Reproductive response and LH secretion in ewes treated with melatonin implants and induced to ovulate with the ram effect

Two experiments were conducted to examine the effects of treating seasonally anoestrous ewes with melatonin before ram introduction on reproductive response, and on LH secretion in anoestrous ewes induced to ovulate by rams.In Experiment 1, a total of 667 ewes from three flocks involving Merino (Flock 1, N = 149), Merino entrefino (Flock 2, N = 325) and Rasa Aragonesa (Flock 3, N = 203) breeds were used. Within each flock, ewes isolated from rams since the previous lambing were assigned at random to receive melatonin implants of Regulin (75, 175 and 105 in Merino, Merino entrefino and Rasa Aragonesa flocks, respectively) or to serve as untreated controls (74 in Merino, 150 in Merino entrefino and 98 in Rasa Aragonesa flocks). Fertile rams were introduced into all flocks 5 weeks after implantation in March (Flocks 1 and 2) or April (Flock 3), and remained with the ewes for a 50 day mating period. Percentage of ewes with luteal activity at ram introduction did not differ between melatonin treated and control ewes in any flock. There were no significant differences in either the mean interval from ram introduction to lambing or the distribution of lambing. Implantation with melatonin resulted in an improvement of prolificacy in all three flocks, although this only reached statistical significance in the Merino flock (1.15 vs. 1.03 in treated and control ewes, respectively, P < 0.05). Fertility was increased significantly (P < 0.05) in the Merino entrefino flock (64.5% in treated vs. 51.3% in control ewes).In Experiment 2, two trials were undertaken utilizing a total of 63 ewes. Trial 1 involved 24 mature Manchega ewes and Trial 2 involved 39 Merino ewe lambs. Half of the animals in each trial received a Regulin implant on 28 February (Trial 1) or 12 March (Trial 2) and the remaining half acted as controls. Rams were introduced 5 weeks after implantation and remained with the ewes for a 25 day period. In both trials, anoestrous ewes at ram introduction were bled at 20 min intervals for 3 h before and 5 h after ram introduction and then at 3 h intervals over the next 24 h for assessment of plasma concentrations of LH. Secretion of LH before or following introduction of rams was not affected by melatonin. Both treated and control anoestrous ewes in each trial responded to introduction of rams with an increase in the frequency of the LH pulses (P < 0.05), but no significant changes were detected in pulse amplitude or mean levels of LH. A preovulatory surge of LH was detected between 8 and 26 h after ram introduction, but neither mean interval from ram introduction to the peak of LH surge, nor the magnitude of the LH peak, was influenced by melatonin treatment.Results from this study show that: (1) melatonin implants administered during early seasonal anoestrus have the potential to improve reproductive performance in Spanish breeds of sheep, but the response is conditioned by breed, management system and environmental factors; (2) melatonin did not modify the secretion of LH in anoestrous ewes induced to ovulate by the ram effect under our experimental conditions.     

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.     

Steroid production and hCG binding by ram-induced ovarian follicles in seasonally anoestrous ewes

The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.     

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.     

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.     

Efficacy of intermittent or continuous administration of GnRH in inducing ovulation in early and late seasonal anoestrus in the Père David's deer hind (Elaphurus davidianus)

