Anoestrous Ewes Research Articles

The effect of paddock size on the response of seasonal anoestrous ewes to the ram effect

Many aspects of the conditions required to maximize the ewe’s response to ram introduction in the late anoestrous season remain unclear. The aim of this research was to determine whether grazing space allowances could influence the efficacy of the ram effect. In August 1995, at Reading (latitude 51°27′N), following a 3-month isolation period from rams, two groups of nulliparous Mule ewes, aged 15 months, were introduced to four rams in a low (12 ewes/ha; treatment L, n=124) or in a high stocking rate (84 ewes/ha; treatment H, n=126). From the beginning of August until the end of August oestrous behaviour was recorded by daily checks of mating marks on ewes. Rams were removed and in October all ewes were scanned (day 50) for pregnancy. No significant differences were found in the parameters investigated. Eighty-two percent of the L and 75.4% of the H ewes exhibited oestrus, with a pronounced peak on day 23 following ram introduction and a compact concentration in the 21–25-day period. The oestrous synchronisation rate in this 5-day period was 69.4 and 68.3%, respectively for L and H. The mean interval from ram introduction to oestrus was 23.17±2.4 days in L and 23.0±2.2 days in the H group. Conception rates were 84.3 and 87.4% for L and H groups, respectively. These results suggest that the response of anoestrous ewes to the introduction of rams was not affected by grazing space allowances and that yearling Mule ewes respond well to the ram effect in the late anoestrus season.

Repeated fence-line ram exposure advances the timing and improves the synchrony of oestrus and lambing in ewes

Introduction of a ram to anoestrous ewes induces an almost instantaneous rise in LH pulse frequency. This is commonly sufficient to overcome the seasonal suppression of the hypothalamic-pituitary axis and induce a synchronous first ovulation. This study investigates the application of repeated fence-line ram exposure during the beginning of the breeding season to synchronize oestrus and subsequent lambing in ewes.

Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.

Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.

Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep

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Oestradiol microimplants in the ventromedial preoptic area inhibit secretion of luteinizing hormone via dopamine neurones in anoestrous ewes.

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.

A note on the effect of individual housing conditions on LH secretion in ewes after exposure to a ram

To determine whether it is feasible to use the stimulus of exposure to a ram to induce sexual activity in individually housed ewes, 33 adult anoestrous ewes were randomly allocated to two treatments: Group 1, n=20, housed in individual pens, with a ram’s pen situated between every two ewes (exposed to rams through the pen’s bars) and Group 2, n=13, housed in a communal pen with five rams (full contact with the rams). The day before the introduction of rams, blood samples were collected from Group 1 ewes, for 6 h at 15 min intervals commencing 10:00 h. Rams were introduced in both groups the following day at 8:00 h. Two hours later, blood samples were collected from Group 1 ewes for 6 h at 15 min intervals. Thereafter, ewes and rams were moved to a communal pen and blood samples collected every 3 h until 10:00 h the next day. In Group 2, blood samples were collected every 3 h for 24 h starting at 10:00 h, 2 h after the rams were introduced. From the samples collected at 15 min intervals, no differences between mean LH concentrations, pulse frequencies and amplitudes before or after the introduction of rams were observed in Group 1. The percentage of ewes presenting a pre-ovulatory surge of LH in the first 24 h after the introduction of rams was similar in both groups (Group 1: 50%, 10 ewes; Group 2: 61.5%, 8 ewes). The pre-ovulatory LH surges began approximately 7 h later ( P=0.05) in Group 1 than in Group 2 (18.5±2.2 h versus 11.8±2.6 h, respectively). Mean plasma LH concentrations were significantly higher in Group 2, during the first 8 h after the introduction of rams, these differences disappeared when all the animals were moved to the communal pen. In conclusion, individual housing together with frequent blood sampling conditions do not induce a significant increase of LH secretion of anoestrous ewes after a ram effect, as compared with that in rams and ewes in communal pens. Further experiments are necessary to separate, on one hand, the effect of housing and on the other hand, the effect of sampling frequency on LH secretion after a ram effect.

