Acquisition of cloned bovines of defined sex by repeated cycles of nuclear transfer

The aim of this study was to confirm the usefulness of and expand multiple generational cloning, so-called repeated cycles of nuclear transfer (RNT). In vitro matured bovine oocytes were enucleated and then parthenogenetically activated. These oocytes were electrically fused with blastomeres from morulae whose sex had been determined by polymerase chain reaction (PCR) detection of a male-specific SRY DNA sequence. The in vitro developmental rate (13.5%, 12 of 89) of the resulting fusion embryos (first generation embryos) to the morula stage was comparable to that (22.1%, 95 of 429) of control oocytes which had been activated but not fused. In each case, sex of the examined first generation clones was identical to that of the donor nuclei. When a blastomere from the first generation clone (morula) was fused to an enucleated oocyte to create a second generation embryo, the rate of development of the resulting fusion embryos to the morula stage was found to be significantly reduced (7.0%, 3 of 43). The sex of each of the second generation clones tested was identical to that of the intial donor nuclei. These findings indicate that PCR-mediated sex detarmination is useful for predicting the sex of RNT embryos.