Identification of an oestrus-associated glycoprotein in oviducal fluid of the sheep

Samples of oviducal fluid were collected daily from sheep with indwelling catheters. Fluid samples taken from both oviducts of 2 sheep for 2 cycles during the middle of the breeding season (April/May) (8 sets of data) were compared with 9 sets of data generated from 2 cycles in 3 sheep later in the breeding season (June/July). Around the period of oestrus, the output of oviducal fluid increased to a peak volume of 1.56 +/- 0.35 ml per day (mean +/- s.d.) compared with a mid-cycle volume of 0.49 +/- 0.29 ml. Later in the breeding season, the flow rates were lower, but showed the same trend (0.91 +/- 0.24 ml at the peak and 0.25 +/- 0.18 ml 7 days later). The total amount of protein secreted by the oviduct each day increased 2-4-fold around the time of oestrus, with higher levels in mid-season ewes. When oviducal fluids were fractionated by SDS electrophoresis, a novel glycoprotein, subunit size of Mr 80-90 000 was identified in samples for 3-6 days of each cycle, coinciding with the period of high fluid flow rate. This protein first appeared in the oviducal fluid on the day of oestrus or the following day and it represented 1 of the 2 major glycoproteins in oviducal fluid as assessed by periodic acid-Schiff (PAS) staining. A PAS-positive protein (Mr 80-90 000) was also detected in fluid taken after oestrus on native highly cross-linked gradient gels after electrophoresis at pH 3.1 but not at pH 8.3. Both gradient gel systems showed an increase in high molecular weight material (Mr greater than 10(6] in fluid taken soon after oestrus.     

Luteinizing hormone, oestrogen and progesterone levels in peripheral serum of anoestrous and cyclic ewes as determined by radioimmunoassay.

Jugular vein blood was collected daily from four mature ewes throughout anoestrus and the first oestrous cycle of the breeding season until 4 days after the second oestrus. The levels of oestrogen, progesterone and LH were determined by radioimmunoassay. There were fluctuations in the LH level throughout most of the observed anoestrous period with a mean plus or minus S.E. value of 2-3 plus or minus 0-9 ng/ml. High LH values of 20-0, 41-2 and 137-5 ng/ml were observed in three ewes on Day - 24 of anoestrus. A brief minor rise in progesterone level was also observed around this period. Progesterone levels were consistently low (0.11 plus or minus 0-01 ng/ml) before Day - 25 of anoestrus. A major rise occurred on Day - 12 of anoestrous and this was followed by patterns similar to those that have been previously reported for the oestrous cycle of the ewe. Random fluctuations of oestrogens deviating from a mean level of 4-40 plus or minus 0-1 pg/ml were observed during anoestrus and the mean level during the period from the first to the second oestrus was 5-2 plus or minus 0-3 pg/ml. A well-defined peak of 13-3 plus or minus 0-7 pg/ml was seen in all ewes on the day of the second oestrus. Results of the present study suggest that episodic releases of LH occur during anoestrus and periods of low luteal activity. The fluctuations in LH levels, as observed during the period of low luteal activity, i.e. before Day - 25 of anoestrus, were less pronounced during the periods of high luteal activity. The view that luteal activity precedes the first behavioural oestrus of the breeding season is supported.     

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.     

Evidence that the onset of the breeding season in the ewe may be independent of decreasing plasma prolactin concentrations

Ten ewes of each of two breeds, Dorset Horn (long breeding season) and Welsh Mountain (short breeding season), were given subcutaneous oestradiol-17 beta implants and then ovariectomized. Another 10 ewes of each breed were left intact. On 3 May 1982, all the ewes were housed in an artificial photoperiod of 16L:8D. After 4 weeks, half of the ewes of each breed and physiological state were abruptly exposed to a short-day (8L:16D) photoperiod while the others remained in long days (16L:8D). The time of onset of the breeding season was significantly (P less than 0.05) advanced in ewes switched to short days (12 August +/- 10 days) compared to those maintained in long days (4 September +/- 14 days). Dorset Horn ewes began to cycle (20 July +/- 7 days) significantly (P less than 0.001) earlier than Welsh Mountain ewes (19 September +/- 6 days). Disparities in the time of onset of cyclic activity in ewes of different breeds and daylength groups were echoed in disparities in the time at which plasma LH and FSH concentrations rose in oestrogen-implanted, ovariectomized ewes of the same light treatment group. Prolactin concentrations showed an immediate decrease in ewes switched to short days, but remained elevated in long-day ewes. Since the breeding season started in the presence of high prolactin concentrations in long-day ewes, it seems unlikely that prolactin is an important factor determining the timing of the onset of cyclic activity.     

