Effect of rumen degradable bolus containing melatonin or progestagen pessary plus pregnant mare serum gonadotropin on estrus response and lambing rate in ewes

Two practical regimens designed to induce estrus and ovulation in ewes in late anestrus were compared. Forty ewes were given a soluble glass rumen bolus containing 150 mg melatonin on July 9 and were joined with two vasectomized rams on July 23 and with three fertile rams on August 6. A second group of 40 ewes was treated with an intravaginal progestagen pessary (60mg medroxy-progesterone acetate) on July 23. Following pessary removal after 12 d, ewes were given 750 IU of pregnant mare serum gonadotropin (PMSG). Five fertile rams were joined with these ewes 48 h after progestagen removal. Melatonin concentrations were determined in single blood samples collected in early afternoon of July 21. Mating dates, lambing dates and litter sizes were recorded. Date of mating was significantly later in ewes treated with melatonin compared with those treated with progestagen plus PMSG (P<0.0001). All ewes given melatonin were mated within 4 wk, and those on progestagen plus PMSG treatment within one day of fertile ram introduction. Thirty-four ewes (85%) allocated to melatonin treatment and 36 (90%) allocated to progestagen plus PMSG treatment lambed (P>0.05). Mean (+/-SEM) lambing date was later in melatonin-treated ewes (January 17+/-1.2 d) compared to those given progestagen plus PMSG (December 30+/-0.6 d; P<0.0001). Mean litter size was lower in melatonin-treated ewes (1.5+/-0.1) compared with those given progestagen plus PMSG (2.0+/-0.1; P<0.001). Plasma melatonin concentrations indicated that 9 of 40 ewes treated with melatonin had circulating melatonin concentrations of less than 16 pg/ml. It is concluded that under conditions that existed in this experiment, treatment with progestagen plus PMSG in late anestrus resulted in more synchronous mating and lambing and a higher litter size than that following administration of a soluble glass rumen-degradable bolus containing melatonin.     

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.     

The effect of melatonin implants on the response to the male effect and on the subsequent cyclicity of Rasa Aragonesa ewes implanted in April

Rasa Aragonesa ewes were used to evalutate whether treatment with melatonin implants in spring could modify: (i) the response to the male effect in terms of oestrous behaviour and ovulation rate; and (ii) the maintenance of sexual activity and ovulation rate at medium term, i.e. over the next 306 days. On 12 April, 42 ewes were divided into two groups, with (M; n = 21) or without (C; n = 21) a subcutaneous implant containing 18 mg melatonin. On 17 May (day 0), three aproned rams were introduced to each group to induce a ram effect. Ewes were observed for oestrus daily. The rams were removed 40 days later after which one aproned ram was introduced daily. Oestrous detection continued until 28 February, 306 days after the first male-female contact. The ovulation rate was determined by endoscopy in the first three cycles after ram introduction and in September-October and January. Progesterone was assayed from blood samples taken on 6 May, 10 and from day 0 to day 22 after ram introduction. Luteal activity before ram introduction was seen in 33% (M) and 29 (C)% of the ewes, respectively. Significantly more M ewes showed oestrous behaviour during the first 40 days after ram introduction (M: 100%; C: 62%; P < 0.01). Similar differences were observed for ewes anovulatory at ram introduction (M: 100%, C: 47%; P < 0.01). These differences were maintained over the three oestrous cycles in both groups. Treatment with melatonin implants was without detrimental effect on cyclic functions in the following breeding season, after seasonal anoestrus. Melatonin treatment significantly increased (P < 0.05) the mean ovulation rate of the first (1.62 +/- 0.11 versus 1.31 +/- 0.13), second (1.78 +/- 0.15 versus 1.36 +/- 0.15) and third cycles (M: 1.73 +/- 0.12 versus C: 1.27 +/- 0.14). There was a significant interaction between the effects of cyclicity at day 0 and melatonin treatment on the ovulation rate in the first cycle (P < 0.05). The mean ovulation rates of both groups were similar at the beginning (September) and middle (January) of the subsequent breeding season. Overall, the results confirmed that melatonin implants, combined with the ram effect, improved the reproductive parameters of reduced-seasonality ewes during spring mating, without impairing sexual activity or ovulation rate during the subsequent breeding season.     

