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J. A. Carnegie, J. J. Morgan, N. McDiarmid, and R. Durnford

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 ± 0.9 pg LIF per 105 cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 ± 6.2 pg LIF per 105 cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1–4) + 10% (v/v) ECS (days 4–10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.

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K. P. McNatty, L. E. Kieboom, J. McDiarmid, D. A. Heath, and S. Lun

Summary. The tissue contents of adenosine cyclic 3′,5′-monophosphate (cAMP) in freshly dissected follicles (0·13–1·00 mm diam.) were significantly higher in Booroola ewes containing a major fecundity gene (FF and F+ ewes) compared to those values in Booroolas with no copy of the gene ( + + animals; P < 0·025). After a 1 h incubation with LH + FSH, the respective proportions of follicles with a diameter of 0·13–0·52 mm (n = 288) and 0·53–1·00 mm (n = 271) that had synthesized ≥0·6 pmol cAMP and ≥1·0 pmol cAMP were significantly influenced by genotype (Booroola ewes homozygous for the F-gene, FF > heterozygous, F+ > ++; P <0·01 for both follicle size ranges).

The contents of progesterone, androstenedione, testosterone and oestradiol-17β in minced ethanolic extracts of freshly dissected follicles (n = 188) were undetectable regardless of Booroola genotype. However, when follicles of 0·53–1·00 mm but not 0·13–0·52 mm diameter were cultured for 48 h with LH + FSH under 70 kPa of a 50% O2, 45% N2 and 5% CO2 gas mixture, the proportions that synthesized high levels of progesterone (≥4·0 ng), androstenedione (≥3 ng), and oestradiol (≥0·8 ng) were significantly influenced by genotype (FF > F+ ≥ ++; P <0·05 for each steroid). No significant genotypic differences were noted for testosterone synthesis.

Collectively, these results show that the Booroola F-gene has an influence on the maturation of ovarian follicles from an early stage of growth.

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K. P. McNatty, N. L. Hudson, K. M. Henderson, S. Lun, D. A. Heath, M. Gibb, K. Ball, J. M. McDiarmid, and D. C. Thurley

Summary. Overall, significantly more antral follicles ≥ 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 ± 3 (mean ± s.e.m.) follicles per ewe, 23 sheep; Day 9–10 of oestrous cycle, 24±1 follicles per ewe, 22 sheep; P < 0·01), although the mean numbers of preovulatory-sized follicles (≥5 mm diam.) were similar (anoestrus, 1·3 ± 0·2 per ewe; oestrous cycle, 1·0 ± 0·1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9–10 of the oestrous cycle there were significantly more (P < 0·001) high-amplitude LH peaks (i.e. ≥ 1 ng/ml) in plasma and significantly fewer (P < 0·001) low amplitude peaks (<1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P < 0·05, prolactin, P < 0·001).

It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.

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K. P. McNatty, S. Lun, D. A. Heath, K. Ball, P. Smith, N. L. Hudson, J. McDiarmid, M. Gibb, and K. M. Henderson

Summary. Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of ≥5 (FF ewes, N = 15), 3–4 (F+ ewes, N = 18) or <3 (++ ewes, N = 18).

Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean ± s.e.m. diameters for the presumptive preovulatory follicles were 3·4 ± 0·3, 4·1 ± 0·2 and 6·8 ± 0·3 mm and in each of these follicles the respective mean ± s.e.m. numbers of granulosa cells (× 106) were 1·8 ± 0·3, 2·2 ± 0·3 and 6·6 × 0·3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4·8 ± 0·4 'oestrogenic' follicles, F+ ewes with 3·2 ± 0·2 'oestrogenic' follicles and ++ ewes with 1·5 ± 0·02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5·4 × 106 cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0·91, P <0.001).

Collectively, these findings support the notion that the maturation of ≥ 5 follicles in FF ewes and 3–4 follicles in F+ ewes may each be necessary to provide a follicularcell mass capable of producing the same quantity of oestradiol as that from 1–2 preovulatory follicles in ++ ewes.

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K. M. Henderson, M. D. Prisk, N. Hudson, K. Ball, K. P. McNatty, S. Lun, D. Heath, L. E. Kieboom, and J. McDiarmid

Summary. Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0·1, 0·5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1·0 ml bFF twice daily (1·9 ± 0·2 ovulations/ewe, mean ± s.e.m. for N = 34) or 5·0 ml once daily (2·0 ± 0·2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1·4 ± 0·1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (≥ 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17β in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17β and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1–2·5 mm diam.) or medium (3–4·5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.