 +/- 0.5 h, P < 0.02). Disruption of the hypothalamus-pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 +/- 4 ng ml-1 versus intact transported 22 +/- 5 ng ml-1, P < 0.02; ovariectomized model controls: 35 +/- 7 ng ml-1 versus model transported 15 +/- 2 ng ml-1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 +/- 0.3 versus 0.3 +/- 0.02 ng ml-1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 +/- 0.4 versus 1.7 +/- 0.6 pulses per 2 h). In conclusion, activation of the hypothalamus-pituitary-adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.     

Effects of breed, ovarian steroids and season on the pulsatile secretion of LH in ovariectomized ewes

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-17 beta 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.     

Effects of time after ovariectomy, season and oestradiol on luteinizing hormone and follicle-stimulating hormone secretion in ovariectomized ewes.

During the breeding season, five groups of three ewes were implanted at ovariectomy with 0.36, 0.5, 1.0 and 6.0 cm oestradiol implants or implants containing no steroid. Eleven days after receiving implants, blood samples were taken every 10 min for 6 h; implants were then removed. Treatments were repeated three times during each of two consecutive breeding seasons and four times during the intervening anoestrus. In ovariectomized ewes without steroid treatment, luteinizing hormone (LH) pulse frequency increased from early to mid-breeding season, decreased to a minimum at mid-anoestrus and increased to reach a maximum at the mid-point of the second breeding season, subsequently declining. LH pulse amplitude was inversely related to frequency. Basal serum LH concentrations decreased gradually from the first breeding season to reach a minimum at mid-anoestrus and gradually increased to reach a maximum at the end of the second breeding season. Mean serum LH and follicle-stimulating hormone (FSH) concentrations were higher at the end of the second breeding season compared with the beginning of the first breeding season. All parameters of gonadotrophin secretion were decreased much more by oestradiol during the anoestrus than during the breeding season. LH pulse frequency was decreased during anoestrus and at high oestradiol concentrations during the first breeding season. Apart from LH pulse amplitude, the decreases in all parameters of gonadotrophin secretion were less during the second compared with the first breeding season. The minimum effective dose of oestradiol required to decrease mean and basal serum concentrations of LH during anoestrus was lower than in the breeding season. The minimum effective dose of oestradiol required to decrease mean serum concentrations of FSH was lower in the first compared with the second breeding season. Oestradiol depression of LH pulse amplitude and mean serum concentrations of LH and FSH showed a dose dependency during the breeding season. During anoestrus dose dependency was seen for basal concentrations of LH and mean serum concentrations of LH and FSH. We conclude that significant chronic changes in gonadotrophin secretion occur in the ewe with time after ovariectomy. Sensitivity to oestradiol also changes, and the effects of oestradiol are not always dose dependent. We suggest that the circannual pattern of LH pulse frequency and basal LH secretion are directly linked to the circannual cycle of photoperiod.(ABSTRACT TRUNCATED AT 400 WORDS)     

Independence of progesterone blockade of the luteinizing hormone surge in ewes from opioid activity at naloxone-sensitive receptors.

Fifteen ovariectomized ewes were treated with implants (s.c.) creating circulating luteal progesterone concentrations of 1.6 +/- 0.1 ng ml-1 serum. Ten days later, progesterone implants were removed from five ewes which were then infused with saline for 64 h (0.154 mol NaCl l-1, 20 ml h-1, i.v.). Ewes with progesterone implants remaining were infused with saline (n = 5) or naloxone (0.5 mg kg-1 h-1, n = 5) in saline for 64 h. At 36 h of infusion, all ewes were injected with oestradiol (20 micrograms in 1 ml groundnut oil, i.m.). During the first 36 h of infusion, serum luteinizing hormone (LH) concentrations were similar in ewes infused with saline after progesterone withdrawal and ewes infused with naloxone, but with progesterone implants remaining (1.23 +/- 0.11 and 1.28 +/- 0.23 ng ml-1 serum, respectively, mean +/- SEM, P greater than 0.05). These values exceeded circulating LH concentrations during the first 36 h of saline infusion of ewes with progesterone implants remaining (0.59 +/- 0.09 ng ml-1 serum, P less than 0.05). The data suggested that progesterone suppression of tonic LH secretion, before oestradiol injection, was completely antagonized by naloxone. After oestradiol injection, circulating LH concentrations decreased for about 10 h in ewes of all groups. A surge in circulating LH concentrations peaked 24 h after oestradiol injection in ewes infused with saline after progesterone withdrawal (8.16 +/- 3.18 ng LH ml-1 serum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus

The effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus were investigated in two experiments. In the first experiment, the introduction of rams induced an increase in the levels of LH in entire ewes. The mean levels increased from 0.68 +/- 0.04 ng/ml (mean +/- s.e.m.) to 4.49 +/- 1.32 ng/ml within 20 min in ewes not treated with progesterone (n = 10). In ewes bearing progesterone implants that provided a peripheral concentration of about 1.5 ng progesterone per millilitre plasma, the LH response to the introduction of rams was not prevented, but was reduced in size so that the concentration was 1.38 +/- 0.15 ng/ml after 20 min (n = 5). Progesterone treatment begun either 2 days before or 6 h after the introduction of rams and maintained for 4 days prevented ovulation. In the second experiment ovariectomized ewes were used to investigate further the mechanism by which the ram evoked increases in tonic LH secretion. In ovariectomized ewes treated with oestradiol implants, the introduction of rams increased the frequency of the LH pulses and the basal level of LH. In the absence of oestradiol there was no significant change in pulse frequency but a small increase in basal levels. Progesterone again did not prevent but reduced the responses in ewes treated with oestradiol. It is suggested that following the withdrawal of progesterone treatment, the secretion of LH pulses in response to the ram effect would be dampened. This effect could be a component of the reported long delay between the introduction of rams and the preovulatory surge of LH in ewes treated with progesterone. Continued progesterone treatment prevented ovulation, probably by blocking positive feedback by oestradiol.     

Gonadotrophin release in ovariectomized ewes fed different amounts of coumestrol

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.     

Ovarian function in ewes during the transition from breeding season to anoestrus

Transrectal ovarian ultrasonography was conducted in six Western white-faced ewes for 35 days from the last oestrus of the breeding season, to record the number and size of all ovarian follicles > or = 3 mm in diameter and luteal structures. Blood samples were collected once a day for estimation of serum concentrations of follicle-stimulating hormone (FSH), oestradiol and progesterone. Each ewe had five follicular waves (follicles growing from 3 to > or = 5 mm in diameter) over the scanning period. The duration of the growth phase of the largest ovarian follicles did not differ (P > 0.05) between waves, but follicular static and regressing phases decreased significantly (P < 0.05) after the decline in serum progesterone concentrations at the end of the last luteal phase of the breeding season. The intervals between the five follicular waves were: 9.2+/-0.4, 5.2+/-0.7, 8.3+/-0.8 and 5.8+/-0.7 days; the two shorter intervals differed (P < 0.05) from the two longer intervals. Using the cycle-detection program, rhythmic increases in serum FSH concentrations were detected in all ewes; the amplitude, duration and periodicity of FSH fluctuations did not vary (P > 0.05) throughout the period of study. The number of identified FSH peaks (7.8+/-0.5 peaks per ewe, per scanning period) was greater (P < 0.05) than the number of emerging follicular waves. Serum concentrations of oestradiol remained low (< or = 1 pg/ml) on most days, in five out of the six ewes studied, and sporadic elevations in oestradiol secretion above the non-detectable level were not associated with the emergence of follicular waves. The ovulation rate was lower than that seen during the middle portion of the breeding season (November-December) in white-faced ewes but the transitional ewes had larger corpora lutea (CL). Maximal serum concentrations of progesterone appeared to be lower and the plateau phase of progesterone secretion appeared to be shorter during the last luteal phase of the ovulatory season in comparison to the mid-breeding season of Western white-faced ewes. During the transition into anoestrus in ewes, the endogenous rhythm of FSH release is remarkably robust but the pattern of emergence of sequential follicular waves is dissociated from FSH and oestradiol secretion. Luteal progesterone secretion is suppressed because of fewer ovulations and diminished total luteal volume, but it may also result from diminished gonadotropic support. These season-related alterations in the normal pattern of ovine ovarian cycles appear to be due to reduction in ovarian responsiveness to gonadotropins and/or attenuation in secretion of luteinizing hormone (LH) occurring at the onset of the anovulatory season in ewes.     

