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G. P. Adams, A. C. O. Evans and N. C. Rawlings

Eleven age-matched (±4 days) Hereford heifers were examined by transrectal ultrasonography daily for 18 days beginning 20 weeks (5 months) before puberty (first ovulation) to determine the suitability of the transrectal ultrasound technique for imaging the ovaries of prepubertal heifers and to test the hypothesis that ovarian follicular development occurs in waves in prepubertal heifers. Satisfactory ovarian images were obtained during preliminary ultrasound examinations conducted 4 weeks before the observational period (that is 32 weeks of age), during which a semirigid probe extension was used to allow external manipulation of the intrarectally placed ultrasound transducer. Daily examinations commencing at 36 weeks of age were accomplished by intrarectal placement of the operator's hand and transducer, without complication, in all 11 heifers throughout the observational period. Periodic increases in the number of follicles detected (day effect, P < 0.02) were inversely related to the diameter of the largest follicle (r = −0.3, P < 0.03). Portions of three anovulatory follicular waves were detected in all heifers during the observational period (first and third waves in part and second wave in whole). Individual follicles destined to assume a dominant or subordinate position in a wave were retrospectively identified and monitored beginning at a diameter of 4–5 mm. The interval between the emergence of dominant follicles of successive waves (interwave interval) was 8.0 ± 0.4 days and the interval between successive maxima in the number of follicles per heifer per day was 8.1 ± 0.5 days. The growing phase of the dominant follicles best fit a quadratic curve. The growing phase of the largest subordinate follicles, and the static and regressing phases of dominant and subordinate follicles best fit simple linear expressions. Periodic surges in serum concentrations of FSH (day effect, P < 0.0001), but not of LH (day effect, not significant), were associated with follicular wave dynamics. FSH surges (increase and decrease, respectively, best fit quadratic curves) spanned a mean of 3 days and reached maximum values 0.9 ± 0.3 days before emergence of the wave. Results supported the hypothesis that follicular development occurs in waves in prepubertal heifers. Mechanisms controlling the well-ordered phenomena of wave emergence, follicle selection and follicle regression, similar to those of sexually mature heifers, were present in 36-week-old prepubertal heifers.

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A. C. O. Evans, G. P. Adams and N. C. Rawlings

Changes in the pattern of follicular growth and development, and the associated endocrine changes, were examined in prepubertal heifers approaching their first ovulation. Ten, age-matched (± 3 days), Spring-born Hereford heifers were examined daily by transrectal ultrasonography for 17 days beginning 12 weeks before the first ovulation, and daily from just before the first ovulation until the completion of one normal duration ovulatory cycle. On each day of ultrasound examination, the position and diameter of corpora lutea and follicles ≥ 3 mm in diameter were recorded, and one blood sample was collected. Blood samples were also collected every 15 min, for 12 h, at 20, 12 and 4 weeks before the first ovulation, to assess the pulsatile nature of LH and FSH secretion. The first ovulation occurred at 56.0 ± 1.2 weeks of age, at a body weight of 391.9 ± 12.0 kg. Waves of follicular development, similar to those of adult cows, were seen at all ages, and in all heifers, the first ovulation was followed by an ovulatory cycle of short duration (7.7 ± 0.2 days) and then by a normal duration ovulatory cycle (20.3 ± 0.5 days). The maximum diameter of the dominant, or largest subordinate, follicles did not increase as the first ovulation approached, or during the subsequent ovulatory cycles. Similarly, there were no differences between follicle growth rates (1.4 ± 0.1 mm day−1) or regression rates (1.2 ± 0.1 mm day−1) as the animals matured, and the interwave interval increased up to the first ovulation. Waves of follicular development were associated with peaks in FSH concentrations at 12 weeks before the first ovulation. The short duration, ovulatory cycle was associated with low progesterone concentrations and small corpora lutea (mean maximum values 2.75 ± 0.66 ng ml−1 and 19.9 ± 2.0 mm, respectively), compared with normal cycle durations (10.15 ± 0.58 ng ml−1 and 25.8 ± 0.8 mm). Mean serum oestradiol and LH concentrations and LH pulse frequency increased as the first ovulation approached, but FSH concentrations did not. We conclude that, in heifers before the first ovulation, growth and regression of large follicles occur in a wave-like pattern, with characteristics and associated patterns of gonadotrophin secretion similar to those seen in adult, cyclic animals. Hence, in late prepubertal heifers, factors controlling follicle growth are in place, and there are no changes in these parameters in the three months preceding the first ovulation.