Père David's deer hinds were treated with GnRH, administered as intermittent i.v. injections (2.0 micrograms/injection at 2-h intervals) for 4 days, or as a continuous s.c. infusion (1.0 micrograms/h) for 14 days. These treatments were given early (February-March) and late (May-June) in the period of seasonal anoestrus. The administration of repeated injections of GnRH increased mean LH concentrations from pretreatment values of 0.54 +/- 0.09 to 2.10 +/- 0.25 ng/ml over the first 8 h of treatment in early anoestrus, and from 0.62 +/- 0.11 to 2.73 +/- 0.49 ng/ml in late anoestrus. The mean amplitude of GnRH-induced LH episodes was greater (P less than 0.01) in late (4.03 +/- 0.28 ng/ml) than in early (3.12 +/- 0.26 ng/ml) anoestrus, but within each replicate (early or late anoestrus), neither mean LH episode amplitude nor mean plasma LH concentrations differed significantly between the four periods of intensive blood sampling. On the basis of their progesterone profiles, 6/12 hinds had ovulated in response to treatment with injections of GnRH (1/6 in early anoestrus and 5/6 in late anoestrus), and oestrus and a preovulatory LH surge were recorded in all of these animals. Oestrus and a preovulatory LH surge were also recorded in one other animal treated in early anoestrus in which progesterone concentrations remained low. The mean times of onset of oestrus (91.0 +/- 1.00 and 62.4 +/- 0.98 h) and of the preovulatory LH surge (85.8 +/- 3.76 and 59.4 +/- 0.25 h) both occurred significantly earlier (P less than 0.001) in animals treated in late anoestrus. Continuous infusion of GnRH to seasonally anoestrous hinds resulted in an increase in mean plasma LH concentrations, but this response did not differ significantly between early (2.15 +/- 0.28 ng/ml) and late (2.48 +/- 0.26 ng/ml) anoestrus. Ovulation, based on progesterone profiles, occurred in 2/7 hinds in early anoestrus and in 4/6 hinds in late anoestrus. Oestrus was detected in all except one of these hinds. The mean time of onset of oestrus occurred earlier in animals treated in late anoestrus (66.2 +/- 0.32 h and 46.7 +/- 0.67 h, P less than 0.01). The administration of GnRH, given either intermittently or continuously, will induce ovulation in a proportion of seasonally anoestrous Père David's deer. However, more animals ovulate in response to this treatment in late than in early anoestrus (75% compared with 23%).     

Controlled sheep breeding: update 1980-1985

This contribution to the Symposium concerns four topics which have been addressed in our laboratory over the past five years. First, the responses to a controlled light environment of Merino ewes and rams have been compared with those of two British breeds. The endocrinological patterns were similar in all breeds but cyclic ovarian activity and ram libido were different. While showing a degree of entrainment to photoperiod, the breeding patterns were much less rigidly controlled in the Merinos than in the others. Second, the effectiveness of establishment of a cervical reservoir of spermatozoa, in ewes in which oestrus and ovulation have been controlled, has been re-examined. This is highly dependent on the time of insemination relative to that of the release of LH. Maximum numbers are found when ewes are inseminated shortly after the LH peak, i.e. some 6-10 h after the onset of oestrus. Third, the quantitative and temporal endocrinological and behavioural events following standard, progestagen-PMSG treatment have been quantified. Contrary to earlier expressed beliefs, these events are remarkably predictable provided an intensive system of mating or detection of oestrus is used. The onset of oestrus in treated anoestrous crossbred ewes has a normal distribution, with a range of 24 h, centred around a mean of 33 h after withdrawal of a 30 mg Cronolone intravaginal sponge and injection of 500 i.u. PMSG. This period of time is dose-dependent. The LH peak occurs 4.5 +/- 0.7 h later and the times of onset of oestrus and of LH release are highly correlated (r = 0.93). Ovulation is some 24 h later again. Fourth, differences in the response of ewes to different batches of PMSG have been defined. While the three commercial preparations studied regularly induced ovulation in anoestrous ewes at doses of 250 i.u. and above, the quantitative responses varied greatly. One preparation would not induce multiple ovulation, even at high doses. There are differences in steroidogenesis and in pregnancy rates, associated with dose of PMSG and the consequent ovulation rate: the ideal would be for every ewe to shed two or three ova. A higher ovulation rate is acceptable, as early embryonic mortality generally reduces the litter size. This is particularly important in deep anoestrus. However, this does not solve the problem of breeding in early lactation.     

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

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

Differences in the reproductive endocrine status of ewes in the early post-partum period and during seasonal anoestrus

More (P less than 0.05) post-partum acyclic ewes (8/9) showed evidence of pulsatile LH release than did seasonally anoestrous ewes (2/8). Mean plasma prolactin concentrations were higher (P less than 0.05) in the post-partum ewes than in the seasonally anoestrous ewes. Hypothalamic and pituitary cytosol oestrogen receptor number, median eminence GnRH content and pituitary LH, FSH and prolactin contents were similar in the two groups of ewes. The number of nuclear-bound oestrogen receptors was greater (P less than 0.01) in pituitaries from seasonally anoestrous ewes than in pituitaries from post-partum ewes. These data suggest that the basis of acyclicity in seasonally anoestrous ewes and in post-partum ewes is probably due to their inability to generate LH pulse frequencies similar to that in the follicular phase of the oestrous cycle. The higher LH pulse frequency in post-partum ewes, compared to that in seasonally anoestrous ewes, suggests that pregnancy may partly negate the reduction in LH secretion that is associated with photoperiod-induced acyclicity. The lower number of nuclear-bound oestrogen receptors in post-partum ewes suggests that there may be lower plasma oestrogen levels and less ovarian follicular growth than in non-suckling anoestrous ewes.     

