Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuc lear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocyt es were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 moru lae by a DC electropulse, and then cultured up to 8 days (non-frozen contro ls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), afte r enucleation and aging culture (group IV), or after activation (group V). In group 1, 91% of IVM oocytes could be used for NT and 89% of them fused s uccessfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups I V and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher tha n those in groups II, ill, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respec tively) were higher than the rate in group II (75%). Rates of development i nto blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V) . Developmental kinetics and cell numbers of the blastocysts were similar a mong the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos i n cattle. (C) 1999 Wiley-Liss, Inc.