Effect of oestradiol treatment during GnRH-induced ovulation on subsequent PGF 2 α release and luteal life span in anoestrous ewes

In sheep, induction of ovulation during anoestrus is accompanied by a high incidence of short luteal phases, though pre-treatment with progesterone can overcome this problem. We have investigated the effects of supplementing oestradiol during GnRH-induced ovulation on subsequent PGF 2 α release and luteal life span. Thirty anoestrous crossbred ewes received 250 ng GnRH i.v. at 2 h intervals for 48 h to induce ovulation either alone (group 1; n=10) or in association with either an i.m. injection of 20 mg progesterone 3 days earlier (group 2; n=10) or 3 i.m. injections of 10 μg oestradiol at 8 h intervals on the second day of GnRH treatment (group 3; n=10). Laparoscopy, performed 3 days following GnRH to confirm ovulation and 8 days later, coupled with plasma progesterone analysis were used to determine luteal life span. On day 4 following GnRH, plasma samples were collected at 20 min intervals for 8 h to monitor PGF 2 α release. One ewe from group 1 failed to ovulate and was excluded from further analysis. All groups showed an increase ( P<0.01) in plasma oestradiol during GnRH treatment, with group 3 showing a marked ( P<0.001) increase over that seen in the other two groups. In group 1 there were 1.4±0.2 PGF 2 α episodes/ewe/8 h. In group 2, pre-treatment with progesterone caused the complete inhibition of PGF 2 α episodes (0 episodes/ewe/8 h) while in group 3, treatment with oestradiol resulted in a significant reduction (0.3±0.1 episodes/ewe/8 h) compared with group 1 ( P<0.01). In group 1, 9/9 ewes exhibited short cycles compared with 2/10 ewes in group 2 ( P<0.01). In group 3 the proportion of ewes showing short cycles 7/10 ewes was not significantly different from the other groups. While treatment with oestradiol caused a significant attenuation of PGF 2 α release, this was associated with only a partial reduction in the incidence of short cycles.

Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes

AbstractThe aim was to examine statistically the changes among days in the numbers of follicles relative to the growth of large follicles to test the hypothesis that follicular development occurs in a wave-like fashion in anoestrous ewes. The relationships among the patterns of circulating concentrations of FSH, oestradiol and inhibin-A and the pattern of follicular growth as well as relationships among follicular fluid steroid and inhibin-A concentrations were also studied. The ovaries of 11 ewes were examined daily using transrectal ultrasonography for 14 days and blood samples were collected every 8 h for 9 days. Five ewes were ovariectomized on the last day of ultrasound scanning. One to three identified follicles (a cohort) emerged every 2 to 5 days (mean 2·9 (s.e. 0·2) days) in individual ewes. The numbers of 4 and 5 mm follicles were fewest (P &lt; 0·05) before and greatest (P &lt; 0·05) 1 day after cohort emergence. This change in the numbers of follicles indicates a wave-like pattern of follicular growth. FSH concentrations were greatest (P &lt; 0·05) on the day before wave emergence and lowest (P &lt; 0·05) on the day of wave emergence. Peripheral concentrations of oestradiol and inhibin-A did not fluctuate (P &gt; 0·05) in association with the emergence of follicular waves. The follicles that were collected at ovariectomy originated from one of three different waves. Oestradiol and inhibin-A concentrations in follicular fluid and the oestradiol-to-progesterone ratio were not different among the largest follicles of successive waves, when follicles were collected at the same time, indicating that new waves of follicles developed before the demise of old waves.In conclusion, waves of follicles emerged about every 3 days in anoestrous ewes (defined as significant changes in numbers of follicles) and were associated with fluctuations in FSH concentrations but not peripheral oestradiol or inhibin-A concentrations. New follicular waves also emerged in the presence of steroidogenically active (positive oestradiol-to-progesterone ratio), inhibin-A producing follicles from a previous wave suggesting that follicles do not exert functional dominance during the non-breeding season.