Diurnal variation in the response of anoestrous ewes to the ram effect

The re-introduction of rams after a period of separation was used to stimulate LH secretion and induce ovulation in seasonally anovulatory ewes maintained under natural photoperiod. In 2 experiments, the rams were introduced in the morning or the evening to test for diurnal variations in responsiveness to the treatment. In the first experiment, with Romanov ewes, the ram-induced increase in tonic LH secretion was significantly earlier in the ewes treated (N = 6) at 07:30 h (mean +/- s.e.m. delay to first pulse: 20 +/- 6 min) than in those (N = 5) treated at 19:30 h (66 +/- 15 min; P = 0.006). The pulse interval after the ram effect was significantly shorter in ewes that subsequently ovulated (120 +/- 10 min) than in ewes that did not ovulate (288 +/- 108 min; P = 0.043). There was a significant decline in pulse amplitude from 6.7 +/- 1.2 to 3.4 +/- 0.6 ng/ml (both groups combined) after the introduction of rams (P = 0.040). Of the 11 ewes, 7 subsequently ovulated and a preovulatory LH surge was observed in 6 of these 30-36 h after ram introduction. In the second experiment, with seasonally anoestrous Préalpes-du-Sud ewes, the effect of the timing of the introduction of rams on the periovulatory events was tested. The delay to the onsets of oestrus and the LH surge was not affected, but the ovulation rate was higher after ram introduction in the morning (1.42) than in the evening (1.14). In the 12-h period before the introduction of the rams in the first experiment, there was a difference between the groups in the secretion of LH, but the existence of diurnal rhythms in the concentrations of LH or FSH were not confirmed in a later study in which 7 ewes were sampled every 20 min for 36 h. In contrast, there was a distinct diurnal variation in the secretion of prolactin, with the highest values being recorded at night and the lowest around midday (P = 0.025). The rise and fall in prolactin values did not appear to coincide with dawn or dusk.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of early and late exposure to estrual ewes on ram sexual performance classifications

This study was conducted to determine whether exposure of ram lambs to estrual ewes during their first autumn and again as adults just before serving capacity tests (SCT) affected the outcome of the sexual performance tests. Treatments were either early exposure of Polypay ram lambs (i.e., 7-8-mo-old rams with ewes for 17 d [n=30] or no early exposure [n=30]), and late exposure (i.e., 16-19-mo-old rams with estrual ewes for 3 d) or no exposure to estrual ewes in a 2x2 factorial arrangement. Three serving capacity tests were conducted immediately after the early exposure period for individual ram lambs that were exposed to ewes early. Three sham sexual performance tests (i.e., four ram lambs placed in test pens for 30-min without ewes) were conducted with ram lambs that were not exposed to ewes early. All rams were evaluated during nine 30-min serving capacity tests over a 2-mo period at 16-19 mo of age to determine sexual performance. Prior to serving capacity tests, one half of the rams from each early exposure treatment were exposed to estrual-induced ewes for 3 d. Specific sexual behaviors (e.g., sniffs, flehmens, foreleg kicks, vocalizations, mount attempts, mounts, and ejaculations) were recorded during serving capacity tests. Number of sniffs, flehmens, foreleg kicks, vocalizations, and mount attempts were summed without estimating the value of importance and analyzed as courtship behaviors. Sexual performance data were analyzed with Mixed model procedures for repeated measures. During serving capacity tests, the early exposed rams exhibited more courtships (40.3+/-8.0 versus 23.4+/-4.6; P<0.05; LSM+/-estimated SE), mounts (11.3+/-1.0 versus 7.7+/-0.9; P<0.01), and ejaculations (3.3+/-0.2 or 2.4+/-0.2; P<0.01) than rams not exposed to ewes as ram lambs, respectively. We conclude that early exposure of 7-8-mo-old ram lambs to estrual ewes improves sexual performance in serving capacity tests at 16-19 mo of age in most rams whereas, late exposure to estrual ewes for 3 d prior to serving capacity tests did not improve sexual performance scores.     

