Effect of transport on pulsatile LH release in ovariectomized ewes with or without prior steroid exposure at different times of year

The initial aim of the present study was to test whether the stress of transport suppresses LH pulsatile secretion in ewes. In a pilot experiment in the late breeding season, transport resulted in an unexpected response in three out of five transported, ovariectomized ewes pretreated with oestradiol and progesterone. Before transport, seasonal suppression of LH pulses had occurred earlier than anticipated, but LH pulsatility suddenly restarted for the period of transport. This finding was reminiscent of unexplained results obtained in ovariectomized ewes infused centrally with high doses of corticotrophin-releasing hormone after pretreatment with low doses of oestradiol with or without progesterone. Hence, an additional aim of the present study was to examine whether these latter results with corticotrophin-releasing hormone could be reproduced by increasing endogenous corticotrophin-releasing hormone secretion by transport. Subsequent experiments used groups of at least eight ovariectomized ewes at different times of the year with or without prior exposure to steroids to assess whether these unexpected observations were associated with season or the prevailing endocrine milieu. In the mid-breeding season, transport for 4 h in the absence of steroid pretreatment for 8 months reduced LH pulse frequency from 7.5 +/- 0.3 to 6.3 +/- 0.4 pulses per 4 h (P < 0.05) and LH pulse amplitude from 2.6 +/- 0.5 to 1.8 +/- 0.3 ng ml-1 (P < 0.05). Similarly, in the mid-breeding season, 34 h after the cessation of pretreatment with oestradiol and progesterone, transport suppressed LH pulse frequency from 6.1 +/- 0.4 to 5.5 +/- 0.3 pulses per 4 h (P < 0.05) with a tendency of effect on amplitude (6.2 +/- 2.7 to 2.61 +/- 0.6 ng ml-1; P = 0.07; note the large variance in the pretransport data). During mid-anoestrus, evidence of a suppressive effect of transport was only observed on LH pulse amplitude (4.7 +/- 0.6 versus 3.0 +/- 0.5 pulses per 4 h; P < 0.05) in ovariectomized ewes that had not been exposed to ovarian steroids for 4 months. Repetition of the pilot experiment with 12 ewes during the transition into anoestrus resulted in one ewe with LH pulses seasonally suppressed but increased by transport; 11 ewes had a distinct pulsatile LH pattern which was decreased by transport in six ewes. In anoestrus, there was no effect of transport on LH pulse frequency or amplitude in intact ewes, or those ovariectomized 2-3 weeks previously, with or without prior oestradiol and progesterone treatment. However, basal concentrations of cortisol were greater in anoestrus than in the breeding season, and the increment in cortisol during transport was similar in anoestrus and the breeding season but greater during the transition into anoestrus (P < 0.05). Progesterone concentrations increased from 0.31 +/- 0.02 ng ml-1 before transport to 0.48 +/- 0.05 ng ml-1 during the second hour of transport (P < 0.05). In conclusion, transport reduced LH pulse frequency and amplitude in ovariectomized ewes that had not been exposed to exogenous steroids for at least 4 months. In most animals, the previously observed increase in LH pulsatility induced by exogenous CRH was not reproduced by increasing endogenous CRH secretion by transport. However, in four ewes, transport did increase LH pulsatility, but only during the transition into anoestrus in ewes with seasonally suppressed LH profiles after withdrawal of steroid pretreatment.     

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.     

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.     

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)     

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.     

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.     

Genetic and environmental variation in the LH response of ovariectomized sheep to LH-RH.

The influence of breed and season on the sensitivity of the pituitary gland of sheep to LH-RH was assessed. Ovariectomized ewes of 3 breeds (Finnish Landrace, Scottish Blackface and Tasmanian Merino) with differing normal breeding seasons and with differing ovulation rates were injected (i.v.) with 3 doses of LH-RH (1.56, 6.25 or 25.0 micrograms) at 3 different times of the year covering the anoestrous and the breeding seasons of intact ewes; 9 ewes of each breed (3 per sub-class) were examined on the first and third occasions, 6 (2 per sub-class) on the second. The response was measured in terms of the concentration of LH in peripheral plasma 20, 40, 60 and 80 min after injection. Time of year, but not the breed of sheep, affected the magnitude of the response; the data indicated that the duration of LH secretion was greater during the breeding season than during anoestrus. It was concluded that changes in the spontaneous activity of the hypothalamus/hypophysis could contribute to seasonal changes in LH secretion independently of the modifying effects of gonadal steroids. Such variation in unmodulated activity apparently does not contribute to the differences in ovulation rate among the 3 breeds.     