Ovulation in the merino ewe in the breeding and anoestrous seasons

The pattern of ovulation of Merino ewes was studied by repeated laparoscopy each 14 days in the anoestrous (n = 97) and breeding (n = 87) seasons. In the anoestrous season the proportion of ewes ovulating did not decrease below 11%, 42% of ewes never ovulated and the remainder fluctuated between the two states. On 20 occasions a clear anovulatory period was interrupted by an isolated spontaneous ovulation. In the breeding season the overall mean proportion of ewes with corpora lutea or albicantia at laparoscopy was 87%, 54% of ewes ovulated regularly throughout while in another 31% absence of corpora lutea or albicantia coincided with the follicular phase of an oestrous cycle as evidenced by an appropriately aged corpora lutea at the next laparoscopy. Of the remaining 15% of the flock 3% had anovulatory periods greater than 14 days while the remainder experienced irregular ovulatory cycles--the majority due to short periods of anovulation but some ewes retained corpora lutea for longer than 14 days while others ovulated twice between successive laparoscopies.     

Ovulation and lambing rates in Karaguniki ewes after treatment with follicular fluid

The aim of the present study was to examine the effect of steroid-free bovine follicular fluid (bFF) on both ovulation and lambing rates. For this purpose, 30 adult ewes of the Karaguniki breed were randomly allocated to three treatment groups (A,B and C; n=10 ewes each) during the breeding season of 1988. The ewes in Group A received bFF (6 ml iv) twice daily during their luteal phase, starting on Day 5 and lasting until Day 9. The ewes in Group B received a mixture of bFF/arachid oil (3 ml sc, 2:1) on Days 3, 4, 5, 10 and 12 of the estrous cycle. The ewes in Group C (Controls) were treated subcutanecusly with a mixture of steroid-free bovine plasma and arachid oil (2:1) on the same days as the ewes in Group B. Plasma concentrations of progesterone showed that the luteal function during the treatment cycle was normal in all treated and control ewes. The ovulation and lambing rates, however, were greater in Group A (2.5 +/- 0.2 and 1.9 +/- 0.3, respectively) and in Group B (2.1 +/- 0.2 and 1.6 +/- 0.1, respectively) than in Group C (1.5 +/- 0.2 and 1.2 +/- 0.3, respectively). Precipitating antibodies were detected in the plasma of Group B ewes only.     

Season influences FSH concentration in ovariectomized Ile-de-France ewes

Ile-de-France ewes were ovariectomized during anoestrus or the mid-luteal phase of an oestrous cycle (day of ovariectomy = Day 0). In a short-term study, FSH concentrations were measured in blood samples collected hourly the day before and on Days 1, 3, 7 and 15 after ovariectomy (10 ewes per group). FSH concentrations increased significantly from 6.1 to 16.5 ng/ml within 1 day of ovariectomy and increased further to 47.1 ng/ml by Day 15. Differences between seasons of ovariectomy were not significant. In a long-term study, FSH concentrations were measured in blood samples collected hourly on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy in anoestrus or the breeding season (10 ewes per group). Further samples were taken (5 ewes/group) at 240 and 365 days after ovariectomy. The pattern of change in FSH after ovariectomy differed between the two seasons and the interaction between season and sampling day was significant. For ewes ovariectomized during anoestrus, FSH concentrations increased to a maximum by Day 180 and remained high thereafter. In contrast FSH increased more slowly in ewes ovariectomized in the breeding season and differences between the groups were significant from Day 90 to Day 270. However, both groups had similar FSH concentrations at Day 365. These results show that FSH concentrations increase rapidly after ovariectomy. There are seasonal differences in FSH concentrations in the absence of ovarian feedback with increases in FSH concentration around the time of the onset of the breeding season. Once FSH concentrations had reached a maximum, major seasonal changes were no longer apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive season affects inhibitory effects from large follicles on the response to superovulatory FSH treatments in ewes