Melanin concentrating hormone (MCH) in the cerebrospinal fluid of ewes during spontaneous oestrous cycles and ram effect induced follicular phases

The melanin-concentrating hormone (MCH) is a neuropeptide synthesized by neurons of the lateral hypothalamus and incerto-hypothalamic area that project throughout the central nervous system. The aims of the present report were: (1) to determine if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal and the oestrous phase of spontaneous oestrous cycles; and (2) to study if MCH levels in CSF of ewes vary acutely during the follicular phase induced with the ram effect in anoestrous ewes. In the first experiment, CSF was collected from 8 adult ewes during spontaneous oestrous and during the mid-luteal phase (8-10 days after natural oestrus). In the second experiment, performed during the mid non-breeding season, a follicular phase was induced with the ram effect. After isolating a group of 16 ewes from rams, CSF was obtained from 5 of such ewes (control group). Three rams were joined with the ewes, and samples were obtained 12h (n=6) and 24h (n=5) later. In Experiment 1, there were no differences in MCH concentrations in CSF measured during the mid-luteal phase and spontaneous oestrus (0.14 ± 0.04 vs. 0.16 ± 0.05 ng/mL respectively). In Experiment 2, MCH concentrations tended to increase 12h after rams introduction (0.15 ± 0.08 vs. 0.35 ± 0.21 ng/mL, P=0.08), and increased significantly 24h after rams introduction (0.37 ± 0.15 ng/mL, P=0.02). We concluded that MCH concentration measured in the CSF from ewes increased markedly during the response to the ram effect but not during the natural oestrous cycle of ewes.     

The influence of breeding intensity on above- and below-average sexual performance rams in single- and multiple-sire breeding environments

Two studies were conducted to evaluate the relationship between serving capacity scores and breeding performance of rams. The first study was conducted to determine whether rams with above or below mean serving capacity scores could perform equally in greater and lesser breeding intensity, single-sire mating schemes. The second study was conducted to determine whether rams with above and below mean serving capacity scores could perform equally well when only one or two ewes were in estrus daily in a multiple-sire breeding scheme (two rams/pen). Rams (n=68) were ranked according to average number of ejaculations recorded in serving capacity tests. Sixteen rams with the greatest scores (above-average) and 16 rams with least scores (below-average) were identified for breeding. Half of above-average and half of below-average rams were used in the two studies. For study 1, each ram was individually introduced to 23 estrus-synchronized ewes for 9 d to simulate high breeding intensity. Rams were given a 5-d rest before they were individually introduced to 23-24 naturally cyclic ewes for 17 d (low breeding intensity). For study 2, 16 rams were paired across ram types, and each pair competed for 20 ewes for 18 d (8 pens). For study 1, ewe fertility (ewes lambing/ewes present at lambing) and number of lambs born were greater (P<0.001) for above-average (0.67+/-0.03 and 27.6+/-1.2, respectively) than for below-average rams (0.39+/-0.07 and 15.3+/-2.7) with greater breeding intensity. Ewe fertility and lambs born did not differ for above-average (0.91+/-0.03 and 37.8+/-1.9, respectively) and below-average rams (0.86+/-0.03 and 39.0+/-1.9) with less breeding intensity. For study 2, number of ewes lambing (99+/-8.0 compared with 72+/-13.6; P=0.12) and number of lambs sired (149+/-18.5 compared with 101+/-22.8; P=0.14) did not differ between above- and below-average rams, respectively, in direct competition. Sexual classifications based on serving capacity tests are related to breeding performance of rams in certain breeding environments. When breeding intensity is greater, above-average rams impregnate more ewes and sire more lambs than below-average rams. When only a small number of ewes are in estrus daily, below-average rams for serving capacity scores perform as well as above-average rams in multiple-sire and single-sire breeding environments. We suggest that above-average rams should be used to reduce number of rams required when breeding intensity is greater.     

