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Freezing ovarian cortex is a new option to preserve the fertility of young patients undergoing cancer treatment or in women facing premature menopause. However, the best way to use this banked tissue remains unclear. The function of heterotopic and orthotopic autografts of frozen-thawed ovarian cortex of sheep was compared in the present study. Fresh and frozen-thawed fragments of ovarian cortex were autografted on the uterine horn of six ewes (orthotopic grafts) and under the skin of the belly in nine ewes (heterotopic grafts). In both orthotopic and heterotopic grafts, the resumption of follicular growth and ovulation was monitored. In orthotopically grafted ewes, fertility was recorded. Oocytes from both types of grafts were collected, matured and fertilized in vitro. In both fresh and frozen-thawed grafts follicular growth resumed normally; preantral and antral follicles were first detectable 4 and 10 weeks respectively following grafting but only 5% of the primordial follicles appeared to have survived. This confirms that grafting procedures are more deleterious for follicle survival than cryopreservation. Although ovulation resumed in most ewes, none of the ewes grafted orthotopically became pregnant at a synchronized mating. Seven months following grafting, oocytes could be collected from heterotopic and orthotopic grafts, matured and some of them fertilized, but none developed to the blastocyst stage. Heterotopic grafting may be an alternative to orthotopic grafting to preserve fertility provided follicle survival in the grafts is markedly improved.

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Recently, we demonstrated the relationship between anti-Mü llerian hormone (AMH) circulating concentrations, ovarian follicles, and embryo production in cattle. However, they have not yet been established in a species with a seasonal breeding activity. Thus, goats were subjected to repeated in vivo embryo production during the breeding season, at the end of the breeding season, and at the end of the anestrus season. Embryo production after FSH treatment was highly repeatable for each goat. Plasma AMH concentrations, measured before the first FSH treatment, were highly correlated with the number of collected, transferable, and freezable embryos, resulting from the three sessions of embryo production. Plasma AMH concentrations transiently decreased after each exogenous FSH treatment, but they showed little change with season, and no relationship was observed between AMH and endogenous FSH concentrations during seasonal transitions. Follicles of 1-5 mm in diameter were the main target of the FSH treatment and were major contributors to circulating AMH concentrations. Granulosa cell AMH expression decreased as the follicle approached terminal development, while the expression of maturation markers (CYP19A1 and FSHR) increased. In conclusion, circulating AMH concentrations can be predictive of the capacity of a donor goat to produce high or low numbers of high-quality embryos. This prediction could be accurately made from a single blood measurement of AMH during either breeding or anestrus seasons. Variability in the number of gonadotropin-responsive follicles of 1-5 mm in diameter between individuals resulted in the differences in circulating AMH concentrations measured between individuals.

Today, although not efficient enough to replace multiple ovulation and embryo transfer, in vitro embryo production for small ruminants is a platform for new reproductive technologies, such as embryo sexing, transgenesis and cloning. The in vitro embryo-production system developed for sheep and goats is more efficient now than 15 years ago, but could still be improved. Laparoscopic collection of oocytes in live animals treated with gonadotrophin indicates a promising future for the application of this technology to genetic improvement programmes. Oocyte maturation in defined medium with epidermal growth factor and cysteamine appears as efficient as oocyte maturation in follicular fluid-supplemented medium and allows future study of the effect of other factors involved in the cytoplasmic maturation of oocytes from these species. Further efforts have to be made to standardise the semen-capacitating process and to improve the quality and freezability of in-vitro-produced (IVP) embryos. The optimisation of IVP procedures for deer species has required the study of the seasonal variation of oocyte competence and the development of a specific methodology to allow the culture of embryos up to the blastocyst stage.

Booroola ewes have a major gene that affects ovulation rate. Gene expression has consequences on ovarian somatic cells but it is unknown whether it also affects germ cells in the adult ovary. Hence, the present study examined (1) whether oocyte growth was similar in FecBFecB and Fec+ Fec+ oocytes during preantral and antral follicular growth, (2) whether the patterns of proteins neosynthesized by oocytes of these two genotypes were identical, (3) whether the ability of the oocytes to resume meiosis was unaffected by genotype and (4) whether, after IVF, oocytes from both genotypes could develop to the blastocyst stage at similar rates. Histological examination of the respective sizes of the oocyte and of the follicle demonstrated that oocytes were larger in FecBFecB versus Fec+ Fec+ preantral follicles. Resolution of the proteins neosynthesized by FecBFecB and Fec+ Fec+ oocytes by one-dimensional PAGE and image analysis demonstrated that quantitative (but not qualitative) differences could be observed between genotypes for bands at 74, 59, 35 and 25 kDa. In addition, a genotype by oocyte size interaction was detected for two additional bands at 45 and 43 kDa. After 24 h of culture in vitro in TCM-199 plus 100 ng ml-1 FSH plus 10% sheep follicular fluid, oocytes from FecBFec+ follicles gained the ability to resume meiosis at a smaller size and a higher proportion of them reached metaphase II irrespective of the size class studied compared with Fec+ Fec+ follicles. In addition, the developmental rate of eggs after IVF was also affected by follicle size and genotype, since FecBFec+ oocytes originating from 1.0-3.5 mm follicles had a greater ability (P < 0.05) to develop to the blastocyst stage than Fec+ Fec+ oocytes. It is concluded that the FecB gene, in addition to its effects on granulosa cell maturation, also affects oocyte development and function. Whether these alterations are related requires further investigation.

were collected by flushing the oviducts 28 h after the LH surge, and were fertilized and cultured in vitro for 7 days. Ovulation and cleavage rates were not significantly different among the three groups but a higher rate of blastocysts (P < 0.01) was obtained after Antarelix treatment when LH pulsatility was re\ x=req-\ established (group B). Oestradiol concentration was strongly depressed (P < 0.0003) after Antarelix treatment in group A, but was maintained after injection of LH pulses in group B, although at a lower value than before the preovulatory surge in the control group. In conclusion, inhibition of endogenous LH pulses 1 day before the preovulatory surge was not essential for ovulation and in vitro fertilization but was associated with a decrease in plasma oestradiol concentrations and inferior embryo development both in vivo and in vitro. When LH pulsatility was re-established, oestradiol concentrations increased and embryo development was restored.

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