Secretion of LH, FSH and oestradiol-17 beta during the follicular phase of the oestrous cycle in the ewe

Plasma concentrations of LH, FSH and oestradiol-17 beta were measured in blood samples taken at 15 min intervals for 48 h during the follicular phase of four Merino ewes. The amplitude of pulses of LH and the mean concentration of LH were higher at the beginning of the follicular phase, 36-24 h before the preovulatory surge of LH (amplitude 2.4 ng ml-1, mean concentration 3.9 ng ml-1), than at the end, 24-0 h before the preovulatory surge (amplitude 1.2 +/- 0.1 ng ml-1; mean concentration 1.4 +/- 0.1 ng ml-1). There was no change in the inter-pulse interval during this time (mean 74 +/- 5 min). Over the same period, oestradiol levels increased from 7-8 pg ml-1 to a peak of 10-15 pg ml-1. Mean FSH concentrations declined (36-24 h: 3.6 ng ml-1 vs 24-0 h: 1.8 +/- 0.3 ng ml-1) before rising at the time of the preovulatory surge of LH and again 24 h later. It was concluded that the biphasic response of LH to oestrogen that is seen in ovariectomized ewes may also operate during the follicular phase of the oestrous cycle in entire ewes.     

Ultrasonographic study of the effects of the corpus luteum on antral follicular development in unilaterally ovulating western white-faced ewes

The objective of this study was to examine the local effects of the corpus luteum (CL) on ovarian antral follicle development by looking at follicle populations and dynamics in ovaries with or without CL, in unilaterally ovulating ewes, using a retrospective analysis of daily ultrasonographic records. The present report summarises the data from the first luteal phase of the breeding season (August-October; n = 4), a luteal phase in the mid-breeding season (November-December; n = 5), the last luteal phase of the breeding season (January-March; n = 5), and the luteal phase after GnRH-induced ovulations in mid-anoestrus (May-June; n = 4) of western white-faced ewes. Mean daily numbers of 3mm follicles that did not grow any larger were significantly reduced in the CL-containing ovaries of ewes at all periods of study except for the transition to anoestrus. With all scanning periods combined, daily numbers of 3mm follicles not growing further increased (P<0.05) between day 6 and 15 after ovulation in the CL-containing ovaries. Based on mean data for the whole periods of observation, the non-CL-bearing ovaries of ewes in the transition to anoestrus had fewer (P<0.05) follicles growing from 3 to > or =5mm in size before regression compared with the mid-breeding season and mid-anoestrus. The lifespan of follicles reaching > or =5mm in diameter was shorter (P < 0.05) in the CL- compared with non-CL-containing ovaries of anoestrous ewes induced to ovulate with GnRH ((6.5+/- 1.3) and (9.0+/- 1.0) days, respectively). Circulating concentrations of progesterone were lower during both transitional periods (into and out of anoestrus) and mid-anoestrus than during the mid-breeding season (P < 0.001), and were less during anoestrus than during both transitional periods (P < 0.05). It was concluded that CL/luteal structures locally suppressed the growth of ovarian antral follicles to the 3mm size-range except during the transition to anoestrus, but that there was no inhibitory effect of the CL on the growth of ovarian follicles to larger diameters. The presence of CL/luteal structures did not affect the length of the lifespan of follicles reaching > or =5mm in diameter nor the number of ovulations per ovary in cyclic ewes, but shortened large follicle lifespan in anoestrous ewes. Variations in peripheral concentrations of progesterone across the breeding season and between the breeding season and anoestrus did not alter the lifespan of large antral follicles. In the transition to anoestrus and during mid-anoestrus, the presence of the CL in an ovary appeared to maintain follicle development to ovulatory sizes and to increase the rate of turnover of large antral follicles, respectively.     

