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.     

Effect of melatonin on daily LH secretion in intact and ovariectomized ewes during the breeding season.

This study was conducted to find out whether daily LH secretion in ewes may be modulated by melatonin during the breeding season, when the secretion of both hormones is raised. Patterns of plasma LH were determined in luteal-phase ewes infused intracerebroventricularly (icv.) with Ringer-Locke solution (control) and with melatonin (100 microg/100 microl/h). Response in LH secretion to melatonin was also defined in ovariectomized (OVX) ewes without and after estradiol treatment (OVX+E2). Basal LH concentrations by themselves did not differ significantly before, during and after both control and melatonin infusions in intact, luteal-phase ewes. However, single significant (P<0.05) increases in LH concentration were noted during the early dark phase in the control and 1h after start of infusion in melatonin treated ewes. In both OVX and OVX+E2 ewes, melatonin decreased significantly (P<0.01, P<0.05, respectively) mean plasma LH concentrations as compared to the levels noted before the infusions. In OVX+E2 ewes, a single significant (P<0.05) increase in LH occurred 1h after start of melatonin treatment, similarly as in luteal-phase ewes. No significant differences in the frequencies of LH pulses before, during and after melatonin infusion were found in all treatments groups. In conclusion, melatonin may exert a modulatory effect on daily LH secretion in ewes during the breeding season, stimulating the release of this gonadotropin in the presence of estradiol feedback and inhibiting it during steroid deprivation. Thus, estradiol seems to be positively linked with the action of melatonin on reproductive activity in ewes.     

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.     

Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season.

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 +/- 0.9 h versus 48.3 +/- 0.8 h, P < 0.02; ovariectomized model: 40.8 +/- 0.6 h versus 42.6 +/- 0.5 h, P < 0.02). Disruption of the hypothalamus-pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 +/- 4 ng ml-1 versus intact transported 22 +/- 5 ng ml-1, P < 0.02; ovariectomized model controls: 35 +/- 7 ng ml-1 versus model transported 15 +/- 2 ng ml-1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 +/- 0.3 versus 0.3 +/- 0.02 ng ml-1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 +/- 0.4 versus 1.7 +/- 0.6 pulses per 2 h). In conclusion, activation of the hypothalamus-pituitary-adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.     

Changes in serum LH concentrations during normal and abnormal sexual development in the pig.

Serum levels of luteinizing hormone (LH) were determined in fetal and prepubertal pigs from Day 49 postcoitum to 25 weeks of age, and also in unilaterally cryptorchid, freemartin and castrated pigs of similar ages. Serum LH was undetectable in the second trimester of pregnancy, but then gradually increased up to 2 weeks after birth in both sexes. 2-week-old pigs showed irregular LH peaks exceeding 2 ng/ml. Serum LH concentrations decreased during the 3rd and 4th weeks of life. Mean LH concentrations were approximately 2 ng/ml in castrated pigs and 1.3 + or -.7 ng/ml in freemartins. The differentiation and functional activity of the testis appeared to be well correlated with the changes in serum LH concentrations. Changes in serum LH could not be correlated with normal and abnormal gubernacular development.     

Plasma progesterone concentrations during early pregnancy in spring- and autumn-bred ewes

The objective of this experiment was to measure blood progesterone concentrations during early gestation to determine if the apparent reproductive failure in ewes bred out-of-season is due to a failure to conceive or embryonic loss. Blood samples were collected from spring- (n=61) and autumn-bred ewes (n=29) from Days 8 to 39 post-oestrus. Serum progesterone concentrations were analysed to ascertain whether ewes were ovulating and failing to maintain pregnancy, or conception was failing. Following pregnancy diagnosis 62 days after ram introduction, ewes were categorised as; no display of oestrus, mated but then identified as non-pregnant, or pregnant. A majority of spring-bred ewes that failed to display oestrus had silent oestrus (86%) and 66% of those ewes had abnormally short-lived corpora lutea. Circulating progesterone concentrations during dioestrus in ewes that had ovulated and displayed oestrus were unaffected by season. Similarly, progesterone concentrations during dioestrus did not differ between pregnant and mated non-pregnant ewes. The results indicated that while early luteylosis, low progesterone secretion from corpora lutea and embryo mortality did occur, these were in only a small proportion of ewes. Progesterone concentrations indicated that a majority of mated non-pregnant ewes had elevated progesterone concentrations necessary for the production of at least one viable embryo/foetus. This may be indicative to the failure of maternal recognition of pregnancy, and it is recommended that events surrounding this stage of pregnancy (Days 12-14) be examined more closely in ewes during the non-breeding season.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Time-course relationships between serum LH, serum progesterone and urinary preganediol concentrations in normal women