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A. C. O. Evans, G. P. Adams and N. C. Rawling

The aim of this study was to characterize changes in ovarian follicle dynamics in relation to changes in hormone secretion in heifer calves from birth to 8 months of age. The position and diameter of ovarian follicles ≥4 mm in diameter were recorded, the number of ovarian follicles ≥2 mm in diameter counted, and blood samples collected daily for periods of 18 days, starting at 2, 8, 14, 24 and 34 weeks of age in ten heifers. The mean age at first ovulation was 52.8 ± 1.6 weeks. At all ages ovarian follicular development occurred in a wave-like manner, as in mature cattle. The maximum diameter of the dominant and the largest subordinate follicles increased between 2 and 34 weeks of age (P < 0.05); however, the greatest increase occurred between 2 and 8 weeks of age. There was a similar increase in the numbers of small and large ovarian follicles (P < 0.05). The duration of detection of dominant follicles (number of days visible at a diameter of ≥4 mm) also increased between 2 and 34 weeks of age (P < 0.05). The emergence of waves of follicular development was preceded by peaks in plasma FSH concentrations (P < 0.05) at 2 weeks of age but this was less clear at other ages. There was a rise in circulating concentrations of gonadotrophins between 4 and 14 weeks of age. We concluded that in heifer calves as young as 2 weeks of age ovarian follicles grew in a wave-like fashion, similar to those of adult cattle. We speculate that the early rise in gonadotrophin secretion stimulated the increase in numbers of follicles and follicle diameters observed, indicating an early critical step in reproductive development.

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J. R. McNeilly, N. P. Evans, T. A. Bramley, P. Brown, A. J. Clark and R. Webb

Selection of the luteinizing hormone (LH) response to exogenous gonadotrophin-releasing hormone (GnRH) in sheep has resulted in the establishment of two lines (High and Low) with a fivefold difference in pituitary sensitivity to GnRH. The effect of selection on gonadotrophin gene expression in the presence or absence of an exogenous gonadotrophin-releasing hormone (GnRH) challenge in twenty-week-old ram lambs from both lines was examined. Before treatment with either GnRH or saline, LH and follicle-stimulating hormone (FSH) concentrations were significantly higher in the High line than in the Low line animals (LH and FSH: P < 0.01). One hour after either GnRH or saline, all animals were slaughtered. In the absence of a GnRH challenge, there were significantly higher concentrations of all three gonadotrophin subunit mRNAs in the High line compared with the Low line, corresponding to the higher basal concentrations of LH and FSH. When comparing treatments between the lines, following a GnRH challenge, LHβ subunit mRNA was significantly (P < 0.001) higher in both lines than before the GnRH, whereas there was no significant change in either α or FSHβ subunit mRNA. These results indicate that the differences in basal gonadotrophin secretion are related to differences in gonadotrophin subunit mRNAs with the High line animals having an inherently greater amount of all three gonadotrophin subunit mRNAs. Selection has not altered the differential amounts of gonadotrophin subunit mRNAs, since there is an overall increase in all three gonadotrophin subunits. GnRH appears to preferentially control LHβ mRNA in both High and Low line animals.

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J. P. Ravindra, N. C. Rawlings, A. C. O. Evans and G. P. Adams

Transrectal ovarian ultrasonography was performed daily in eight ewes during one interovulatory interval, using a 7.5 MHz, rigid, human prostate transducer, and a realtime B-mode scanner to record the numbers, diameters and position of all follicles ≥ 2 mm in diameter and the corpora lutea in both ovaries. Blood samples were taken once a day and were analysed for concentrations of FSH, progesterone and oestradiol. During the interovulatory interval of 17.2 ± 0.4 days, antral follicles (follicles > 2 mm in diameter) emerged on all days except for days 1, 5, 15, 16 and 17. A significant increase in the numbers of follicles emerging was seen on days 2 and 11. The ovulatory follicle (6.9 ± 0.1 mm diameter) was retrospectively traced to emergence on day 11.1 ± 0.3 and grew over a period of 4.1 ± 0.1 days at a growth rate of 1.2 ± 0.04 mm day−1. The largest nonovulatory follicles of the same period grew at the same rate as ovulatory follicles and regressed over a period of 2.6 ± 0.2 days at a rate of 1.2 ± 0.07 mm day−1. The mean diameter of the largest follicles seen on each day of the oestrous cycle was lowest on the day of ovulation (2.9 ± 0.2 mm), increased from day 3 to day 5 (4.1 ± 0.4 mm) and again from day 11 to the day before ovulation (6.9 ± 0.1 mm; P < 0.05). The mean number of antral follicles ≥2 mm in diameter increased over the oestrous cycle from 4.5 ± 0.4 on day 3 to 7.2 ± 0.7 on day 11 and showed a sharp decline starting on day 15, to a low of 3.5 ± 0.3 on the day of ovulation (day 17.2 ± 0.4). The corpus luteum could be identified by day 3 of the oestrous cycle in all the ewes, at a mean diameter of 11.5 ± 0.3 mm. The diameter increased to 13.3 ± 0.6 mm on day 5, and declined from day 11 to a diameter of 7.5 ± 0.3 mm on the day of ovulation. Apart from increases before ovulation, there were no clear associations between serum concentrations of FSH or oestradiol and the pattern of follicular growth and regression. We concluded that follicle emergence appeared on many days of the oestrous cycle of ewes, with two phases of increased emergence. There was no discernible connection between follicle emergence and FSH secretion; the overall pattern of growth and regression of follicles was not as distinctly wave-like as in cattle. Follicular dominance was noted only just before ovulation, again, in contrast to cattle.