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 3 mm 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 3 mm 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 ≥5 mm in size before regression compared with the mid-breeding season and mid-anoestrus. The lifespan of follicles reaching ≥5 mm 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 3 mm 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 ≥5 mm 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.

Ovarian responses in gonadotrophin-releasing hormone-treated anoestrous ewes: follicular and endocrine correlates with luteal outcome.

The relationships between the development of antral follicles (growing from 3 to > or = 5 mm diameter), hormone secretion (luteinizing hormone (LH), follicle-stimlating hormone (FSH), oestradiol and progesterone), ovulation and the formation of luteal structures in response to gonadotrophin-releasing hormone (GnRH) were examined in 24 anoestrous Western White Face ewes (May-July). Ewes were monitored by transrectal ovarian ultrasonography for 34 days, commencing 15 days before the administration of GnRH. Following treatment with GnRH, 83% (20/24) of ewes ovulated. Twenty-five per cent of all ewes (6/24) subsequently had normal (full-life span) corpora lutea (CL), 37% (9/24) had inadequate CL, 17% (4/24) had both normal and inadequate CL, 17% (including three of four anovular ewes and one ewe with inadequate CL) formed luteinized follicles and only 4% (1/24) did not ovulate or produce any luteal structure. None of the variables of follicular growth (follicles reaching > or = 5 mm diameter) differed between follicles that either ovulated or failed to ovulate and there was no evident correlation between the age or stage of development of ovulatory sized antral follicles and the type of luteal structure formed, except for luteinized unovulated follicles; these follicles all emerged within 3 days of GnRH injection. Mean serum concentrations of FSH and oestradiol before treatment did not differ (P>0.05) between ewes with different ovarian responses, but peaks of fluctuations in serum concentrations of FSH in daily samples were higher in ewes that produced normal CL compared with ewes with inadequate CL. After GnRH treatment, oestradiol secretion was higher in ewes that formed luteinized unovulated follicles than in all ewes with inadequate CL (P<0.05). The peak concentration of the GnRH-induced LH surge was higher and the interval from GnRH to peak LH discharge was shorter in ewes with inadequate CL compared with ewes that had normal CL after ovulation (P<0.05). In conclusion, ovulatory sized antral follicles at a similar stage of their life span can give rise to either normal or inadequate CL and a proportion of these follicles do not ovulate in response to GnRH in seasonally anoestrous ewes. This suggests differences in ovarian follicular responsiveness to gonadotrophic stimuli. Both the amplitude of episodic elevations in daily serum FSH concentrations and the characteristics of the pre-ovulatory LH surge may be important for luteogenesis following ovulation.

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

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

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

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

Isoforms and half-life of FSH from sheep with different reproductive states.

The glycoprotein hormone FSH comes in many different isoforms. In humans and rats the charges of the FSH isoforms vary with reproductive state and these affect the half-life of FSH in plasma. In this study we examined the charge heterogeneity of FSH in pituitary extracts from sheep with different reproductive states. Also the half-life of clearance of pituitary FSH from the different reproductive states was determined in mice. Pituitaries were collected from: anoestrous, luteal phase, follicular phase, early-pregnant and late-pregnant ewes, ewe lambs, ram lambs, rams during the breeding and non-breeding seasons and wethers (5 per group). After extraction, FSH isoforms were fractionated by HPLC anion exchange chromatography. The volume at which half of the FSH had eluted from the ion exchange column was determined (HP(50)). It was found that FSH isoforms from ewes (HP(50)=96.7+/- 1.3 ml (s.e.m. )) eluted later (P<0.01) than those from rams (HP(50)=82.3+/-1.3 ml) indicating that FSH isoforms in the ewes were more acidic than those from rams. There was a seasonal difference in ewes, with ewes in anoestrus (HP(50)=101.6+/-2.6 ml) having more-acidic (P<0.01) FSH isoforms than the ewes during the oestrous cycle (HP(50)=95.3+/-0.7 ml). There was an effect of age, with the FSH isoforms from cycling ewes (HP(50)=95.3+/- 0.7 ml) being more acidic (P<0.01) than those from ewe lambs (HP(50)=88.3+/-1.9 ml). There was an effect of pregnancy, with late-pregnant ewes (HP(50)=107.3+/- 1.6 ml) having more-acidic FSH isoforms (P<0.05) than those from anoestrous ewes (HP(50)=101.6+/-2.6 ml) and there was an effect of castration with the breeding season rams (HP(50)=80.7+/-1.4 ml) having more-acidic (P<0.05) FSH isoforms than wethers (HP(50)=74.0+/-0.5 ml). The half-life of pituitary FSH from animals in the different reproductive states was found to be negatively correlated with HP(50) (r(2)=0.56, P<0.01). The FSH isoforms from wethers were the least acidic and had the longest half-lives. Collectively, these findings show that in sheep, age, sex and reproductive state are all factors which influence the forms of FSH that are extracted from the pituitary gland. Moreover, these results demonstrate that FSH from sheep with the most-acidic FSH isoforms have the shortest half-life in plasma.