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in the ewe: relationship of frequency of LH pulses to day length and response to estradiol negative feedback

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in ovariectomized ewes were examined over the course of 2 yr in relation to annual changes in environmental photoperiod, shifts in response to estradiol negative feedback control of LH secretion, and timing of the breeding season. Under natural environmental conditions, the frequency of LH pulses in individual ovariectomized ewes changed gradually and in close association with the annual cycle of day length. As days became shorter in late summer and autumn, LH pulse frequency increased; conversely, as day length increased in late winter and spring, frequency declined. Under artificial conditions in which ovariectomized ewes were exposed to different photoperiods, a similar inverse relationship was observed between day length and LH pulse frequency. The seasonal changes in frequency of LH pulses in ovariectomized ewes, although symmetric with the annual photoperiodic cycle, were not temporally coupled to the dramatic shifts in response to estradiol feedback inhibition of LH secretion at the transitions between breeding season and anestrus. The feedback shifts occurred abruptly and at times when LH pulse frequency in ovariectomized ewes was at, or near, the annual maximum or minimum. The tight coupling between LH pulse frequency and photoperiod leads to the conclusion that there is a photoperiodic drive to the LH pulse-generating system of the ewe. The temporal dissociation between changes in this photoperiodic drive and the seasonal shifts in response to estradiol negative feedback support the hypothesis that the neuroendocrine basis for these two phenomena is not one and the same.     

Suppression of follicular development after chronic LHRH immunoneutralization in the ewe

Active immunization of 6 Damline ewes against LHRH during seasonal anoestrus resulted in an inhibition of ovarian cyclicity throughout 2 subsequent breeding seasons. This was associated with a significant suppression of plasma LH and FSH concentrations but no significant effect on prolactin. The ovaries of LHRH-immunized ewes 30 months after primary immunization contained no follicles greater than 2.5 mm in diameter and a greater proportion of follicles between 1 and 2 mm were atretic than in control ewes (N = 8). In-vitro production of testosterone and androstenedione were similar in follicles 1-2 mm in both control and LHRH-immunized ewes (N = 6) and all had little or no ability to secrete oestradiol. However, basal and hCG-stimulated progesterone secretion was suppressed in the follicles from LHRH-immunized ewes. These results show that follicular development beyond 2.5 mm in the ewe is dependent on adequate stimulation by both LH and FSH.     

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.     

Effects of pentobarbital anesthesia on tonic luteinizing hormone secretion in the ewe: evidence for active inhibition of luteinizing hormone in anestrus

In the ewe, seasonal anestrus appears to result from two effects of inhibitory photoperiod: 1) estradiol gains the capacity to suppress luteinizing hormone (LH) pulse frequency and hence becomes a potent inhibitor of tonic LH secretion and 2) a steroid-independent decrease in LH pulse frequency occurs in ovariectomized ewes. In this study, we have obtained evidence, using pentobarbital anesthesia, that both these actions of photoperiod reflect the activation, in anestrus, of an inhibitory neural system. Administration of pentobarbital to intact anestrous ewes produced a dramatic, 3-fold increase in LH pulse frequency during the 6 h of anesthesia. In contrast, during the breeding season, pentobarbital inhibited LH pulse frequency in luteal phase animals. There was also a seasonal variation in the effects of pentobarbital in ovariectomized ewes. During the breeding season this drug again suppressed LH secretion, inhibiting both LH pulse amplitude and frequency. In anestrus, pentobarbital also suppressed pulse amplitude, but it produced a transitory increase (lasting 3 h) in pulse frequency. To account for the stimulatory actions of pentobarbital, we propose that in anestrus, but not the breeding season, LH pulse frequency is held in check by a set of estradiol-sensitive inhibitory neurons. Further, we suggest that these neurons are activated by inhibitory photoperiod and account for both the steroid-dependent and steroid-independent actions of photoperiod.     