The relationship between ram breeding capacity and flock fertility

The effect of breeding capacity of rams on flock fertility was studied by exposing each of 15 rams to 1 of 15 flocks of 200 naturally-cycling ewes for 17 days. Five of the rams were arbitrarily designated as being of high breeding capacity (mean+/-SEM = 14.7+/-0.5 services in 2 3-hour pen breeding tests); five rams were designated as being of medium capacity (7.3 +/- 0.2); and the remaining five were designated as being of low breeding capacity (1.7 +/- 0.5). Breeding capacity was shown to be positively correlated with the number of services during flock mating, to the number of ewes mated and to insemination success. Breeding capacity as measured in a pen test of shorter duration (1 hour) was shown to be similarly related to flock fertility. Further, the number of services during flock mating was also positively correlated with the number of ewes mated and impregnated, as well as to the number of fetuses conceived and to insemination success. Breeding frequency of the ram is, therefore, closely related to flock fertility, but breeding capacity, as measured in the present study, is only a moderately accurate indicator of breeding frequency.     

Effects of shortened daylength and melatonin treatment on plasma prolactin and melatonin levels in pinealectomised and sham-operated ewes

Comparisons have been made between the effects of shortened daylength and melatonin treatment on plasma prolactin and melatonin levels in pinealectomised (Px) and sham-operated (Sh) ewes. Twenty-two anoestrous Merino crossbred ewes, maintained under normal grazing conditions, were assigned to four groups for a period of 9 weeks. Group 1 remained untreated (control), Group 2 was herded into a dark shed at 1600 h each day until dark (approx 4 h), ewes in Group 3 were injected with 100 μg melatonin s.c. at 1600 h each day and ewes in Group 4 were implanted with a melatonin capsule releasing 125–200 μg/day. Another group (Group 5) of 4 Px and 4 Sh ewes from the same flock was maintained in an animal house and subjected to shortened daylength (10. 5 h L : 13. 5 h D, lights off 1600 h). Three weeks after the treatments began, ewes in Groups 1–4 were exposed to a fertile ram and ewes in Group 5 to a vasectomised ram and the day of mating noted. No differences were evident between Groups 1–4 in the ewes' response to the ram, time taken to conceive, duration of gestation or number of lambs born. In untreated Px ewes no plasma melatonin (< 20 pg/ml) was found in either day or night samples, whereas intact animals showed the characteristic night-time rise. The silastic implants produced stable daytime blood levels of 90–120 pg/ml, whereas a single injection of 100 μg melatonin caused a transitory (2–3 h) rise. Shortened daylength (Group 2) or a single daily injection of melatonin (Group 3) lowered prolactin levels but only in ewes with an intact pineal gland, whereas melatonin implants (Group 4) caused a reduction in plasma prolactin in both Px and Sh sheep. The results indicate that light-induced alterations in prolactin production in sheep involve both the pineal gland and melatonin. Continuous melatonin release from implants caused changes in plasma prolactin levels similar to those seen following exposure to short days.     

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.     

Seasonal changes in LH secretion in normal ewes and ewes which grazed oestrogenic clover

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.     

The effects of ram exposure during progestagen oestrus synchronisation and time of ram introduction post progestagen withdrawal on fertility in ewes

Three experiments were undertaken to investigate the effect of a pre-mating ram exposure during progestagen synchronisation treatment on time of breeding, ovulation rate, embryo quality and fertility and any interaction with time of ram introduction for breeding post sponge withdrawal. Crossbred ewes in experiment 1a (n = 348), 1b (mule; n = 133) and 2 (n = 58) underwent a 12-14 days synchronisation protocol. Three days prior to sponge withdrawal ewes were divided into Control (ewes in continued isolation from rams) or +Ram (ram-exposed) groups. Rams were introduced to +Ram ewes and remained with ewes until sponge withdrawal. Ewes in experiments 1a and 2 received eCG at sponge withdrawal and were reintroduced to rams at either 36 or 48 h post sponge removal (PSR). In experiment 1b, ewes did not receive eCG and were reintroduced to rams at 24 h PSR. In experiments 1a and 1b time of breeding, date of lambing and litter size were recorded. In experiment 2, ewes were slaughtered 5 days post breeding, reproductive tracts flushed and corpora lutea, ova and embryos assessed. Fewer +Ram ewes were mated by 96 h PSR (P < 0.001) than Control ewes in experiment 1a but not when rams were introduced earlier in experiment 1b. In experiment 1a, ram introduction at 36 h PSR improved conception to first service compared to introduction at 48 h PSR (P < 0.01) in both +Ram and Control groups. In experiments 1a and 1b, +Ram ewes had reduced litter size caused by more single births (1a; P < 0.001, 1b; P < 0.01). In experiment 2, +Ram ewes had fewer corpora lutea than Control ewes (P < 0.001) but embryo quality was similar. However, more good embryos were produced when rams were introduced for breeding at 36 h compared to 48 h PSR (P < 0.001). We conclude that a pre-mating ram exposure during the synchronisation treatment reduced the number of ewes mated at and conceiving to the first service. This was partially overcome by introducing rams for breeding earlier (24 or 36 h compared to 48 h PSR) but the most dramatic decrease in fertility was due to a reduction in ovulation rate in the ram-exposed ewes.     