Effects of an opioid antagonist on pulsatile luteinizing hormone secretion in the ewe vary with changes in steroid negative feedback

In ewes during the breeding season, estradiol (E) and progesterone (P) synergistically regulate pulsatile luteinizing hormone (LH) secretion. E primarily inhibits LH pulse amplitude and P inhibits LH pulse frequency. To determine if endogenous opioid peptides (EOP) mediate these negative feedback effects, we administered the long-acting opioid antagonist WIN 44,441-3 (WIN) to intact ewes during the luteal and follicular phases of the estrous cycle and to ovariectomized ewes treated with no steroids, E, P, or E plus P. Steroid levels were maintained at levels seen during the estrous cycle by Silastic implants placed shortly after surgery. WIN increased LH pulse frequency, but not amplitude, in luteal phase ewes. In contrast, during the follicular phase, LH pulse amplitude was increased by WIN and pulse frequency was unchanged. Neither LH pulse frequency nor pulse amplitude was affected by WIN in long-term ovariectomized ewes untreated with steroids. In contrast, WIN slightly increased LH pulse frequency in short-term ovariectomized ewes. WIN also increased LH pulse frequency in ovariectomized ewes treated with P or E plus P. WIN did not affect pulse frequency but did increase LH pulse amplitude in E-treated ewes. These results support the hypothesis that EOP participate in the negative feedback effects of E and P on pulsatile LH secretion during the breeding season and that the inhibitory effects of EOP may persist for some time after ovariectomy.     

Changes in pulsatile LH secretion after ovariectomy in Ile-de-France ewes in two seasons

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.     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.     

Fluctuation in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe

In ewes in the mid-luteal phase, LH pulse frequency (P less than 0.01) and amplitude (P less than 0.05) increased during a 24 h infusion of naloxone (0.5 mg/kg/h) compared to a 24 h infusion of vehicle (mean +/- s.e.m.; 0.25 +/- 0.03 vs 0.14 +/- 0.01 pulses/h and 0.84 +/- 0.08 vs 0.55 +/- 0.08 ng/ml serum, respectively). The increase in pulse amplitude was immediate, but was less (P less than 0.05) during the second 12 h, compared to the first 12 h, of naloxone infusion (0.52 +/- 0.14 vs 0.98 +/- 0.08 ng/ml serum). Oestradiol concentrations were higher (P less than 0.01) during naloxone than during control infusion (5.63 +/- 0.26 vs 4.13 +/- 0.15 pg/ml serum). In ovariectomized ewes in the breeding season, LH pulse frequency was lower (P less than 0.01) during a 24 h infusion of morphine (0.5 mg/kg/h) than during a 24 h infusion of vehicle (mean +/- s.e.m.; 1.17 +/- 0.08 vs 1.71 +/- 0.06 pulses/h). We conclude that long-term infusion of naloxone results in a sustained increase in LH pulse frequency but only a transient elevation in pulse amplitude. No effects on FSH secretion were noted. LH secretion was sensitive to morphine in the absence of ovarian steroids, suggesting that ovarian steroids are not required for the presence of functional opioid receptors capable of modulating LH release.     

Changes in pulsatile LH secretion and the positive and negative feedback actions of oestradiol after administration of a GnRH agonist in the ovariectomized ewe

Administration of a GnRH agonist (5 micrograms) every 12 h to long-term ovariectomized ewes for 5 or 10 days during the breeding season suppressed mean LH levels from around 6 to 1 ng/ml on Days 1 and 4 after treatment; on Day 1 after treatment LH pulse frequency and amplitude were lower than pretreatment values. On Day 4 after treatment LH pulse frequency was restored to pretreatment levels (1 per h) whereas LH pulse amplitude had only slightly increased from 0.5 to 1 ng/ml, a value 25% of that before treatment. This increase in amplitude was greater the shorter the duration of treatment. Ovariectomized ewes treated with the agonist for 5 days exhibited both negative and positive feedback actions after implantation of a capsule containing oestradiol; however, compared to control ewes treated with oestradiol only, the positive and negative feedback actions of oestradiol were blunted. These results suggest that the recovery of tonic LH concentrations after GnRH agonist-induced suppression is limited primarily by changes in LH pulse amplitude. The results also demonstrate that the feedback actions of oestradiol are attenuated, but not blocked, by GnRH agonist treatment.     