The main objective of this study was to compare the effect of the presence of large follicles at the start of FSH treatment on the superovulatory response in ewes in the breeding and nonbreeding seasons. A second objective was to verify the effect on the superovulatory response of the presence of a corpus luteum at the start of the FSH treatment during the breeding season. Fifteen ewes in breeding season (October) and 14 in nonbreeding season (May-June) were treated with 40 mg FGA sponges (Chronogest) for 14 days, together with a single dose of 125 microg cloprostenol on Day 12, considering Day 0 as day of progestagen insertion. Superovulatory treatments consisted of eight decreasing doses (1.5 ml x 3, 1.25 ml x 2 and 1 ml x 3) of Ovagen twice daily from 60 h before to 24h after sponge removal. Ovarian structures were assessed by transrectal ultrasonography using a 7.5 MHz linear array probe. Luteal activity at progestagen insertion (Day 0) and presence of corpus luteum and of large follicles at first FSH dose (Day 12) were determined. There were no significant differences between the breeding season and nonbreeding season for ovulation rate (11.6+/-1.4 versus 11.6+/-1.3), number of recovered embryos (8.0+/-1.1 versus 9.6+/-1.3) or number of viable embryos (7.2+/-1.1 versus 5.8+/-1.2). During the breeding season, there were fewer recovered embryos in ewes with a large follicle (> or =6mm) at first FSH dose (6.9+/-1.1 versus 12.3+/-1.8, P<0.05) and fewer viable embryos (5.0+/-1.2 versus 10.5+/-0.5, P<0.05) than in ewes without such a follicle. During the nonbreeding season, however, there were no significant differences between ewes with or without a large follicle for either recovered (9.0+/-2.5 versus 11.3+/-1.2) or viable embryos (6.3+/-2.3 versus 8.1+/-1.2). Analysis of seasonal differences in ewes with a large follicle showed a lower number of recovered embryos in the breeding season (P<0.05) due to a lower recovery rate (65.7% versus 92.3%, P<0.05), since mean number of corpora lutea in response to the FSH treatment was similar (10.9+/-1.3 versus 10.0+/-2.5). These results indicate that, in sheep, the inhibitory effects of large follicles during the nonbreeding season are not as obvious as during the breeding season.     

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.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Effects of recent sexual experience and melatonin treatment of rams on plasma testosterone concentration, sexual behaviour and ability to induce ovulation in seasonally anoestrous ewes

The aim of this study was to determine whether advancing the seasonal changes associated with rams by treatment with exogenous melatonin and allowing the rams previous sexual experience would increase the proportion of anoestrous ewes ovulating in early July. North Country Mule ewes (n = 225) were grouped by live body weight and body condition score and allocated randomly to the following treatments: (i) isolated from rams (control; n = 25); (ii) introduced to rams (treatment 2); (iii) introduced to rams that had mated with ewes during the previous 2 days (treatment 3); (iv) introduced to rams implanted with melatonin (treatment 4); and (v) introduced to rams that were implanted with melatonin and had mated with ewes during the previous 2 days (treatment 5). Treatments 2-5 were replicated (2 x 25 ewes) and two rams were introduced to each replicate group. Introductions began on 4 July and were completed by 11 July. The rams were withdrawn from the ewes after 8 days. Melatonin was administered as a subcutaneous implant (Regulin((R))) on 22 May and again on 20 June. Blood samples were taken from all rams to determine plasma melatonin and testosterone concentrations (19 samples in 6 h). The behaviour of the sheep was videotaped continuously during the first 3 h after the ram was introduced. Ovulation was detected by an increase in plasma progesterone concentrations from < 0.5 ng ml(-1) to > 0.5 ng ml(-1). Mean +/- SE plasma melatonin concentrations were 649.7 +/- 281.4 and 18.3 +/- 2.4 pg ml(-1) in rams with and without melatonin implants, respectively (P < 0.001). Melatonin implants also increased plasma testosterone concentrations from 4.30 +/- 1.88 to 10.10 +/- 1.10 ng ml(-1) (P < 0.01), the libido of the rams and the proportion of ewes that ovulated in response to the rams (43 and 56% (treatments 4 and 5) versus 24% (treatments 2 and 3)). In conclusion, implanting rams with melatonin before introducing them to seasonally anoestrous ewes increases the proportion of ewes that ovulate in response to introduction of a ram, but previous sexual experience of rams appears to have little or no effect.     

