A series of related experiments and studies has been conducted to further enhance the breeding of the chimpanzee and to develop methods for the recovery, maturation and preservation of primate gametes. These studies are important for continued preservation of the chimpanzee and other primates; for development of methods for increasing the population of the bonobo; and for application to the human with regard to problems of infertility. The effects of certain agents known to stabilize oocyte cytoskeletal elements were evaluated for freezing of immature and mature rhesus monkey oocytes. These agents, cytochalasin-B and EGTA (ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetracetic acid, produced a significantly higher (77.6 vs 50%, p<0.01) morphological survival rate than did control media when tested in three replicated experiments. The fertilization rate and development rate of fertilized oocytes were not different between the control and test groups. These results sug gest that use of these stabilizing agents will improve freeze preservation of immature and mature oocytes. A second series of experiments was designed to assess the effects of Antifreeze Protein (AFP) and Insulin-Transferrin-Selenium (ITS) on chimpanzee (Pan troglodyte) spermatozoa during the freeze-thaw process. The AFP or ITS were added to the semen sample at varied concentrations of 0, 1, 10 and 100 mg/ ml. The semen from each animal was processed independently prior to exposure to the AFP or ITS. The fresh semen samples were collected by artificial vagina, analyzed for sperm count, viability, and motility using a computer assisted motion analysis (CAMA). The Curvilinear Velocity, Linearity, and Straight Line Velocity declined somewhat from the original pre-freeze values but remained constant among the different concentrations of ITS. The Lateral Head Movement remained close to the pre-freeze value. A concentration of 100 mg/ml gave no recovery of sperm motility. Best re sults were obtained after addition of 10 mg/ml AFP and 1 mg/ml ITS. These results have significance for the storage of semen at wet ice temperatures for transportation. With respect to collection and maturation of oocytes, preantral follicles have been isolated from four ovaries by two methods; (i) collagenase dissociation 5 mg/ml collagenase enzyme alone was used to dissociate two ovaries. It was possible to collect a clean preparation of up to 150 preantral follicles (with denuded basement membrane of theca cells i.e., oocyte-granulosa cell complex) and 50 or more free cumulus-enclosed oocytes from a single animal. The ages of animals that supplied these follicles were 3 to 18 years. (ii) microdisection, using microdisection we were able to mechanically dissect up to 20 intact preantral follicles from a single animal. This work will be extended in order to isolate a larger number of viable follicles and to devise media which will support their maturation in vitro. Studies on in vitro fertilization have been extended to include the use of oocytes from the pygmy chimpanzee (P. paniscus) and the sooty mangabey (C.atys) in accord with the goa ls of ongoing, funded work. P51RR00165-36 1/1/96 - 12/31/96 Yerkes Regional Primate Research Center
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