Early pituitary LH response to injections of GnRH in ewes

In the deep anoestrous period (June), five intact ewes and five ovariectomized ewes received 50 ug synthetic gonadotrophin-releasing hormone (GnRH). In the mid-breeding season (October), the GnRH administrations were repeated in five intact and four ovariectomized ewes; the former were in the luteal phase of the cycle. Blood samples were collected every 30 sec for 15 min, then at 15-min intervals. Release of luteinizing hormone (LH) occurred as soon as the second minute after injection in all ewes. This early response was earlier and more abrupt in the ovariectomized ewes than in the intact animals. In a second experiment three intact ewes that were in deep anoestrus received 50 ug GnRH followed 5 h 20 min later by a second identical injection. Another three intact ewes in deep anoestrus received two injections of 1 ug GnRH. Blood samples were taken every 15 sec for 15 min, then every 20 min until the next injection, and for a further 5 h after the second injection. This regimen was repeated in mid-breeding season during the luteal phase. There was again a very early release of LH; the magnitude of response was similar after the first injection of either 50 ug or 1 ug GnRH to intact ewes either in the breeding season or during deep anoestrus. However, a greater early release of LH was obtained at the lower dose only after the second injection of GnRH. Apart from this exception, the similar early release of LH occurred in spite of different amounts of LH released thereafter in response to the two doses of GnRH. It is suggested that the early response to GnRH consists of LH stored in a "readily releasable" pool in the pituitary, whereas the main release of LH may be a result of increased synthesis and/or release of a more stable pool.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

Changes in LH pulse frequency and serum progesterone concentrations during the transition to breeding season in ewes

To characterize the changes in LH pulse frequency during the transition to breeding season. LH pulse patterns and serum progesterone profiles were determined in 8 intact ewes from mid-anoestrus to the early breeding season. Overall, 8 increases in LH pulse frequency were observed and these were restricted to 5 ewes. Of the 8 increases, 7 occurred during the 4 weeks before the first cycle, 5 of them within 1 week after a pulse frequency typical of anoestrus (0-2 per 8 h). Six of them occurred less than 1 week before either a full-length luteal phase (n = 2) or a 1-3-day increment in progesterone (n = 4). Seven of these brief progesterone increases were observed in 6 ewes, 5 of them immediately preceding the first full-length luteal phase. These results are consistent with the hypothesis that the seasonal decrease in response to oestradiol negative feedback at the beginning of the breeding season causes an increase in GnRH, and thereby LH pulse frequency. In addition, they demonstrate that the first increase in tonic LH secretion occurs in less than 1 week and, in most ewes, initiates either the first full-length cycle or a transient increase in progesterone, the latter occurring more often.     

FACTORS AFFECTING THE SECRETION OF LUTEINIZING HORMONE IN THE EWE

(1) Luteinizing hormone (LH) is secreted as discrete pulses throughout all stages of the reproductive cycle of the ewe, including pre-pubertal, seasonal and lactational anoestrus, and the luteal and follicular phases of the oestrous cycle. Secretion is probably also pulsatile during the preovulatory surge of LH. (2) The secretion of LH is affected by the ovarian steroids, oestradiol and progesterone, both of which act principally to reduce the frequency of the pulses. During the luteal phase the two steroids act synergistically to exert this effect, and during anoestrus oestradiol acts independently of progesterone. Androstenedione secreted by the ovary apparently has no role in the control of LH secretion. (3) The amplitude of the pulses may also be affected by the steroids but there are conflicting reports on these effects, some showing that amplitude is lowered by the presence of oestrogen and others showing increases in amplitude in the presence of oestrogen and progesterone. (4) The secretion of LH pulses is affected by photoperiod, social environment and nutrition. Under the influence of decreasing day-length, oestradiol alone cannot reduce the frequency of pulses and the ewe experiences oestrous cycles. When day-length is increasing, the hypothalamus becomes more responsive to oestradiol which reduces the frequency of the pulses. (5) A hypothetical pheromone secreted by rams can increase the frequency of the LH pulses in anoestrous ewes and thereby induce ovulation, possibly by inhibiting the negative feedback exerted by oestradiol. (6) The relationships between nutrition and reproduction are poorly understood, but it seems likely that the effects of nutrition are mediated partly through the hypothalamus and its control of the secretion of LH pulses. (7) The pulses of LH secreted by the anterior pituitary gland are evoked by pulses of GnRH secreted by the hypothalamus. The location of the centre controlling the GnRH pulses and the neurotransmitter involved are not known.     

