Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer

Nuclear transfer has the potential to produce large numbers of identical progeny. Current limitations of the technique are associated with the use of micromanipulation for the demanding enucleation and reconstitution procedure. With the overcoming of this limitation, increased numbers of nuclear transfer embryos could be produced. Centrifugation of bovine oocytes at 15,000 x g for 2 min resulted in the stratification of organelles within the cytoplasm which positioned the metaphase II spindle for enucleation. After removal of the zona pellucida with Pronase, the oocytes were centrifuged in a Percoll density gradient so that the oocytes were stretched apart to form cytoplasts and the metaphase II spindle was separated from the majority of oocytes. Enucleation by centrifugation efficiently produced a consistent population of enucleated cytoplasts from bovine in vitro-matured oocytes. The population of enucleated cytoplasts was enriched by exclusion of the cytoplasts that exhibited an extrusion cone containing metaphase II chromosomes 6 h after centrifugation. The enucleated oocyte cytoplasts were aggregated with blastomeres isolated from in vivo-collected morulae. The aggregated embryonic cells were electrofused to obtain nuclear transfer embryos that were placed into a sodium alginate false zona and were capable of cleavage and development in vitro. The development of nuclear transfer embryos produced through use of centrifugation and aggregation techniques was comparable with that of nuclear transfer embryos produced by micromanipulation techniques.