Effects of duration and timing of progesterone priming on the incidence of corpora lutea with a normal life-span in Merino ewes induced to ovulate by the introduction of rams

Three experiments investigated the effects of progesterone on the incidence of corpora lutea with normal life-span in seasonally anovulatory Merino ewes induced to ovulate by the introduction of rams.Ewes in Experiments 1 and 2 received a single i.m. injection of 20 mg progesterone in oil at the time of, or up to 5 days before introduction of rams. Control ewes received oil alone. Progesterone priming had no effect on the proportion of ewes induced to ovulate, and the beneficial effect of progesterone on the proportion of corpora lutea with a normal life-span (P < 0.001, Experiment 1; P < 0.01, Experiment 2) was independent of its time of administration. Injection of progesterone delayed the time of the surge of LH compared to control ewes (P < 0.001), but only when given at the time of introduction of rams.Administration of progesterone by intravaginal devices for 6 h, 12 h or 24 h before introduction of rams (Experiment 3) increased the incidence of corpora lutea with normal luteal life-span compared to control ewes, but had no effect on the proportion of ewes ovulating. The incidence of corpora lutea with a normal life-span was significantly and positively correlated (R=0.977; P < 0.05) with duration of progesterone priming before introduction of rams.These results indicate that duration but not timing of progesterone priming is critical to its ability to eliminate short life-span corpora lutea. Such findings support the concept of a mode of action of progesterone at the ovarian level.     

Effects of the presence of rams during pregnancy on lambing performance in ewes

Ewes of the Préalpes-du-Sud breed (n=112) were mated with fertile rams and were used to investigate the effect of the presence of vasectomized rams during pregnancy on reproductive outcomes. Ewes in the control group (n=56) were isolated from rams during the whole period of pregnancy, whereas those in the experimental group (n=56) were kept with vasectomized rams from day 10 post-mating until lambing. At day 10 post-mating, a series of blood samples was collected every 15 min for 8 h from five control ewes and five experimental ewes to determine their patterns of LH secretion. The introduction of the ram was associated with a rapid increase of pulsatile LH release. The lag between the introduction of the ram and the onset of the first episodic LH release was less than 15 min. The mean(±sem) number of LH pulses/4 h after the introduction of the ram (2.8±0.4) was significantly higher (P<0.01) than that observed/4 h before the introduction of the ram (1.4±0.2). Although more ewes were pregnant in the control group (87.5%) than in the ram-exposed group (82.1%), the difference was not significant. The presence of rams did not affect gestation length (145.8 days), overall lamb mortality (3.5%) or birth weights of single (3.96 kg), twin (3.24 kg) or triplet (2.59 kg) lambs. The proportion of ewes with multiple births in the control group (69.4%) was significantly greater (P<0.05) than that in the ram-exposed ewes (47.8%). The ewes in the control group had significantly more (P<0.01) twin lambs born alive (72.3%) than the ewes in the ram-exposed group (50.0%). In conclusion, the presence of vasectomized rams during early pregnancy affected the incidence of multiple births but did not affect pregnancy rate or gestation length. The altered fertility of ewes exposed to vasectomized rams may reflect changes in embryonic loss during early pregnancy.     

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

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

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.     

Effect of the introduction of rams during the anoestrous season on the pulsatile secretion of LH in ovariectomized ewes

Introduction of rams to ovariectomized ewes treated with oestradiol implants (N = 10) increased the frequency of LH pulses from 4 X 8 to 10 X 6 pulses per 12 h. This effect was reflected by increases in mean levels of LH and the basal levels upon which the pulses were superimposed. In ewes that had not been treated with oestradiol (N = 5), there was no significant increase in pulse frequency but mean and basal levels of LH increased slightly after the introduction of rams. In a second experiment, similar effects of the introduction of rams were seen in ovariectomized ewes treated with oestradiol or oestradiol + androstenedione (N = 16), but no significant effects of the rams were observed in untreated ewes (N = 8) or ewes treated only with androstenedione (N = 7). No preovulatory surges of LH were observed in the 30-h period after the introduction of rams. It was concluded that the ram stimulus probably evokes the increase in pulse frequency by inhibiting the negative feedback action of oestradiol, and that the surge normally observed in entire ewes is dependent on the ovarian response to these pulses. However, the observation of responses in some ewes not treated with oestradiol also raises the possibility that the ram stimulus can act directly on the hypothalamic neurones that control the secretion of LH, and that this effect is enhanced in the presence of oestrogen.     