The pattern of cervical penetration and the effect of topical treatment with prostaglandin and/or FSH and oxytocin on the depth of cervical penetration in the ewe during the peri-ovulatory period

Artificial insemination in sheep has two major limiting factors: the poor quality of frozen-thawed ram semen and the convoluted anatomy of the sheep cervix that does not allow transcervical passage of an inseminating catheter. It has been demonstrated that in the ewe during estrus, there is a degree of cervical relaxation mediated by ovarian and possibly gonadotrohic hormones, and we set out to investigate factors that might enhance cervical relaxation. Five experiments were conducted on ewes of different breeds to determine: 1) the pattern of cervical penetration during the periovulatory period in ewes of several breeds (Welsh Mountain, Île-de-France, Vendéenne, Romanov and Sarda); 2) the effect of the “ram effect” a socio-sexual stimulus, on cervical penetration; and 3) the effects of the intracervical administration of follicle-stimulating hormone (FSH), oxytocin and a prostaglandin E agonist (misoprostol) on the depth of cervical penetration during the periovulatory period. The results showed that during the periovulatory period in all breeds examined, there was increased penetration of the cervical canal (P < 0.05) by an inseminating catheter. Cervical penetration increased to a maximum 54 h after the removal of progestagen sponges and then gradually declined. Furthermore, the depth of cervical penetration but not its pattern, was affected (P < 0.05) by the breed of ewe. The maximum depth of cervical penetration was lower (P < 0.05) in the Vendéenne breed compared to the Île-de-France and Romanov breeds, which did not differ from one another. In the presence of rams, the depth of cervical penetration was increased at 48 and 54 h after removal of sponges (P < 0.05) and reduced at 72 h (P < 0.05). The local administration of hormones FSH, misoprostol (a PGE agonist) and oxytocin alone and in various combinations did not have any significant effect on the depth of cervical penetration during the periovulatory period. In conclusion, the natural relaxation of the cervix observed in ewes of several breeds occurs at a time during estrus, 54 h after the removal of progestagen sponges, which is the most suitable for artificial insemination. The effect was enhanced by the presence of a ram but not by the local intracervical administration of FSH, misoprostol and oxytocin even though oxytocin and PGE₂ are involved in cervical function. The time of maximum cervical penetration in the preovulatory period (54 h) coincides with high LH and estradiol concentrations suggesting they might be responsible for the relaxation of the cervix probably through an oxytocin-PGE mediated pathway.     

Response of seasonally anoestrous ewes to six-hour periods of GnRH infusion administered on six consecutive days

Twelve seasonally anoestrous Clun Forest ewes were infused i.v. with either 500 or 1000 ng GnRH/h for 6 h on each of six consecutive days in early or mid-anoestrus. Plasma LH concentrations were elevated during each GnRH infusion but returned to pretreatment levels when infusion ceased. The response to the first infusion was significantly greater (p<0.001) than that to subsequent infusions. In addition, both a GnRH dose and a seasonal influence were evident in the LH response, but these failed to reach statistical significance. Although 7 12 ewes ovulated, only two produced functionally normal corpora lutea.     

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.     