Seasonal and hormonal control of pulsatile LH secretion in the dairy goat (Capra hircus)

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe

The long-term negative feedback effects of sustained elevations in circulating estradiol and progesterone on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were evaluated in the ewe following ovariectomy during the mid-late anestrous and early breeding seasons. GnRH secretion was monitored in serial samples of hypophyseal portal blood. Steroids were administered from the time of ovariectomy by s.c. Silastic implants, which maintained plasma concentrations of estradiol and progesterone at levels resembling those that circulate during the mid-luteal phase of the estrous cycle; control ewes did not receive steroidal replacement. Analysis of hormonal pulse patterns in serial samples during 6-h periods on Days 8-10 after ovariectomy disclosed discrete, concurrent pulses of GnRH in hypothalamo-hypophyseal portal blood and LH in peripheral blood of untreated ovariectomized ewes. These pulses occurred every 97 min on the average. Treatment with either estradiol or progesterone greatly diminished or abolished detectable pulsatile secretion of GnRH and LH, infrequent pulses being evident in only 3 of 19 steroid-treated ewes. No major seasonal difference was observed in GnRH or LH pulse patterns in any group of ewes. Our findings in the ovariectomized ewe provide direct support for the conclusion that the negative-feedback effects of estradiol and progesterone on gonadotropin secretion in the ewe include an action on the brain and a consequent inhibition of pulsatile GnRH secretion.     

Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes

Daily transrectal ultrasonography of ovaries was done in seven Finn ewes during three 17-day periods from May to July. Blood samples were collected each day for estimation of the serum follicle-stimulating hormone (FSH), oestradiol and progesterone concentrations, and also every 15 min for 6 h, halfway through each period of ultrasonographic examination, to determine the patterns of gonadotropic hormone secretion. Four ewes ceased cycling from March to mid-April (ewes entering anoestrus early) and three in May (ewes entering anoestrus late). In all ewes cyclicity resumed during the period from mid-August to mid-September. The growth of ovarian antral follicles to periovulatory sizes of >/=5 mm in diameter was seen at all stages of anoestrus. An average of four waves of follicular development (follicles growing from 3 to >/=5 mm in diameter before regression) with a periodicity of 4 days were recorded during each of the three scanning periods. There was a close temporal relationship between days of follicular wave emergence and peaks of successive FSH fluctuations. Ewes entering anoestrus late exceeded ewes that became anoestrus early in numbers of large (>/=5 mm in diameter) ovarian antral follicles and maximum follicle diameter. Peak concentrations of transient FSH increases were higher (P<0.05) in ewes entering anoestrus late than in ewes entering anoestrus early. The secretion of luteinising hormone, (LH; mean and basal level, and LH pulse frequency, but not amplitude) was lowest during the month of June in all ewes. Oestradiol production was markedly suppressed throughout anoestrus. Peaks of progesterone secretion appeared to occur at regular intervals and were associated with the end of the growth phase of the largest follicles of sequential waves. In conclusion, the growth of ovarian follicles to ostensibly ovulatory diameters is maintained throughout anoestrus in Finn ewes and periodic emergence of follicular waves is correlated with an endogenous rhythm of FSH secretion. The present study also provides evidence for the inverse relationship between the time of the onset of seasonal anoestrus and the number and size of antral follicles developing throughout anoestrus in Finn ewes, and indicates that differences exist in both the secretion of and ovarian responsiveness to gonadotropic hormones among early and late anoestrous ewes.     

Social cues can play a role in timing onset of the breeding season of the ewe

Female Suffolk sheep were pinealectomized around the vernal equinox to eliminate the major environmental input to the reproductive system (photoperiod) and then either isolated from, or maintained with, pineal-intact gonad-intact sheep. The ewes were ovariectomized and treated with constant-release oestradiol implants and reproductive state was monitored by measuring serum LH concentrations. Pinealectomized ewes that were isolated from the normal flock showed a 2 1/2-month delay in onset of the seasonal rise in LH values compared with that of pineal-intact controls (18 November vs 5 September). On the other hand, pinealectomized ewes that were maintained with the flock showed an onset of the seasonal rise in LH that was not delayed. These results suggest a timekeeping role for social cues for timing onset of the breeding season in an animal that normally relies on photoperiodic signals for temporal regulation of the seasonal reproductive cycle.     

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.