Oestrous cycles and the breeding season of the Père David's deer hind (Elaphurus davidianus)

Reproductive cycles were studied in a group of tame Père David's deer hinds. The non-pregnant hind is seasonally polyoestrous and, in animals studied over 2 years, the breeding season began in early August (2 August +/- 3.3 days; s.e.m., N = 9) and ended in mid-December (18 December +/- 5.7 days; N = 8) and early January (6 January +/- 3.2 days; N = 11) in consecutive years. During the anoestrous period, plasma progesterone concentrations were low (0.2 +/- 0.01 ng/ml) or non-detectable. There was a small, transient increase in progesterone values before the onset of the first cycle of the breeding season. In daily samples taken during an oestrous cycle in which hinds were mated by a marked vasectomized stag, progesterone concentrations remained low (less than 0.5 ng/ml) for a period of about 6 days around the time of oestrus, showed a significant increase above oestrous levels by Day 4 (Day 0 = day of oestrus) and then continued to increase for 18 +/- 2.8 days to reach mean maximum luteal levels of 3.5 +/- 0.6 ng/ml. The plasma progesterone profiles from a number of animals indicated that marking of the hinds by the vasectomized stag did not occur at each ovulation during the breeding season and therefore an estimate of the cycle length could not be determined by this method. In the following year, detection of oestrus in 5 hinds was based on behavioural observations made in the absence of the stag. A total of 19 oestrous cycles with a mean length of 19.5 +/- 0.6 days was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Does inadequate luteal function limit the establishment of pregnancy in the early post-partum ewe?

In Exp. 1 the effect of lactation versus early weaning on luteal function was examined in seasonally anoestrous Finn Dorset ewes that were induced to ovulate at 21 (N = 14) or 35 (N = 14) days post partum by using a CIDR device and PMSG. Prolactin concentrations were significantly higher (P less than 0.001) in lactating compared with early weaned ewes throughout the study. The proportion of lactating ewes with inadequate luteal function (as assessed by daily progesterone concentrations) in the 21-day group was 0.43 (3 or 7) compared with 0.67 (4 of 6) for those weaned within 2 days after parturition. Corresponding values for the 35-day group were 0 (0 of 4) and 0.14 (1 of 7) respectively. There was no evidence of abnormal luteal function in standard ewes (N = 8) for which the interval from parturition was greater than 150 days. In Exp. 2 we examined whether pregnancy can be successfully established during the breeding season following transfer of embryos into lactating or early weaned ewes in the early post-partum period. Embryos were donated from Border Leicester x Scottish Blackface ewes for which the interval from previous parturition was greater than 150 days. These embryos were transferred synchronously on Day 5 after behavioural oestrus to recipient ewes with the same breeding history as the donors (standard ewes, N = 15) or to lactating or early weaned recipients that had been induced to ovulate on Day 21 (N = 16) or 35 (N = 24) post partum. In the 21-day group inadequate luteal function was observed in 2 of 7 (0.28) lactating and 4 of 9 (0.44) early weaned ewes compared with corresponding values of 1 of 13 (0.08) and 2 of 11 (0.18) in the 35-day post-partum group. Luteal function was normal in all standard ewes. The proportion of successful pregnancies in the standard ewes was 0.80 (12 of 15) compared with 0 in lactating and early weaned ewes in the 21-day group and 0.08 (1 of 13) and 0.36 (4 of 11) respectively in the 35-day group. The incidence of inadequate luteal function is therefore independent of the suckling stimulus and is higher in ewes induced to ovulate on Day 21 than Day 35 post partum during breeding and non-breeding seasons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.     