Effects of medroxyprogesterone acetate (MAP) on ovarian antral follicle development, gonadotrophin secretion and response to ovulation induction with gonadotrophin-releasing hormone (GnRH) in seasonally anoestrous ewes

When ovulation is induced with gonadotrophin-releasing hormone (GnRH) in anoestrous ewes, a proportion of animals fail to form normal (full-lifespan) corpora lutea (CL). Progesterone treatment before GnRH prevents luteal inadequacy. It remains uncertain whether a similar effect, achieved with medroxyprogesterone acetate (MAP) from intravaginal sponges, is mediated by influences on growing ovarian follicles and/or secretion of gonadotrophic hormones, before and after GnRH treatment. Two experiments were performed, on 13 and 11 anoestrous Western white-faced ewes, respectively. Seven and six ewes, respectively, received MAP-containing sponges (60 mg) for 14 days; the remaining ewes served as untreated controls. To test the effect of timing of GnRH administration after pre-treatment with MAP-releasing sponges, GnRH injections (250 ng every 2h for 24h followed by a bolus injection of 125 microg of GnRH i.v.) were given either immediately (Experiment 1) or 24h after sponge removal in the treated ewes (Experiment 2). Ovarian follicular dynamics (follicles reaching >or=5mm in size) and development of luteal structures were monitored using transrectal ultrasonography. In Experiment 1, the mean ovulation rate (0.7+/-0.3 and 1.0+/-0.4) and proportion of ovulating ewes (57 and 67%, respectively) did not vary (P>0.05) between MAP-treated and control ewes. Normal (full-lifespan) CL were detected in 29% of treated and 67% of control ewes (P>0.05). In Experiment 2, the mean ovulation rate (2.3+/-0.2 and 1.2+/-0.6; P<0.05) and percentage of ewes with normal (full-lifespan) CL (100 and 40%, respectively; P<0.10) were greater in the treated compared to control ewes. In Experiment 1, the mean peak concentration of the GnRH-induced LH surge was lower (P<0.05) in MAP-treated than in control ewes. There were no significant differences between MAP-treated and control ewes in the characteristics of follicular waves, mean daily serum FSH concentrations, and secretory parameters of LH/FSH, based on intensive blood sampling conducted 1 day before sponging and 1 day before sponge removal. It is concluded that treatment with MAP has no effect on the tonic secretion of LH/FSH or follicular wave development in anoestrous ewes. However, the GnRH-stimulated LH discharge was attenuated in the ewes that received MAP-impregnated sponges for 14 days and were treated with GnRH immediately after sponge withdrawal. Ovulatory response and CL formation were increased when GnRH was administered 24 h after sponge removal.     

Reproductive and endocrine effects of active immunization against a testosterone conjugate in the heifer

Yearling heifers were actively immunized against 8 mg testosterone-3-carboxymethyloxime-ovalbumin in Non-Ulcerative Freund's Adjuvant, with or without the addition of Corynebacterium parvum (Groups A and B, respectively; N = 4 for each group). After the priming injection, Groups A and B were boosted twice at 4-monthly intervals. Control heifers (N = 9) were not injected. All treated animals except one gave a measurable antibody response, and all responding animals became anoestrous and displayed ovarian cysts after the first booster injection. There were no apparent differences between treatments, and so results for Groups A and B were pooled. At 25 weeks after the second booster 3 of the 7 responding, anoestrous heifers resumed cyclicity; one with two consecutive double ovulations, and one with one double ovulation. The 3rd heifer showed 4 corpora lutea, then became anoestrus again. The 4 remaining acyclic heifers, and the control heifers, were intensively blood sampled; the anoestrous heifers showed significantly higher mean LH and significantly lower mean FSH concentrations and higher LH pulse frequency than did the control animals. These heifers remained anoestrous for 11 months after the second booster, at which point they were injected with GnRH and PGF-2 alpha; only 1 heifer resumed ovarian cyclicity. These results indicate that it is possible to increase ovarian activity in cattle by active immunization against testosterone, but that there is a high incidence of anoestrus.     

Use of gonadotropin releasing hormone to improve reproductive responses of ewes introduced to rams during seasonal anestrus

