Role of gonadal negative feedback on the gonadotrophin responses to gonadotrophin-releasing hormone (GnRH) in ram lambs from two lines of sheep selected for their luteinizing hormone response to GnRH.

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 micrograms 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.

Direct responses in males and correlated responses for reproduction in females to selection for testicular size adjusted for body weight in young male lambs.

Selection based upon testicular diameter adjusted for body weight at 6, 10 and 14 weeks of age was used to produce two lines of sheep, with either high or low testicular size. Ten generations of selection were carried out and the estimate of the realized heritability of the selection criterion was 0.53 +/- 0.01. There were significant positive correlated responses to selection for testicular diameter at 6, 10 and 14 weeks of age, but the correlated responses in body weight at these ages were negative. In mature females, there were significant negative correlated responses to selection in premating body weight in the 1st, 2nd and 3rd breeding season and in the day of the first oestrus in the 2nd breeding season. Litter size per ewe mated had a small positive correlated response to selection in the second breeding season. This latter response appeared to be due to a positive correlated response in fertility, ewes from the High-line having a significantly higher probability of conceiving to a single mating than those from the Low-line. There was no significant correlated response in ovulation rate or litter size per ewe lambing and the genetic correlation between these traits and the selection criterion is likely to be close to zero. This may be due to the adjustment for body weight used, but it is possible that, in any event, body weight in young rams may be a better predictor of female ovulation rate than testicular diameter. These results do not rule out the possibility that testicular size in rams older than those selected would provide a good predictor of genetic merit for female ovulation rate.

Study of LH response to GnRH in the young male as a criterion of genetic merit for female reproduction in sheep.

A high and a low response line in sheep were selected on the basis of the mean concentration of LH in 10-week-old Finn-Dorset ram lambs after an i.v. injection of 5 micrograms GnRH. After 8 male generations the mean LH response of the high line was more than 5-fold that of the low line and the heritability of the selected trait was estimated at 0.44 +/- 0.015. Highly significant line differences in mean LH response to GnRH were also found in males at 20 weeks of age and females at 10 and 20 weeks of age and the genetic correlations between the four LH response traits appear to be close to unity. Large line differences in the mean FSH response to GnRH were also found in both males and females at 10 and 20 weeks of age. Selection had little effect on the physical characteristics of lambs. High-response line ewes entering their first breeding season at about 7 months of age showed oestrus earlier in the season and had higher ovulation rates and numbers of lambs born per ewe lambing than did low-response line ewes. In the second breeding season, at about 19 months of age, the only line difference was a higher ovulation rate early in the breeding season in high-line ewes. It is suggested that these changes may be mediated by a more rapid response in high-line ewes to increased GnRH stimulation at puberty or at the beginning of the breeding season.

Changes in the feedback control of gonadotrophin secretion in ewes from lines selected for testis size in the ram lamb.

The dynamics of FSH and LH secretion were studied in sheep genetically selected for High (H) and Low (L) rates of testis growth. Gonadotrophin secretion had previously been shown to be affected in the ram lamb with H-line lambs more sensitive to steroid feedback than L. While there were significant differences in mean LH concentrations during the luteal and follicular phases of the oestrous cycle, mean LH values were essentially similar in the two lines in response to ovariectomy, the effect of oestradiol implants on the response to ovariectomy and the response to LHRH. However, the frequency of LH pulses in the H line was similar during both phases of the oestrous cycle, showing a surprising insensitivity to steroid feedback. By contrast, LH pulse frequency was markedly lower in the L-line ewes in the luteal than the follicular phase (0.6 vs 1.1 pulses/h) as expected from the literature. Mean FSH concentrations were significantly higher in the L-line ewes during the follicular phase of the oestrous cycle and after ovariectomy but no significant differences were detected at the other sampling periods. There were no differences in ovulation rate between the lines. It was concluded that selection for testis size had affected the feedback control of gonadotrophin release in the ewe, as in the ram, and hence the expression of the genes controlling this is not sex limited.

Transgenic livestock.