Effects of antibodies to progesterone on reproduction of ewes

Two experiments were conducted to determine the effects of immunization of ewes with progesterone-11alpha hemisuccinate coupled to bovine serum albumin (P-BSA) on estrous cycles, serum progesterone and fertility. In experiment I, ewes were immunized during the first estrous cycle in September and observed through January. Immunization against progesterone increased (P<.01) the proportion of estrous cycles of abnormal length. Two general patterns were evident in the ten ewes which were immunized against progesterone: 4 continued to show cyclic patterns of estrous activity throughout the experimental period and 6 entered periods of anestrus characterized by presence of corpora lutea. Apparent, aberrant, estrous activity and shortened luteal phases were also observed in ewes which were immunized against progesterone. In experiment II, immunization against progesterone caused serum progesterone concentrations to be 4 to 8 times higher (P<.01) than ewes which were immunized against bovine serum albumin. Fertility was reduced (P<.01) by immunization with P-BSA. In experiment II, immunization against progesterone shortened (P<.01) the second estrous cycle post-immunization, and at day 13 of the third cycle corpora lutea in P-BSA-immunized ewes were regressing and were lighter (P<.05) than in ewes which were immunized with bovine serum albumin.     

The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season

Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n=3) [corrected] and late luteal (LL; n=6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentration [corrected] of LH (at least P<0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P<0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P<0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle.     

Increased plasma LH levels in seasonally anovular merino ewes following the introduction of rams

Seven seasonally anovular Merino ewes, isolated from rams, were bled every 10 min for 12 h. Rams were introduced the following day, after a further 2 h of sampling every 10 min, and samples were taken for the following 10 h. Three days after the introduction of rams, the ewes underwent laparoscopy, and four had ovulated.Radioimmunoassay of plasma samples revealed pulsatile rhythms in the concentration of LH, with the frequency of pulses varying between ewes, from about once hourly to once every 10 h. Upon the introduction of rams, LH secretion increased within 10 min in six ewes, of which four ovulated within 3 days. There is apparently a relationship between the pattern of LH secretion after introducing the rams, and the ovulatory response that can be expected.     

Stimulation of seasonally anovular merino ewes by rams. I. Time from introduction of the rams to the preovulatory LH surge and ovulation

Two experiments were performed on seasonally anovular Merino ewes to determine the intervals between time of introduction of rams, the preovulatory surge of LH and the first ovulation. Ovulation was determined by laparoscopy and LH was measured by solid phase radioimmunoassay. In Experiment 1 the interval between the introduction of rams and the beginning of the LH surge was 27 ± 4 h (mean ± S.E., range 6–52 h), and in Experiment 2 probit analysis shows that 50% of teased ewes ovulated within 41 h of being exposed to rams.     

Effects of heat stress on serum progesterone in cyclic ewes and on progesterone and cortisol response to ACTH in ovariectomized ewes

The daily mean of serum progesterone in cyclic ewes (N = 5) as well as the profile characteristics of progesterone and cortisol in response to an acute single dose (5 i.u./kg liveweight 0.75) of adrenocorticotrophic hormone (ACTH) into ovariectomized ewes (N = 4) was investigated during exposure to a constant thermoneutral temperature of 18 +/- 1 degree C or to a daily cyclic heat stress temperature of 18 degrees C-35 degrees C-18 degrees C, in an environmental chamber. Serum collected daily from the cyclic ewes was assayed for progesterone, while serum collected more frequently for 10 h, on the 14th day of exposure to the respective temperature, from the ovariectomized ewes was assayed for progesterone and cortisol by RIAs. In cyclic ewes, heat stress increased the area under the daily progesterone curve (P less than 0.09) but had no effect on progesterone concentration after the regression of the CL. In ovariectomized ewes, ACTH significantly elevated the response of both cortisol and progesterone (r = 0.75, P less than 0.001) within 10-15 min of injection. In the ovariectomized ewes and during heat stress, the responses of progesterone and cortisol to ACTH were characterized by an initial acute rise, a transient drop, a steep elevation and a gradual but prolonged decline. During thermoneutral temperatures, this biphasic response pattern was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

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

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

LH response of seasonally anovular Corriedale ewes acutely exposed to rams and estrous ewes

Serial blood sampling before and after exposing four anovular Corriedale ewes to a group of rams and estrous ewes during the non-breeding season revealed a pattern of LH secretion similar to that previously observed in Merinos. Mean LH values doubled (P<0.001) from 0.24+/-0.06 microgL(-1) (mean+/-s.e.m.) before to 0.55+/-0.05 microgL(-1) after 2h of visual, auditory, and odor exposure to rams and estrous ewes in an indoor facility. A non-significant (P<0.17) increase of LH pulses per hour was also observed (0.7+/-0.3 pulses per hour before compared with 1.3+/-0.3 during stimulation). All four ewes had recently formed corpora lutea by five days after stimulation. Results are consistent with the pattern of sudden increase and sustained release of LH observed in other sheep breeds, particularly the Merino.     