The effect of exposure to oestrous ewes on rams' sexual behaviour, plasma testosterone concentration and ability to stimulate ovulation in seasonally anoestrous ewes

Previous research has shown that the proportion of seasonal anoestrous ewes that ovulate in response to the introduction of rams (‘ram effect’) is dependent upon pheromonal and sexual behavioural stimuli emitted by the rams. Close contact with oestrous ewes is likely to increase the rams' libido and level of testosterone secretion, which in turn has been suggested to positively influence the production of pheromones. Thus, the sexual stimulation of rams could be used to improve the efficacy of the ‘ram effect’. In the present experiment, 272 ewes were introduced (1) to rams without recent experience of oestrous ewes, (2) to rams recently exposed to oestrous ewes, (3) and (4) to oestrous ewes and rams with or without recent experience with oestrous ewes, or (5) remained isolated from rams. Serum testosterone concentration of rams was elevated equally ( P<0.05) when oestrous or anoestrous were introduced. Exposure to oestrous ewes before or after introduction increased ( P<0.05) the various measures of ram sexual behaviour directed towards anoestrous ewes. However, there was no statistical evidence that increased sexual activity resulted in an improvement in the ability of rams to stimulate ovulation in anoestrous ewes.

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.

Ovarian function in ewes at the onset of the breeding season.

Transrectal ultrasonography of ovaries was performed each day, during the expected transition from anoestrus to the breeding season (mid-August to early October), in six Western white-faced cross-bred ewes, to record ovarian antral follicles > or = 3 mm in size and luteal structures. Jugular blood samples were collected daily for radioimmunoassay (RIA) of follicle-stimulating hormone (FSH), oestradiol and progesterone. The first ovulation of the breeding season was followed by the full-length oestrous cycle in all ewes studied. Prior to the ovulation, all ewes exhibited a distinct increase in circulating concentrations of progesterone, yet no corpora lutea (CL) were detected and luteinized unovulated follicles were detected in only three ewes. Secretion of FSH was not affected by the cessation of anoestrus and peaks of episodic FSH fluctuations were associated with the emergence of ovarian follicular waves (follicles growing from 3 to > or = 5 mm). During the 17 days prior to the first ovulation of the breeding season, there were no apparent changes in the pattern of emergence of follicular waves. Mean daily numbers of small antral follicles (not growing beyond 3 mm in diameter) declined (P < 0.05) after the first ovulation. The ovulation rate, maximal total and mean luteal volumes and maximal serum progesterone concentrations, but not mean diameters of ovulatory follicles, were ostensibly lower during the first oestrous cycle of the breeding season compared with the mid-breeding season of Western white-faced ewes. Oestradiol secretion by ovarian follicles appeared to be fully restored, compared with anoestrous ewes, but it was not synchronized with the growth of the largest antral follicles of waves until after the beginning of the first oestrous cycle. An increase in progesterone secretion preceding the first ovulation of the breeding season does not result, as previously suggested, from the ovulation of immature ovarian follicles and short-lived CL, but progesterone may be produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum concentrations of progesterone does not alter the pattern of follicular wave development, hence it seems to be important mainly for inducing oestrous behaviour, synchronizing it with the preovulatory surge of luteinizing hormone (LH), and preventing premature luteolysis during the ensuing luteal phase. Progesterone may also enhance ovarian follicular responsiveness to circulating gonadotropins through a local mechanism.