Single genes can now be added routinely to the genome of mice by molecular manipulation as simple Mendelian dominants; this complements the normal process of reproduction to give 'transgenic' animals. Success in ruminants is limited to a few examples in sheep and although gene expression has yet to be documented, there is every reason to expect that it will be achieved. The application of this technology to livestock improvement depends on the identification of circumstances in which the phenotype is limited by the deficiency of a single protein. While there is little evidence to indicate that single dominant genes are in general likely to have favourable effects, it is argued that there are likely to be exceptions. These include particular combinations of promoter and structural gene sequences to alter feedback control, for example through a change in tissue specificity, and the alteration of definitive proteins such as those of milk. A mouse model has been established to study the molecular manipulation of sheep milk proteins. The sheep beta lactoglobulin gene has been incorporated and the sheep whey protein is secreted by the mammary gland of transgenic mice. For the future, means to delete or reduce the expression of existing genes are likely to be important, as are more effective means of incorporation such as retroviral based methods and the incorporation of multigene constructs. The resources required to test transgenic livestock will, however, be greater than those required to create them.

Endocrine differences in rams after genetic selection for testis size.

Testis diameter and body weight were recorded from 6 to 76 weeks of age in ram lambs from two established lines selected for high (H) and low (L) testis size. While testis growth was greater in the H line up to 14 weeks of age (P less than 0.001), body weight was significantly lower, with the L line rams being 10 kg heavier by 76 weeks. There were no differences in plasma LH up to 20 weeks of age, but FSH concentrations were significantly lower at 14 and 20 weeks in the H line. Testosterone concentrations were not significantly higher in the H line from 6 to 20 weeks. In lambs castrated at birth, significantly higher FSH values were recorded from 6 to 20 weeks of age in the H line (P less than 0.001) whereas there was no difference in LH concentration at 6 and 10 weeks of age between the lines. At 14 and 20 weeks, however, the concentrations of LH were greater in the H than L line lambs (P less than 0.05). After hemicastration at 6 weeks of age, the rate of growth of the remaining testis in the L line lambs was significantly faster than in entire lambs of that line from 10 to 20 weeks (P less than 0.05 at 10 weeks to P less than 0.001 at 20 weeks). There was no difference in the rate of testis growth between the the entire and hemicastrated lambs from the H line from 6 to 12 weeks of age. It can be concluded that there is an underlying genetic difference in pituitary gland and/or hypothalamic activity in ram lambs from the two selected lines.

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.

Concentration of gonadotrophins in the plasma of sheep given gonadal steroid antisera to raise ovulation rate.

The concentration of FSH and LH in peripheral plasma was studied in sheep from 8 h before to 17.5 h after injection (i.v.) with antisera to the steroids androstenedione, oestradiol, oestrone and testosterone. The fitted mean concentration of LH increases after all treatments and the increase was associated with a higher frequency of LH pulses. The greater concentration was evident for all groups by the period 3.5-6.5 h after injection, but by the end of the sampling period the concentration had returned to or towards the values in the controls. For FSH, significant change was limited to those animals given anti-oestrogen sera but it was more rapid than for LH, both groups receiving anti-oestrogen sera showing an increase during the period 0.5-3.0 h after injection. The ovulation rate was increased by treatment and an effect close to 0.75 corpora lutea per ewe was maintained by treatment in subsequent oestrous cycles. This declined to 0.25 corpora lutea after two oestrous cycles without treatment.

Effect of season of birth and of hemicastration on the histology of the testis of 6-month-old lambs.

Season but not hemicastration affected the cellular composition of the testis. Despite similar weight, the testicular histology differed markedly with season of birth. The number of Leydig cells and of Sertoli cells was greater in summer- than in winter-born lambs by factors of 2 and 1.5 respectively. Similarly the number of spermatogonia and their rate of production increased substantially in summer-born lambs. The rate of spermatid production was affected by both hemicastration and season. Season of birth exerted more modifications to testicular histology than did hemicastration.

Fertility of sheep given antisera to steroids during anoestrus.

