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  • Author: A. J. Springbett x
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J. R. McNeilly, M. Fordyce, R. B. Land, G. B. Martin, A. J. Springbett and R. Webb

Summary. 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.

Keywords: ewes; gonadotrophins; selection; testis diameter

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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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I. Wilmut, C. J. Ashworth, A. J. Springbett and D. I. Sales

Summary. Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1·10 ± 0·06 g; Day 6, 1·15 ± 0·06 g; Day 8, 1·41 ± 0·08 g; P < 0·05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development.

The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.

Keywords: embryo transfer; embryo survival; embryo growth; sheep

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M. I. Boulton, C. S. Haley, A. J. Springbett and R. Webb

The aim of this study was to investigate the role of FSH in the control of ovulation rate by the Booroola gene. Three Booroola genotypes (FecBFecB, FecBFec+ and Fec+Fec+) of the F2 population, from a cross between Booroola Merino and Scottish Blackface, and two Booroola genotypes (FecBFec+ and Fec+Fec+; 25% Booroola Merino and 75% Scottish Blackface), from the backcross of FecBFec+ sires to Scottish Blackface ewes, were compared. During seasonal anoestrus significant differences (P < 0.05) in hCG-stimulated ovulation rates were obtained between FecBFecB and Fec+Fec+ ewes from the F2 population, and FecBFec+ ewes were intermediate. No significant difference in hCG-stimulated ovulation rate was observed in the backcross population between FecBFec+ ewes and Fec+Fec+ ewes. There were no significant differences between genotypes in mean serum FSH concentrations during seasonal anoestrus in either backcross or F2 populations. During the breeding season, two separate experiments confirmed the expected ovulation rate differences between genotypes (FecBFecB > FecBFec+ > Fec+Fec+). In both experiments, mean peripheral FSH concentrations in the F2 population were similar in FecBFec+ and Fec+Fec+ ewes, but were significantly higher (P < 0.05) in FecBFecB ewes. In the backcross population, mean peripheral FSH concentrations during the oestrous cycle were not significantly different between FecBFec+ and Fec+Fec+ ewes, despite significant differences in ovulation rate. Ovariectomy during the breeding season resulted in significantly higher (P < 0.001) mean peripheral FSH concentrations in all three genotypes. After ovariectomy, mean FSH concentrations between FecBFec+ and Fec+Fec+ ewes, from both the backcross and F2 populations, were not significantly different. However, mean FSH concentrations in the F2 population were significantly higher in FecBFecB ewes than in the other two genotypes. The pattern of differences between genotype in peripheral FSH concentrations and ovulation rates suggest that FSH is not wholly responsible for differences in ovulation rate between genotypes. The results support the hypothesis that the FecB gene is operating both within the ovary and at the level of the hypothalamus and pituitary gland.

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R. Webb, G. Baxter, D. McBride, M. Ritchie and A. J. Springbett

Summary. Three experiments were carried out during seasonal anoestrus in Finnish Landrace and Scottish Blackface ewes, to establish whether the differences between the breeds in ovulation rate are functional during the non-breeding season and are therefore independent of the mechanism controlling ovulation.

In Expt 1, follicles ⩾2 mm in diameter were dissected from the ovaries of both breeds and incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. In both breeds, follicles producing ⩾500 pg oestrogen/ml/h (oestrogen-active) were readily identifiable from a population producing less (oestrogen-inactive). The number of oestrogen-active follicles in each breed was similar to the number of ovulations near the end of the breeding season. Oestrogen-active follicles also had more luteinizing hormone (LH) receptors and larger diameters than oestrogen-inactive follicles. There were, however, no significant differences between the two follicle types in follicular fluid or in-vitro testosterone concentrations.

In Expt 2, seasonally anoestrous Scottish Blackface ewes were unilaterally ovariectomized; the second ovary was removed 7 days later. Follicles from both ovaries were processed as described for Expt 1; oestrogen-active follicles were categorized according to their ability to produce >500 pg/ml/h. There were twice as many oestrogen-active follicles in the second ovary as in the first ovary; the number of oestrogen-active follicles in the second ovary was also similar to the total number of oestrogen-active follicles in both ovaries of the Scottish Blackface ewes in Expt 1. There were no significant differences between the first and second ovaries for any of the other parameters measured in oestrogen-active follicles. There were no significant changes in peripheral gonadotrophin concentrations measured 24 h after removal of the first ovary.

In Expt 3, seasonally anoestrous ewes of both breeds were challenged with an ovulatory dose of human chorionic gonadotrophin (hCG) (750 iu). There was a significant difference in the mean number of ovulations between the breeds and it was representative for the breed (Finnish Landrace 2·6 ± 0·2; Scottish Blackface 1·6 ± 0·2 mean ovulations per ewe). None of the saline-treated controls ovulated.

The results demonstrated that the mechanism controlling the number of mature, oestrogen-active follicles, and hence ovulation rate, is functional during seasonal anoestrus. This conclusion was confirmed by the observation that compensatory ovarian hypertrophy also occurs during seasonal anoestrus.

Keywords: sheep; follicle; ovulation rate; steroidogenesis; seasonal anoestrus

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R. Webb, G. Baxter, R. D. Preece, R. B. Land and A. J. Springbett

Summary. 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 ∼ 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 < 0·01). FSH and LH concentrations increased significantly in Groups 3 and 4, but values in Group 4 were significantly lower (P < 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 < 0·05), but higher pulse amplitude (P < 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.

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S J Martins da Silva, J O Gardner, J E Taylor, A Springbett, P A De Sousa and R A Anderson

The ability of an oocyte to support early embryonic development requires both nuclear and cytoplasmic maturation. We have investigated the effects of brain-derived neurotrophic factor (BDNF) on maturation of the bovine oocyte and embryo development after parthenogenetic activation. By RT-PCR and immunohistochemistry, cumulus and oocytes were shown to express mRNA and protein for BDNF and the p75 common neurotrophin receptor. However, mRNA for the BDNF-specific full length and truncated isoforms of the TrkB receptor are only detected in cumulus, suggesting that oocytes and cumulus differ in their capacity to respond to neurotrophin signalling. In in vitro maturation experiments, the proportion of cumulus oocyte complexes maturing to metaphase II was not altered by BDNF in groups lacking fetal calf serum (FCS), but was significantly lower than the positive control containing 10% FCS (P < 0.01). However, after maturation, the proportion of parthenogenetically activated oocytes forming blastocysts was highest for 10 ng/ml BDNF (24%, n = 95) followed by 100 ng/ml BDNF (18%, n = 91) and 10% FCS (15%, n = 103), which in turn were greater than no serum (10%, n = 83; P < 0.01). Maturation in the presence of a BDNF blocking antibody resulted in a blastocyst yield that was comparable to the absence of serum, and lower than in the presence of BDNF (P < 0.01). Similar effects on progression to metaphase II and blastocyst formation were observed using oocytes matured without cumulus. Together, these results provide the first evidence for a role for neurotrophins in promoting oocyte cytoplasmic competence to support embryonic development, despite being insufficient in the absence of serum to enhance nuclear maturation.