Efficiency of asynchronously in vitro-matured oocytes as recipients for nuclear transfer and of blind enucleation in zona-free bovine cloning.

We studied the dynamics of in vitro maturation of bovine oocytes, the efficiency of asynchronously matured oocytes as recipients for the generation of embryos produced by nuclear transfer, and the potential for using blind enucleation of zona-free bovine oocytes in bovine cloning. At 15 h after the initiation of maturation (hpm), oocytes were freed from both cumulus cells and the zona pellucida, and the dynamics of oocyte maturation were monitored every 30 min through the criterion of extrusion of the first polar body (PB1). More than 41% of bovine oocytes had extruded PB1 by 16.5 hpm, and were designated as representing a group of rapidly maturing oocytes. A second group, comprising about 25% of all oocytes, had extruded PB1 by 18.5-20.0 hpm. Examination of Hoechst 33342-stained samples demonstrated that PB1 on the surfaces of zona-free bovine oocytes were always located near the maternal chromosomes. Zona-free oocytes were enucleated by removing PB1 and about 3% of the adjacent oocyte cytoplasm without chromatin staining. Successful enucleation of zona-free bovine oocytes was achieved in 96.9% of cases. The rate of development to the blastocyst stage was significantly greater in embryos reconstructed from rapidly maturing oocytes (47.8%) than with oocytes maturing at 18.0-20.0 hpm (33.3%). Overall, two large groups of bovine oocytes could be distinguished during in vitro maturation by the time required to reach the second stage of metaphase. Bovine embryos reconstructed from rapidly maturing enucleated oocytes had a significantly greater rate of development to the blastocyst stage than did embryos derived from later-maturing oocytes. We conclude that blind enucleation is a simple and efficient method for preparing cytoplasts in zona-free bovine cloning.