Studies on Preservation of Enucleated Oocytes and Nuclear Transplantation in Cattle and Horses

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Enucleated oocytes can support reprogramming of transferred nuclei and development of the zygotes produced by the nuclear transfer. The fitst aim of this study was to improve cloning efficiency in cattle, throughout the development of cryopreservation methods for enucleated oocytes. Results are summarized as follows:( 1 ) Addition of linoleic acid-albumin, which improved freezing tolerance of in vitro-produced bovine embryos, to the media during the preparation process of enucleated oocytes ( IVM, enucleation, and activation ) was effective to produce oocytes tolerable to freezing ( Hochi et al., J. Vet. Med. Sci., 2000 ).( 2 ) Vitrification of in vitro-matured bovine oocytes at a cooling rate of 3,000-5,000℃/min resulted in no loss of the viability when judged from routine IVF ( Hochi et al., Cryobiology, 2001 ). The second aim of this study was to establish the system for in vitro production of equine!embryos, for the future progress in horse cloning by nuclear transfer. Results are summarized as follows:( 3 ) Using sperm penetration assay into zona-free hamster oocytes, treatment of stallion spermatozoa with ionophore A23187 at 0.1 μM for 2 min was found to induce sperm capacitation and acrosome reaction effectively ( Matsukawa et al., J. Reprod. Dev., 2002 ).( 4 ) Intracytoplasmic injection of stallion spermatozoa following the A23187 treatment resulted in the normal fertilization rate of 65 % and successful production of blastocyst-stage embryos in vitro ( Matsukawa et al., Poster presentation at IETS meeting, 2002 ).