Effect of progesterone on the GnRH-induced secretion of oestradiol and androstenedione from the autotransplanted ovary of the anoestrous ewe.

Two experiments were conducted during the anoestrous period in Border Leicester x Merino ewes with ovarian autotransplants to study the effects of a single injection of 20 mg progesterone on follicular steroid secretion. The aim of these experiments was to determine whether pretreatment with a 20 mg intramuscular injection of progesterone could reduce GnRH-induced ovarian steroid secretion in anoestrous ewes. In both experiments, an injection of 150 ng GnRH induced an LH pulse in all ewes with a maximum concentration 10 min (the first post-injection sample) after injection. Oestradiol and androstenedione secretion increased progressively after the GnRH-induced LH pulse and reached maximum rates of secretion between 60 and 90 min before decreasing slowly to pre-injection rates at 150 min. There were no differences in the pattern of secretion of oestradiol (measured in both experiments) or androstenedione (measured only in Expt 2). In Expt 1, the injection of progesterone 72 h before the challenge with GnRH had no effect on the maximum rate of oestradiol secretion from the autotransplanted ovary. However, in Expt 2, when progesterone was given either 36 or 60 h before GnRH, there was a significant suppression in the maximum rate of secretion of both oestradiol and androstenedione between 60 and 90 min after GnRH injection. These data show that pretreatment of anoestrous sheep with progesterone can suppress LH-stimulated steroid secretion from the ovary and indicate that progesterone may have a direct effect on oestrogenic follicles in sheep.

Effectiveness of short-term progestogen primings for the induction of fertile oestrus with eCG in ewes during late seasonal anoestrus

AbstractTwo hundred and seventy-seven ewes were used in three experiments during late seasonal anoestrus to evaluate short-term progestogen primings for equine chorionic gonadotropin (eCG)-induced oestrus. In experiment 1, medroxy-progesterone acetate (MAP) sponges were inserted for 6, 9 or 13 days in nulliparous ewes. Percentage of ewes in oestrus (92.3, 85.7 and 96.4%, respectively) and conception rates (66.7, 75.0 and 63.0%, respectively) did not differ among groups (T &gt; 0·05). In experiment 2, MAP sponges were inserted for 1, 2, 3, 6 or 12 days in multiparous ewes. Percentages of ewes in oestrus were higher with 3, 6 or 12 days than with 1 or 2 days of priming (72.2, 93.8 and 87.5% v. 25.0 and 33.3% respectively, P &lt; 0.05) and maximum response was with 6-day priming. From both experiments we conclude that short-term progestogen (6 days) priming is as effective as long-term traditional primings for inducing oestrus. In experiment 3, level of progestogen priming was compared among treatments using a new progesterone releasing device (CIDR) for 6 days, a 6-day used CIDR for 6 days, or an 11-day used CIDR for 6 days. While percentages of oestrous response were similar among groups (95.9, 93.6 and 88.9%, respectively), pregnancy rates were higher with a new CIDR than with one that had been used for 11 days (57.1% v. 27.8%, P &lt; 0.05), with the 6-day used CIDR being intermediate (51.1%). We conclude that a 6-day progestogen priming combined with eCG can be used successfully to induce fertile oestrus in seasonal anoestrous ewes if progestogen levels during priming are kept sufficiently high.

Oestrogen and progesterone receptor immunoreactivity and c-fos expression in the ovine cervix.

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0-3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0-3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0-3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.