The incidence of oestrus (6/46) and ovulation (14/46) in ewes given antisera to androstenedione, oestrone, oestradiol and testosterone either separately or as a mixture of these sera at the time of treatment with progestagen sponges alone or progestagen sponges followed by LH-RH was similar to that of control ewes (2/13 and 6/13 respectively). The number of corpora lutea (CL) recorded for those ewes that did ovulate was, however, greater in the antiserum-treated ewes (22 CL/14 ewes) than in the controls (6 CL/6 ewes) at the first ovulation after sponge withdrawal. This superiority persisted to the second ovulation (53 CL/42 treated ewes compared to 13 CL/13 controls). The results for groups treated with antisera did not differ amongst themselves.

Improvement of sheep fecundity by treatment with antisera to gonadal steroids.

Sera from sheep immunized against oestrone (Group E1), oestradiol (Group E2), androstenedione (Group A) and testosterone (Group T) were given to ewes singly or as a mixture (Group M) of all 55 types as a single intravenous injection at the time of the start of mating. The number of lambs produced, the numbers of eggs shed and the display of oestrus were recorded. The ovulation rates were 1.8 in Group E1, 2.1 in Group E2, 1.6 in Group A, 1.8 in Group T and 2.1 in Group M compared with 1.3 for the controls (P, variation among groups, less than 0.001) in the first oestrous cycle. The effect persisted in those animals not conceiving to the first mating--1.3 in Group A, 1.8 in Group E1, 1.9 in Group E2 and 2.0 in Group M compared with 1.3 for the controls; all of the ewes in Group T conceived to mating at a single oestrus. The mean number of lambs born alive per ewe treated was 1.1 for Group A, 1.3 for Group E1, 1.3 for Group E2, 1.5 for Group T, 1.5 for Group M and 1.0 for the controls. The increase in the number of lambs born was due to a higher proportion giving birth to twins (P less than 0.01); no ewe gave birth to triplets. High conception rates were recorded for all treatments.

Inadequate corpus luteum function after the induction of ovulation in anoestrous ewes by LH-RH or an LH-RH agonist.

Scottish Blackface ewes were given LH-RH (3 x 30 micrograms i.v., at 90 min intervals) or D-Ser-(But)6-des Gly10 LH-Rh ethylamide (LH-RH agonist) as a single injection (8 or 40 micrograms) during anoestrus. Ovulation as judged by laparoscopy occurred in 8 of the 27 animals. Despite the fact that the LH-RH agonist induced a greater release of LH and FSH the different treatments had no effect on the number of ewes ovulating and within each treatment group there was no apparent difference in the amounts of gonadotrophins released between the ewes that did or did not ovulate. All ovulations resulted in the formation of CL associated with plasma progesterone concentrations of less than 1 ng/ml (1--5 ng/ml in the normal luteal phase). In comparison with CL of the normal cycle the induced CL were of lower weight and had reduced progesterone content and ability to secrete progesterone in vitro. However, the binding of hCG was equivalent to that of normal CL. These results suggest that the inadequate CL formed in anoestrous ewes after a single LH-RH injection have not developed the ability to synthesize and secrete progesterone in spite of the presence of normal amounts of LH receptors.

Increased testicular growth of Tasmanian Merino ram lambs treated with antisera to oestrogens.

Passive immunization against oestrone and oestradiol increased the rate of testicular growth (P less than 0.01) without a statistically significant change in the concentrations of LH and FSH at the time of measurement. Although the concentration of testosterone was higher in the group with the largest testes, the difference was not statistically significant. Within groups, the concentration of FSH was correlated with the oestrone antibody titre (r = 0.5), suggesting that oestrone may have a particular role to play in the feedback control of FSH release in the ram; in addition, the concentration of testosterone was correlated with that of LH (r = 0.65). The data indicate a role of oestrogens in the control of testicular growth and are compatible with earlier reports that the testes of Merino lambs grow more quickly when gonadal feedback is reduced.

Plasma FSH, LH, the positive feedback of oestrogen, ovulation and luteal function in the ewe given bromocriptine to suppress prolactin during seasonal anoestrus.

