Bovine Nuclear Transfer Using Fresh Cumulus Cell Nuclei andIn Vivo- orIn Vitro-Matured Cytoplasts

Abstract

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.