Cryopreservation of bovine pre-morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before use in nucleus transfer.

We have determined that the tolerance of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos to cryopreservation at the pre-morula stage can be improved by removal of cytoplasmic lipid droplets by centrifugation. Nucleus transfer was also performed using cryopreserved, delipated (lipid droplets removed) 8- to 16-cell-stage blastomeres of IVM/IVF embryos as donor nuclei. In vitro developmental ability of the delipated embryos to the blastocyst stage (20 of 126) was found to be equal to that of undelipated embryos (35 of 176); and of 53 delipated embryos cryopreserved at the 8- to 16-cell stage, 12 developed into blastocysts in vitro after thawing. On the other hand, only 2 of 43 undelipated embryos and 5 of 59 sham-operated embryos survived (p < 0.05). When blastomeres isolated from cryopreserved, delipated 8- to 16-cell-stage embryos were used for nucleus transfer, 57 of 80 successfully fused with enucleated oocytes, which was significantly lower than the fusion rate obtained with blastomeres of unfrozen, undelipated embryos (93 of 104, p < 0.01). However, the developmental rate to the blastocyst stage for nucleus transfer embryos reconstituted with frozen, delipated blastomeres (9 of 57) was not different from that of the nucleus transfer embryos with unfrozen, undelipated embryos (23 of 93). These results confirm that removal of cytoplasmic lipid droplets from bovine IVM/IVF zygotes allows for successful cryopreservation at the 8- to 16-cell stage and that blastomeres from these embryos can be used as donors of karyoplasts for nucleus transfer.