Temporal variation in LH and testosterone responses of rams after the introduction of oestrous females during the breeding season

The response of sexually experienced Ile-de-France rams to the presentation of oestrous females in October at sunrise (Subgroup S) or at 11:00 h (Subgroup N) was studied and compared with unstimulated controls (Subgroup C). Animals (12 per group) were bled for 7 h at 20-min intervals, starting 3 h before stimulation by oestrous females (3 per group). Eight rams from Subgroup S showed an increase of LH pulse frequency and only 3 in Subgroup N (P less than 0.03). In Subgroup S the introduction of females led to 2- and 3-fold increases in LH pulse frequency during the stimulation period compared with values in Subgroup C or before the stimulation period (3, 1.6 and 1 peaks/rams/6 h respectively; P less than 0.05). The presence of females also led to an increase in mean testosterone concentrations, and small increases in basal and mean LH values. No differences were found in LH peak amplitudes. In Subgroup N only inconsistent evidence of increases in mean LH and testosterone values was found. No differences between Subgroups S and N in behavioural patterns during stimulation were detected. We conclude that the presence of females affects LH pulse frequency at sunrise but not at noon during the breeding season and this effect is at least partly independent of sexual behaviour. These results suggest a possible circadian variation in CNS sensitivity involving the hypothalamic regulation of LH secretion in response to the presence of oestrous females.     

Effect of litter size on blood haematocrit and liveweight in Booroola Merino and Merino ewes during pregnancy and the post-partum period

Blood haematocrit and liveweight were determined throughout pregnancy and the post-partum period in 217 Booroola Merino and Merino ewes in order to relate these parameters to litter size at birth. In pregnant ewes, haematocrit declined from three until five months gestation, rose immediately after parturition then declined until two months post-partum. During the third to fifth month of gestation, haematocrit decreased in proportion to litter size. Nonpregnant ewes, measured at similar intervals, did not show the same pattern. Haematocrit of nonpregnant animals was higher than that of triplet-bearing ewes at three, four and five months gestation, but was only significantly different to single- and twin-bearing ewes at five months. The liveweight of pregnant ewes increased up to parturition and then declined until two months post-partum. The liveweight of nonpregnant ewes increased over the experimental period. It was concluded that the number of foetuses a ewe carried had significant effects on the decline in haematocrit during pregnancy. Haematocrit was not a precise indicator of litter size in sheep. Haematocrit, ewe liveweight and ovulation rate together in a multiple regression only accounted for 37% of the variation in litter size.     

Mechanism controlling ovulation rate in ewes in relation to seasonal anoestrus.

Three experiments were carried out during seasonal anoestrus in Finnish Landrace and Scottish Blackface ewes, to establish whether the differences between the breeds in ovulation rate are functional during the non-breeding season and are therefore independent of the mechanism controlling ovulation. In Expt 1, follicles greater than or equal to 2 mm in diameter were dissected from the ovaries of both breeds and incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. In both breeds, follicles producing greater than or equal to 500 pg oestrogen/ml/h (oestrogen-active) were readily identifiable from a population producing less (oestrogen-inactive). The number of oestrogen-active follicles in each breed was similar to the number of ovulations near the end of the breeding season. Oestrogen-active follicles also had more luteinizing hormone (LH) receptors and larger diameters than oestrogen-inactive follicles. There were, however, no significant differences between the two follicle types in follicular fluid or in-vitro testosterone concentrations. In Expt 2, seasonally anoestrous Scottish Blackface ewes were unilaterally ovariectomized; the second ovary was removed 7 days later. Follicles from both ovaries were processed as described for Expt 1; oestrogen-active follicles were categorized according to their ability to produce greater than 500 pg/ml/h. There were twice as many oestrogen-active follicles in the second ovary as in the first ovary; the number of oestrogen-active follicles in the second ovary was also similar to the total number of oestrogen-active follicles in both ovaries of the Scottish Blackface ewes in Expt 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.