Effect of a melatonin implant on the circadian variation of plasma prolactin and rectal temperature in newborn sheep.

Plasma prolactin and rectal temperature show a circadian rhythm in newborn sheep raised under continuous light. Melatonin lowers the concentration of plasma prolactin but it is not known if it affects its circadian rhythm. To detect whether melatonin acts on the circadian system we studied the effect of a subcutaneous melatonin implant in the circadian rhythms of prolactin and rectal temperature in newborn lambs raised under continuous light. We placed catheters in the pedal artery and vein in 9 newborn lambs (2-5 days of age). A subcutaneous melatonin implant was placed in 4 of the lambs at 9-12 days of age. Blood samples and rectal temperature measurements were obtained hourly for a period of 24 h, 11-15 days after the implant, at 20-27 days of age. To avoid interferences of heparin in our melatonin assay, serum melatonin concentration was measured before and during the implant in three additional newborns. Prolactin and melatonin were measured by RIA. Melatonin concentrations were 52.8 +/- 45.9 pg/ml (day) and 315.5 +/- 77.0 pg/ml (night) before treatment (SEM, P less than 0.001), and increased to 594.1 +/- 54.5 pg/ml after placing the implant (there was no difference in melatonin concentration between day and night during the time that the implant was in place). Melatonin had no effect on rectal temperature or its rhythm, but decreased basal plasma prolactin concentration (control: 97.5 +/- 11.3 ng/ml; treated: 25.1 +/- 2.4 ng/ml, P less than 0.001) and abolished the prolactin circadian rhythm, (Cosinor analysis): control: log prolactin (ng/ml) = 1.8 + 0.26 cos 15 (t - 11.16), p = 0.05; treated: log prolactin (ng/ml) = 1.2 + 0.14 cos 15 (t - 9.43), P = 0.36.     

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.     

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.     

Response of anestrous ewes to the ram effect after follicular wave synchronization with a single dose of estradiol-17beta

Anestrous ewes respond to the introduction of rams with either an ovulation within 2-3 days that may be followed by luteal phases of normal or short length, with delayed ovulations (5-6 days later), or with the luteinization of follicles. The aim of this work was to study the relationship between the growth status of the largest follicle present when rams are introduced and the type of ovarian response in non-treated ewes and in ewes treated with estradiol-17beta before ram introduction. Thirteen anestrous Corriedale ewes were divided into 2 groups: E2 (n = 7) and C (n = 6). The E2 ewes received a single dose of 50 microg estradiol-17beta 5 days before the introduction of the rams to synchronize the onset of their follicle waves, while C ewes remained untreated. When the rams were introduced, all E2 ewes had the largest follicle in a growing stage in contrast with the C ewes (3 out of 6; P < 0.05). Five C and 4 E2 ewes ovulated after the introduction of the rams (Day 3.4 +/- 0.4 for C vs. 4.8 +/- 0.3 for E2 ewes, respectively, P < 0.05). Only one ewe from each group developed a normal luteal phase: 4 C and 3 E2 ewes had short luteal phases. One C ewe and 2 E2 ewes had short luteal phases originating from follicles that did not ovulate. After the first luteal phase, all ewes returned to anesirus without a second ovulation or luteal phase. The remaining E2 ewe did not ovulate or show any changes in progesterone serum concentrations. We conclude that the growth status of the largest follicle alone does not determine the ovarian responding pattern of anestrous ewes to the ram effect.     