Ovarian function in ewes at the onset of the breeding season

Transrectal ultrasonography of ovaries was performed each day, during the expected transition from anoestrus to the breeding season (mid-August to early October), in six Western white-faced cross-bred ewes, to record ovarian antral follicles > or = 3 mm in size and luteal structures. Jugular blood samples were collected daily for radioimmunoassay (RIA) of follicle-stimulating hormone (FSH), oestradiol and progesterone. The first ovulation of the breeding season was followed by the full-length oestrous cycle in all ewes studied. Prior to the ovulation, all ewes exhibited a distinct increase in circulating concentrations of progesterone, yet no corpora lutea (CL) were detected and luteinized unovulated follicles were detected in only three ewes. Secretion of FSH was not affected by the cessation of anoestrus and peaks of episodic FSH fluctuations were associated with the emergence of ovarian follicular waves (follicles growing from 3 to > or = 5 mm). During the 17 days prior to the first ovulation of the breeding season, there were no apparent changes in the pattern of emergence of follicular waves. Mean daily numbers of small antral follicles (not growing beyond 3 mm in diameter) declined (P < 0.05) after the first ovulation. The ovulation rate, maximal total and mean luteal volumes and maximal serum progesterone concentrations, but not mean diameters of ovulatory follicles, were ostensibly lower during the first oestrous cycle of the breeding season compared with the mid-breeding season of Western white-faced ewes. Oestradiol secretion by ovarian follicles appeared to be fully restored, compared with anoestrous ewes, but it was not synchronized with the growth of the largest antral follicles of waves until after the beginning of the first oestrous cycle. An increase in progesterone secretion preceding the first ovulation of the breeding season does not result, as previously suggested, from the ovulation of immature ovarian follicles and short-lived CL, but progesterone may be produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum concentrations of progesterone does not alter the pattern of follicular wave development, hence it seems to be important mainly for inducing oestrous behaviour, synchronizing it with the preovulatory surge of luteinizing hormone (LH), and preventing premature luteolysis during the ensuing luteal phase. Progesterone may also enhance ovarian follicular responsiveness to circulating gonadotropins through a local mechanism.     

Control of gonadotrophin release in Scottish Blackface and Finnish Landrace ewes during seasonal anoestrus

The patterns of LH and FSH secretion were measured in 4 experimental groups of Finnish Landrace and Scottish Blackface ewes: long-term (18 months) ovariectomized ewes (Group 1), long-term ovariectomized ewes with an oestradiol implant, which has been shown to produce peripheral levels of approximately 5 pg/ml (Group 2), long-term ovariectomized ewes with an oestradiol implant for 18 months which was subsequently removed (surgery on Day 0) (Group 3) and short-term ovariectomized ewes (surgery on Day 0) (Group 4). LH and FSH concentrations were monitored in all groups at approximately weekly intervals, before and after Day 0. Finnish Landrace ewes in Groups 1, 2 and 3 had significantly higher mean FSH concentrations than did Scottish Blackface ewes (P less than 0.01). FSH and LH concentrations increased significantly in Groups 3 and 4, but values in Group 4 were significantly lower (P less than 0.01) than those in Group 1 ewes even up to 30 days after ovariectomy. In Group 3, LH concentrations increased to levels similar to those in Group 1. The pattern of LH release was, however, significantly different, with a lower LH pulse frequency (P less than 0.05), but higher pulse amplitude (P less than 0.05). This difference was maintained at least until 28 days after implant removal. We suggest that removal of negative feedback by ovariectomy demonstrates an underlying breed difference in the pattern of FSH secretion and that ovarian factors other than oestradiol are also involved in the negative-feedback control of hypothalamic/pituitary gland function. Furthermore, negative-feedback effects can be maintained for long periods, at least 28 days, after ovariectomy or oestradiol implant removal.