Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer.

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.