The effects of pharmacological reduction of the high plasma prolactin concentration typical of seasonal anoestrus in sheep were assessed with respect to positive feedback of oestrogen on LH release, ovulation, and progesterone secretion. Treatment of 16 Scottish Blackface ewes with 1 mg bromocriptine, i.m. twice daily for 12 days, reduced prolactin concentrations in peripheral plasma from 64 +/- 10 ng/ml before treatment to < 4 ng/ml. This treatment had no effect on the proportion of ewes discharging LH and FSH in response to 12.5 microgram oestradiol benzoate (3/8 before compared with 5/16 during treatment) or the proportion of ewes ovulating in response to oestrogen treatment. Plasma progesterone concentrations remained low even in ovulating ewes. It is concluded that treatment with bromocriptine alone is unlikely to restore oestrous cycles to ewes in seasonal anoestrus.

Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in LH-RH-treated anoestrous ewes.

Nineteen Scottish Blackface ewes were given LH-RH (3 X 30 micrograms i.v., 90-min intervals) during anoestrus when prolactin levels were elevated. Plasma levels of prolactin were suppressed with CB 154 (twice daily, i.m.) on Days -5 to 0 (N = 5), 0 to +5 (N = 5) or -5 to +5 (N = 5) around the day of LH-RH treatment (Day 0). Control animals (N = 4) received saline on Days -5 to +5. Nine animals ovulated forming corpora lutea as judged by laparoscopy on Day +7. No difference in FSH or LH levels was found between treatments and ovulations occurred equally in all treatment groups. Progesterone levels were less than ng/ml in all animals up to Day 14. It is concluded that short-term suppression of prolactin does not affect the incidence of ovulation or corpus luteum progesterone production in LH-RH-treated anoestrous ewes.

Genetic and environmental variation in the LH response of ovariectomized sheep to LH-RH.

The influence of breed and season on the sensitivity of the pituitary gland of sheep to LH-RH was assessed. Ovariectomized ewes of 3 breeds (Finnish Landrace, Scottish Blackface and Tasmanian Merino) with differing normal breeding seasons and with differing ovulation rates were injected (i.v.) with 3 doses of LH-RH (1.56, 6.25 or 25.0 micrograms) at 3 different times of the year covering the anoestrous and the breeding seasons of intact ewes; 9 ewes of each breed (3 per sub-class) were examined on the first and third occasions, 6 (2 per sub-class) on the second. The response was measured in terms of the concentration of LH in peripheral plasma 20, 40, 60 and 80 min after injection. Time of year, but not the breed of sheep, affected the magnitude of the response; the data indicated that the duration of LH secretion was greater during the breeding season than during anoestrus. It was concluded that changes in the spontaneous activity of the hypothalamus/hypophysis could contribute to seasonal changes in LH secretion independently of the modifying effects of gonadal steroids. Such variation in unmodulated activity apparently does not contribute to the differences in ovulation rate among the 3 breeds.

Oestradiol levels in sheep plasma during the oestrous cycle.

The concentration of oestradiol was measured by radioimmunoassay in ovarian venous plasma collected from ewes of three breeds (Finnish Landrace, Scottish Blackface and Tasmanian Merino) on Day 9 of the oestrous cycle and in jugular venous plasma collected daily around oestrus in two of these breeds. The mean +/- s.e.m. concentration in the ovarian venous plasma of the Merino (44.1 +/- 7.6 pg/ml) was lower than that in Blackface (72.2 +/- 10.2 pg/ml) and Finn ewes (66.8 +/- 10.2 pg/ml). The overall fitted mean concentration in peripheral venous plasma was 1.7 pg oestradiol-17beta/ml, with no difference between the Finn and Blackface ewes, in which the highest preovulatory values were 3.9 +/- 0.5 and 3.4 +/- 0.5 pg/ml respectively.

Reproduction in young sheep: some genetic and environmental sources of variation.

"The extent of genetic and environmental variation" in the development of reproduction in sheep is illustrated by examples with particular reference to variation among breeds and to the effects of photoperiod. The interactions between genetic and environmental effects are introduced; these may be so great that genetic groups may reverse their ranking for rate of development in different environments. The "physiology of puberty" is then discussed. The difficulty of separating puberty from seasonal variation is stressed, and a possible contrast is drawn between the physiological characteristics of genetic variation and those of environmental variation in reproductive development. Finally the physiological factors associated with sterility in young females are discussed; most studies, however, have been conducted during the time of year when adult females would also be expected to be sterile, so that conclusions are difficult and a "missing link" cannot be identified.