Pulsatile release of oxytocin into the circulation of the ewe during oestrus, mating and the early luteal phase

Two experiments were designed to investigate release patterns of oxytocin into plasma during oestrus and the early luteal phase. In Exp. 1, blood samples were collected from 5 ewes every 30 min for 10 h during 6 days around oestrus and the early luteal phase. During oestrus concentrations of oxytocin were generally low (1.27 +/- 0.54 pg/ml; mean +/- s.d.) but with occasional pulses up to 6 pg/ml. By Day 5 mean basal concentrations had risen to 4.5 +/- 2.1 pg/ml with a fluctuating release pattern. In Exp. 2, a method was developed for continuous blood sampling from conscious, unrestrained ewes. On the predicted day of oestrus following an untreated oestrous cycle, 8-ml blood samples were collected every minute for two 35-min periods (8 ewes: 16 sampling periods). For 6 ewes a ram was introduced to the pen for part of this time, and resulting behaviour was recorded. Additional blood samples were assayed for LH and progesterone to determine the stage of the cycle. Overall mean oxytocin concentrations ranged from 1.5 +/- 0.53 to 6.8 +/- 5.25 pg/ml in different animals. Ewes which were both in oestrus and exposed to the ram showed a pulsatile oxytocin release pattern consisting of low baseline concentrations with short-duration pulses superimposed (duration 1-4 min; amplitude 2.5-31.7 pg/ml; frequency 3.18/h). Coitus was not temporally associated with pulsatile release. However, the importance of the presence of the ram was indicated by total separation of 2 oestrous ewes from the ram until after experimentation. In these animals only 1 pulse of oxytocin was detected in 2.7 h of sampling. It is concluded that, although mean oxytocin concentrations at oestrus were low, short duration pulses were released into the plasma at this time. This effect may be dependent on the presence of a ram.     

Effect of GnRH administration, combined with the ram effect, on the occurrence of ovulation and pregnancy during the transition period from anoestrus in crossbred ewes

The effect of a GnRH analogue (buserelin) combined with the ram effect on the reproductive efficiency of ewes was investigated in 105 cross-bred fat tailed ewes, during the transition period from anoestrus to the natural breeding season. Plasma progesterone concentration was used in the assessment with regard to ovulation and pregnancy. Ewes were maintained on natural pastures composed of medium to low quality forages, and received supplementation (40% alfalfa hay: 60% wheat straw) ad libitum, plus 100–300 g barley grain per head per day. Ewes were isolated from the rams for at least two months and then kept in close proximity of the rams for one week, before the introduction of the rams. The ewes were randomly divided into three equal groups (n = 35 per group). With the introduction of the rams into the flock (day 1) one group was considered as the control and the other two groups were treated with 4.2 μg (low dose) and 8.4 μg (high dose) buserelin on days 5 and 19, respectively. Blood samples were collected on days 5, 12, 26 and 120 after ram introduction for determining the plasma progesterone levels. On day 12 (one week after treatment) the high dose GnRH group recorded significantly lower plasma P4 concentrations (P < 0.05), compared with the control group (1.0 ± 0.1 ng/ml versus 2.4 ± 0.4 ng/ml). On the same day the low GnRH dose group recorded intermediate P4 concentrations, recording no significant differences with the other two groups. The high dose group recorded a significantly (P < 0.05) higher proportion of non-ovulated ewes (61.8%), compared to the control (32.3%) and low dose (31.4%) groups on day 12 of the study. At days 5 and 26 these differences were not significant, but the proportion of non-ovulated ewes was higher in the high dose buserelin treatment group. The percentage of pregnant (plasma P4 > 2.5 ng/ml) and non-pregnant (plasma P4 ≤ 2.5 ng/ml) ewes at day 120 of the study was not statistically different between the treatment groups. The pregnancy rate was highest in the control group (97.1%), when compared to the treated ewes (94.3% and 88.6% in low dose and high dose treatment groups, respectively). Treatment with buserelin combined with the male effect during the breeding season negatively affected the plasma P₄ concentration, reducing the reproductive performance of the ewe treatment groups.     