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N. P. Evans, J. R. McNeilly, A. J. Springbett and R. Webb

Summary. Divergent selection in 10-week-old Finn-Dorset ram lambs was based on the luteinizing hormone (LH) response to a pharmacological dose of GnRH (5μg). After eight generations of selection, the LH responses of the two lines (low and high) to GnRH differed by a factor of five. This study investigates the pituitary sensitivity of the two lines to exogenous GnRH. Initially, two pilot studies were performed: one to determine the range of doses of GnRH which would stimulate LH pulses of similar amplitude to those seen endogenously, and the other to confirm that sodium pentobarbitone prevents pulsatile LH secretion in prepubertal ram lambs. The results indicated that barbiturate anaesthesia suppressed pulsatile LH secretion in castrated and intact ram lambs. A model system was therefore constructed in 18 10-week-old intact ram lambs (high n = 7, low n = 11), whereby endogenous pulsatile LH secretion was prevented by sodium pentobarbitone anaesthesia and the amplitudes of LH pulses produced in response to different doses of exogenous GnRH could be measured. The GnRH dose–response curves demonstrated that there was a five-fold difference in the sensitivity of the pituitary glands of the two lines to stimulation with GnRH. The projected minimum concentration of GnRH required to produce a measurable pulse of LH was 4·75 ng for the high-line animals and 26·6 ng for the low-line animals. The results indicated that the low-line animals required five times more GnRH than the high-line lambs to stimulate LH pulses of similar amplitude (high line 43·67 ng; low line 206·55 ng).

These results demonstrate that selection has produced two lines of sheep which differ in the control of LH secretion at the level of the hypothalamus–pituitary gland.

Keywords: pituitary sensitivity; GnRH; sodium pentobarbitone; ram lambs

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N. P. Evans, R. B. Land, J. R. McNeilly and R. Webb

Summary. Divergent selection has resulted in two lines of lambs (high and low) that have a 5-fold difference in their ability to release luteinizing hormone (LH) in response to 5 μg of gonadotrophin-releasing hormone (GnRH).

Baseline gonadotrophin concentrations, the gonadotrophin responses to a GnRH challenge and the concentrations of testosterone and oestradiol were compared in lambs which were castrated at birth and intact lambs from both selection lines at 2, 6, 10 and 20 weeks of age. The pattern of LH and follicle-stimulating hormone (FSH) secretion was similar in the two lines, but differed between the intact and the castrated lambs. Basal LH and FSH secretion were significantly higher in the castrates than in the intact lambs from both selection lines. The high-line lambs had significantly higher basal FSH concentrations at all ages tested and significantly higher basal LH concentrations during the early postnatal period.

The magnitude of the gonadotrophin responses to GnRH differed significantly between the intact and the castrated lambs within each line, the amount of gonadotrophins secreted by the castrated lambs being significantly greater. The removal of gonadal negative feedback by castration did not alter the between-line difference in either LH or the FSH response to the GnRH challenge. Throughout the experimental period, the concentration of testosterone in the intact lambs was significantly greater than in the castrated lambs in both selection lines, but no significant difference was seen in the concentrations of oestradiol. No significant between-line differences were found in the peripheral concentrations of testosterone or oestradiol in the intact lambs from the two selection lines.

Therefore, despite similar amounts of gonadal negative feedback in the selection lines, there were significant between-line differences in basal gonadotrophin concentrations, at 2 and 6 weeks of age, and in the LH and FSH responses to an exogenous GnRH challenge, at all ages tested. Removal of gonadal negative feedback did not affect the magnitude of the between-line difference in the response of the lines to GnRH stimulation. The results indicate that the effects of selection on gonadotrophin secretion are primarily at the level of the hypothalamo–pituitary complex.

Keywords: GnRH; ram lambs; castration; gonadal negative feedback