Three experiments were conducted on anestrous ewes of Suffolk, Dorset, and Katahdin breeding to examine the potential value of GnRH to improve ovulation and pregnancy in response to introduction of rams. In Experiment 1, treatment with GnRH 2 d after treatment with progesterone (P₄; 25 mg i.m.) at introduction of rams was compared to treatment with P₄ alone at the time of introduction of rams. Treatment with GnRH did not increase percentages of ewes with a corpus luteum (CL) 14 d after introduction of rams, pregnant 32 d after treatment with PGF₂α 14 d after introduction of rams, or percent of treated ewes lambing to all services. In Experiment 2, treatments with GnRH on day 2, 7, or both after introduction of rams were compared. Treatments did not differ in mean estrous response, percentages of ewes with a detectable CL or number of CL present on day 11, or mean pregnancy and lambing rates. Therefore, neither one nor two injections of GnRH at these times appeared to be effective to induce anestrous ewes to breed. In Experiment 3, treatments compared included GnRH 4 d before introduction of rams, GnRH 4 d before and 1 d after introduction of rams, ram introduction alone, and treatment with P₄ (25 mg i.m.) at the time of introduction of rams. Percentages of ewes with concentrations of P₄ greater than 1 ng/mL (indicating formation of CL had occurred) 7 d after ram introduction tended to be greater (P < 0.07) in ewes treated with GnRH or P₄ than in control ewes treated with ram introduction alone. However, there was no difference in P₄ concentrations between groups by day 11 or 12 after introduction of rams. Estrous response rates and percentages of ewes pregnant 95 d after PGF₂α was administered (on day 12 after introduction of rams) tended to be greater (P = 0.08 and 0.06, respectively) in ewes treated with GnRH or P₄ than in ewes exposed to rams only. There was no difference in response variables between ewes treated with GnRH 4 d before introduction of rams and ewes treated with GnRH 4 d before and 1 d after introduction of rams. In conclusion, treatment with GnRH 4 d before ram introduction showed promise as an alternative to treatment with P₄ to improve the ovulatory response and reproductive performance of ewes introduced to rams during seasonal anestrus.     

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

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

Ovulation rate in induced oestrous cycles of anoestrous ewes given bromocriptine

The suppression of prolactin in anoestrous ewes by daily injections of bromocriptine reduced the average number of corpora lutea on Day 8 after induced oestrus from 2.8 to 1.6 (P less than 0.02). There was a significant correlation (r = 0.87, P less than 0.05) between the preovulatory peak values for prolactin and ovulation rate in treated but not in control ewes. Bromocriptine failed to prevent the ewes from returning to anoestrus after the induced oestrus.     

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.     

Reproductive potential and endocrinological responses of sheep kept under controlled lighting. II. Pituitary and gonadal responses of ewes and rams to a six-monthly light cycle

Forty entire ewes of mixed breeds were kept in environmentally-controlled rooms with a 6-monthly light cycle. Six mature spayed Border Leicester × Merino ewes and four mature intact Poll Dorset rams were kept under the same conditions.Over a period of 2 years (four light cycles) estimates were made of ovarian, testicular and pituitary activity in response to the artificial light regime. Non-pregnant ewes were bled twice weekly: peripheral plasma progesterone levels > 1 ng/ml were taken as indicative of ovarian activity. Testicular activity was estimated by weekly tests for peripheral plasma testosterone and scrotal sac volume. Pituitary activity was estimated monthly by the response to the injection (i.v.) of 75 μg gonadotrophin releasing hormone (GnRH) to rams and of 18.75, 37.5 or 75 μg to ewes, using peripheral plasma luteinising hormone (LH) estimations from the time of GnRH injection.Data for ovarian and testicular measurements were classed into categories according to week and for pituitary response to month of the light cycle.Cubic regression analyses were conducted on the percentage of ewes with progesterone levels > 1 ng/ml and on ram testosterone and scrotal measurements.Despite the irregularity of the curve for the light cycle, the ovarian and testicular responses of rams and ewes followed an alternating curve with peaks and troughs of activity separated by approximately 13 weeks in the 26-week cycle. The peak of ovarian activity occurred during the long daylength period which followed a 22-week period of decreasing daylength and was preceded by 1 month by peak ram testosterone and scrotal sac volume.The pattern of pituitary response was related to that of the actual light cycle and the data were subjected to time-lag regression. This econometrical technique revealed that there was a delay in pituitary response to daylength changes of 2 months for rams, and between 2 and 3 months for the spayed ewes. The peak pituitary response to the GnRH test occurred one month earlier for rams than for the spayed ewes, and coincided with the corresponding troughs of gonadal activity of each sex.The results showed that the breeding season of sheep can be compressed into 6 months and that the pattern of pituitary response follows the daylength pattern more closely than do measurements of gonadal activity. Peak reproductive activity in rams, as measured by pituitary and gonadal activity, precedes that of ewes by approximately 1 month.     

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.