Production of cloned calves embryos by somatic cells nuclear transfer to enucleated oocytes maturated in vitro

A technology for production of cloned calves embryos by somatic cells nuclear transfer to enucleated oocytes maturated in vitro was developed. The cumulus cells of adult animals were used as a source of karyoplasts. Conditions and parameters for the basic stages of technology are determined. Cytoplast to karyoplast fusion was carried out using electric pulses (1.5-2.0 kV/cm for 10 micro seconds each) in Zimmerman's medium. Reconstructed cells were activated by treatment with Ca++-ionophore and 6-DMAP in 0.5-1.5 hours after the fusion. In vitro cultivation of reconstructed cells was resulted in 20.3-23.6 % of embryos developed up to a blastocyst. In two cultivation system (KSOM medium enriched with replaceable and irreplaceable amino acids under a gas phase of 5 % CO2, 5 % O2 and 90 % N2, and the cumulus cells feeding layer in DMEM medium under a gas phase 5 % CO2 in air) the efficiency of development of the reconstructed calves embryos was the same. Thus, the developed technology allows obtaining up to 23.0 % of cloned calves embryos at the stage of blastocyst.