104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P > 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.