Effect of breed and age on sexual behaviour of rams

The objective of this study was to highlight the problems that arise during the reproduction between thin-tailed rams and fat-tailed ewes. At the same time, particular emphasis laid on the influence of sheep breed, sheep age, time after ram introduction and day of the ewe estrus cycle on ram and ewe sexual behaviour. Rams were subjected to sexual performance tests by being individually exposed to 12 ewes for 3 h daily, 19 consecutive days. The 16 rams of the experiment were separated according to their age (9 and 21 months old) and breed (Chios and Karagouniki), and the 96 ewes of Chios fat-tailed breed, were divided by age (9 and 21 months old). The main characteristics of courtship behaviour, like sniffing, nudging, flehmen response and following were recorded and studied in detail. Mature Chios rams, which were the only one with previous experience of Chios ewes, exhibited higher rates of sexual interest per ewe than the other rams (P < 0.05). On the other hand, rams sniffed and nudged more young than mature ewes (P < 0.05), probably due to the fact that young ewes did not express intense symptoms of estrus. Young rams exhibited substandard sexual interest towards mature ewes, when they first came in contact with them (P < 0.05). In general, Karagouniki thin-tailed rams exhibited reduced rates of mating behaviour when they courted with Chios fat-tailed ewes in comparison with Chios rams (P < 0.05). Moreover, as the time after ram introduction passed, the frequency and duration of sexual behaviour components decreased (P < 0.001). Finally, the effect of the day of the experiment was only significant in the case of sniffing, which increased during the first 2 days and then declined and stabilized (P < 0.01). As it was demonstrated, ram age and ram breed played a fundamental role in the exhibition of sexual interest elements.     

Developmental programming in sheep: administration of testosterone during 60-90 days of pregnancy reduces breeding success and pregnancy outcome

Evidence suggests that exposure to excess steroids during critical periods of fetal development leads to reproductive disorders. Exposure of female lambs to excess testosterone (T) from Days 60 to 90 of gestation (T60-90; term, 147 days) delayed onset of the LH surge and resulted in to male-typical reproductive behavior. The objectives of this study were to test the ability of T60-90 ewes to mate, conceive and lamb during the first three breeding seasons (Years 1, 2 and 3). Pregnant Suffolk ewes were injected with T propionate in cottonseed oil (100mg, im twice weekly) or vehicle (control; C) from Days 60 to 90 of gestation. In Year 1, ewes (C=12, T60-90=12) were kept with a vasectomized ram for 3 months and markings/visual observation of copulations were recorded. Rams had paint applied to their chest to facilitate detection of estrus and mating. All C but only three T60-90 ewes were marked (P<0.001). All ewes were then estrus-synchronized with two injections of prostaglandin F2alpha (20mg, im) given 11 days apart and allowed to mate with a painted, fertile ram. Nine of 12 C and 4 of 12 T60-90 ewes (P=0.1) were mated. Based on estrus and long-term monitoring of progesterone, more C than T60-90 became pregnant (82 and 18%, respectively; P<0.01). In Year 2, to maximize ram exposure, two C and two T60-90 estrus-synchronized ewes were placed with a painted, fertile ram at a time and mated ewes were removed to a nearby pen to force mating with others. Twenty-four hour video monitoring revealed the rams mated more C than T60-90 ewes (83 and 25%, respectively; P=0.01). In both Years 1 and 2, the rams preferred C over T60-90 ewes; therefore in Year 3 rams were given access only to T60-90 ewes. Only four T60-90 estrus-synchronized ewes were placed with a painted ram at a time. Not given an option, 91% of the T60-90 ewes were marked resulting in 4 of 11 (36%; first-service pregnancy rate in the breeding herd was 91%) ewes becoming pregnant to the synchronized estrus. Collectively these studies showed that fertility in T60-90 females was severely compromised, even after overcoming ram preference for controls.