A specific and sensitive gas chromatographic method was used to investigate the concentration of pregnanediol glucuronide in urine in relation to the time of ovulation. Serum LH and progesterone concentrations in the same subjects were used as evidence for the occurrence of ovulation. The urinary concentration of pregnanediol glucuronide in 24-hour collections and in overnight specimens increased 2-fold or more from the day of the midcycle LH peak to the time of predicted ovulation (24-48 hour after the LH peak) in parallel with the rise in serum progesterone concentration.     

Plasma concentrations of progesterone in female red deer (Cervus elaphus) during the breeding season, pregnancy and anoestrus

Concentrations of progesterone in peripheral plasma of red deer hinds were basal (less than 1 ng/ml) during lactation/seasonal anoestrus, but increased abruptly at the onset of the breeding season. Lactating hinds (N = 19) started ovarian cycles 10 days later (P less than 0.01) and conceived 16 days later (P less than 0.001) than did 13 weaned hinds. There was no evidence, from plasma progesterone values, of silent oestrus at the start of the season. Progestagen/PMSG treatment induced early ovulations in 8 anoestrous hinds but fertility was low, only 2 conceiving and giving birth. Pregnant hinds (N = 42) had high plasma concentrations of progesterone (mean 3-5 ng/ml) which declined just before parturition.     

Ram novelty and the duration of ram exposure affects the distribution of mating in ewes exposed to rams during the transition into the breeding season

This study compared the affect of short-term and continuous exposure to rams during the transition between anoestrus and the breeding season on the distribution of mating and subsequent lambing. Further, within ewes continuously exposed to rams we investigated the effect of replacing these rams every 17 days with 'novel' rams. During August (late anoestrus, Northern Hemisphere), multiparous, North of England mule ewes were allocated to one of four groups: SVR ewes were exposed to vasectomised rams for 24h on Day 0 (short term; n=109), RVR ewes were exposed to vasectomised rams for 24h on Days 0, 17 and 34 (short term; n=113); PVR ewes were exposed to vasectomised rams on Day 0 and remained with the same rams for the duration of the pre-mating period (continuous; n=104); NVR ewes were continuously exposed to vasectomised rams from Day 0 with the rams replaced with 'novel' rams every 17 days (continuous; n=113). Blood samples were collected from a subset of ewes (n=22 per group) to monitor progesterone. On Day 50, harnessed, entire rams were introduced for mating and raddle marks recorded daily for the first 17 days. The median date of mating occurred 1 day earlier in NVR ewes than PVR ewes (P<0.05). A synchrony score calculated from the blood sampled ewes showed that the distribution of mating was more synchronised in PVR and NVR ewes than SVR and RVR ewes (P<0.001). PVR and NVR ewes had an earlier onset of cyclic activity than RVR ewes (P<0.01). However, only NVR ewes differed from SVR ewes in this variable (P<0.05). Within ewes lambing to first service, the median date of lambing of PVR, NVR and SVR ewes occurred at least 2 days earlier than RVR ewes (at least P<0.05). Further, PVR and NVR ewes had a more compact distribution of lambing than SVR and RVR ewes (P<0.05) and lambing was more compact in NVR ewes than PVR ewes (P<0.05). In conclusion, ewes in continuous contact with rams prior to mating had a more synchronised distribution of mating and lambing than ewes given only short-term exposure to rams. This distribution of mating in continuous ram exposed ewes can be further enhanced by periodic exposure to novel rams.     

Plasma progesterone and LH concentrations in ewes after injection of an analogue of prostaglandin F-2alpha

Plasma progesterone and LH concentrations were monitored throughout a natural oestrous cycle in 12 Clun Forest ewes and compared to those following treatment with a single i.m. injection of 100 microng ICI 80,996, an analogoue of prostaglandin F-2alpha, given during the luteal phase of the cycle. After injection of the analogue there was a high degree of synchrony in the return of oestrus (440 +/- 1-9 h; mean +/- S.E.M.) and the timing of the LH peak (48-5 +/- 2-0 h) from injection. There were no significant differences in the plasma progesterone concentrations or in the height and duration of the preovulatory LH peak between control and treatment cycles. The technique offers the possibility of controlled ovulation in the ewe.     

