This project will eventually cover induced superovulation, oocyte recovery and culture, semen collection and in vitro sperm capacitation, in vitro fertilization, embryo transfer, and cryopreservation of gametes and early embryos. The preliminary studies on superovulation, oocyte recovery, and sperm cryopreservation were conducted this year. Recombinant gonadotropins were used to stimulate follicular development in rhesus monkeys. Eight monkeys were injected with the gonadotropins starting on Day 2 or 3 of their menstrual cycle. Four of these monkeys had received progesterone to control the timing of their cycles. Oocytes were recovered from 60% of the follicles aspirated. The aspiration was done, using a 1 cc syringe with a 21 gauge needle, under laparoscopic visualization. The presence of follicles 4 mm or larger were detected by ultrasonography. All oocytes appeared normal and none had a first polar body. This method of follicular aspiration was not completely satisfactory and a vacuum system was developed during the nonbreeding season. Follicular stimulation using the same hormonal schedule during the nonbreeding season resulted in some follicular growth but the follicles did not mature. A similar results was previously reported when squirrel monkeys were used for induced ovulation studies. Extending the period of gonadotropin stimulation may result in mature follicles but may also cause a premature LH surge and ovulation. Preliminary studies with sperm cryopreservation involved the use of serial dilutions of an egg yolk-buffer cryoprotectant medium with glucose enriched Tyrode's solution to wash the sperm away from the cryoprotectant medium following cooling, but not freezing. Previous studies in which the sperm remained in the cyoprotectant medium had shown that we could get post-thaw recovery of motility (60% of pre-freeze) but that the sperm did not survive for more than 90 to 120 minutes. The next studies will involve the cryopreservation procedure from cooling to freezing to post-thaw washing with serial post-thaw washing.
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