Seasonal variations in prolactin, growth hormone and thyroid hormones and the prolactin surge at ovulation do not affect litter size of ewes during pregnancy in the oestrous or the anoestrous season

Injection of bromocriptine from 5 days before until 5 days after mating clearly suppressed the periovulatory prolactin surge in ewes in the anoestrous and oestrous season but did not change the litter size significantly. Progesterone, GH, TSH or thyroid hormone concentrations were not influenced by the bromocriptine treatment. The progesterone concentrations were lower during the first weeks after mating in the anoestrous season compared to the oestrous season, while there was no difference between pregnant and non-pregnant ewes. During later gestation this seasonal difference was only observed in the non-pregnant ewes. At the same time there was a clear difference between pregnancy and non-pregnancy in both seasons. The prolactin, GH and thyroid hormone values also varied significantly during gestation. Since these patterns are identical in pregnant and non-pregnant ewes, the fluctuations are due to environmental factors and not to pregnancy or altered progesterone concentrations. In the anoestrous season prolactin, GH, T4 and T3 levels were higher than in the breeding season, while rT3 showed the opposite pattern. The TSH concentration did not differ between the two seasons. These results suggest that seasonal variations in prolactin, GH and thyroid hormones or the periovulatory prolactin surge do not affect litter size of ewes during pregnancy in the oestrous or the anoestrous season.     

Effect of recombinant alpha interferons on fertility and interestrous interval in sheep

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 mug of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 mug recombinant human interferon alpha1 (rhlFN); or 3) 1500 ug of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 mug/conceptus) than from rblFN-treated (56 +/- 12 mug/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 mug/conceptus) and rblFN-treated (147 +/- 17 mug/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.     

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 ± 0.64 vs. 7.27 ± 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients. Mol. Reprod. Dev. 47:164–169, 1997. © 1997 Wiley-Liss, Inc.     

Intrauterine infusion of ovine conceptus secretory proteins reduces prostaglandin F2α release without affecting endometrial oxytocin receptor concentrations

Chronically ovariectomized ewes were pretreated with progesterone and oestradiol to induce oestrus and randomly allocated into four treatment groups. Progesterone injections were given to Groups 1 and 2 on Days 1–12 and Groups 3 and 4 on Days 1–15. Ewes in Groups 2 and 4 were infused with conceptus secretory proteins (oCSP), via an intrauterine catheter, twice daily on Days 13–15. Ewes in Groups 1 and 3 were similarly infused, but with serum proteins (oSP). Endometrial oxytocin receptor (OTr) concentrations and oxytocin-induced 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) release were measured on Day 16.Progesterone concentrations in ewes receiving 12 days of progesterone treatment declined after Day 12, reaching a nadir on Day 14. In contrast, plasma progesterone concentrations remained elevated until Day 16 in ewes receiving the extended progesterone treatment. On Day 16, endometrial OTr concentrations were significantly higher in ewes given 12 days of progesterone treatment than in ewes given 15 days of progesterone irrespective of the presence of oCSP or oSP. Treatment with oCSP significantly decreased oxytocin-induced PGFM release in ewes given 12 days of progesterone treatment compared with those ewes receiving oSP infusions. The extended 15 day progesterone treatment resulted in a further decrease in oxytocin-induced PGFM release in both oCSP and oSP infused ewes.These data indicate that, in steroid treated ovariectomized ewes, intrauterine infusion of oCSP will reduce oxytocin-induced PGFM response but not OTr concentrations. Progesterone appears to play a dominant role in the regulation of OTr as well as oxytocin-induced PGFM release.