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

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

Changes in plasma estradiol, progesterone and LH level in the menstrual cycle of the adult female Japanese monkey during the mating season

Adult 15 female Japanese monkeys showing regular menstrual cycles were subjected to the daily blood sampling for the measurement of estradiol (E2), progesterone (P) and biological LH in the mating season. Monkeys were maintained under controlled conditions in a standardized environment. Of the 35 cycles observed, 18 (51.4%) were estimated as anovulatory cycles and 17 (48.6%) were ovulatory cycles. The anovulatory cycles were classified into three types according to the peak level of E2 (Type I: E2 less than 50 pg/ml 3 cycles, Type II: E2 less than 170 pg/ml 7 cycles, Type III: E2 greater than 170 pg/ml 8 cycles). The ovulatory cycles were classified into two Types according to the peak level of P (Type IV: P less than 5.0 ng/ml 5 cycles, Tyep V: P greater than 5.0 ng/ml 12 cycles). The menstrual cycle was 27.5 +/- 7.8 days. The differences between mid cycle LH surge and P level in Type IV and in Type V were statistically significant. It was revealed that female Japanese monkeys kept under controlled condition in the mating season showed high incidence of various types of anovulatory cycles and that the ovulatory cycles with low P elevation in the mid luteal phase showed low LH and P secretions on the mid cycle date.     

Pattern of LH and testosterone secretion of adult male fallow deer (Dama dama) during the transition into the breeding season

Pituitary secretion of LH and testicular secretion of testosterone were investigated during the transitional period from the non-breeding to breeding season of mature male fallow deer exhibiting either normal transitional patterns or shortened transitional patterns in response to summer melatonin treatment. Melatonin implants were administered to 4 bucks for a 150-day period starting 130 days after the winter solstice. Four contemporary bucks served as controls. Melatonin treatment advanced rutting activity, testis development and neck muscle hypertrophy by 6-8 weeks. Profiles of plasma LH and testosterone, based on a 30-min sampling frequency over 24 h, were obtained from 3 treated and 3 control bucks on 4 occasions over the period spanning the transition into the breeding season. In control bucks, LH and testosterone pulse frequency were low (0-2 pulses/24 h) in January and increased (5-7 pulses/24 h) in February. By March and April (pre-rut and rut periods respectively) there was a two-fold increase in basal plasma LH concentrations, a decline in LH pulse frequency (0-1 pulse/24 h) and episodic surges in plasma testosterone concentrations. Melatonin treatment resulted in a shift in hormone profiles, with highly pulsatile patterns of LH and testosterone secretion (7 pulses/24 h) occurring earlier in January. The subsequent post-rut profiles of treated bucks were characterized by lower basal plasma LH concentrations, and reduced frequency and amplitude of plasma testosterone surges.     

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.     

Elevated peripheral concentrations of LH block ovulation and increase thecal and serum progesterone in the hamster

Insertion of osmotic minipumps containing 1 mg ovine LH on Day 1 (oestrus) elevated circulating serum concentrations of LH, progesterone and androstenedione when compared with values at pro-oestrus. Ovulation was blocked for at least 2 days at which time there were twice the normal numbers of preovulatory follicles. Follicular and thecal progesterone production in vitro was elevated when compared with that in pro-oestrous controls. Follicular and thecal androstenedione production in vitro was lower than in controls even though serum concentrations of androstenedione were elevated; the higher androstenedione values may be due to the increase in number of preovulatory follicles when compared with pro-oestrous controls. Follicles from LH-treated hamsters aromatized androstenedione to oestradiol and follicular production of oestradiol was similar to that in pro-oestrous follicles despite low follicular androstenedione production in the LH-treated group. Treatment with 20 i.u. hCG on Days 4 or 6 after insertion of an LH osmotic minipump on Day 1 induced ovulation of approximately 30 ova, indicating that the blockade of ovulation was not due to atresia of the preovulatory follicles. Serum progesterone concentrations on Days 2, 4 and 6 in LH-treated hamsters were greater than 17 nmol/l, suggesting that the blockade of ovulation might have been due to prevention of the LH surge by high serum progesterone concentrations.