EFFECTS OF SUPEROVULATION, CULTURE AND MICROINJECTION ON DEVELOPMENT OF RABBIT EMBRYOS IN-VITRO

Factors influencing the developmental potencial of cultured rabbit zyg otes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n=1053) were recov ered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated g roup (18 vs 9 ova). However, the quality of recovered zygotes (presenc e of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor -supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transfe rrin and sodium selenite) the percentage of morula/blastocyst stage em bryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fe tal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and we re examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, res pectively) than in; microinjected embryos (31.0 and 21.5%, respectivel y). Summarized data of the PCR assay of microinjected zygotes demonstr ated positive signals fdr the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes. (C) 1998 by Elsevier Sci ence Inc.