Skin Fibroblast Cell Culture And Heterogeneous Embryo Reconstruction In Horse

Horse skin fibroblasts were cultured in different ways and induced to quiescence (Go Phase) by serum starvation culture. The cells were injected into the enucleated bovine oocytes to reconstruct embryos.Horse skin tissue fibroblasts were dissociated and cultured in vitro. The results of experiment showed that: The primary cultured cells of horse skin tissue were obtained by explant tissue culture and typsin digestion at 37℃,100% humidity and 5% CO2; The collegenase I solution (in DMEM supplemented with 100-150 IU/ml and 10% NCS) was suitable for the dissociation of horse skin tissue, and trypsin digestion was feasible for separating homogeneous fibroblasts from the mixture of fibroblasts and epithial cells after repeatitial attachments for two or three times of subcultures; Desirable passaged cells were obtained when the primary or passaged cells were treated for 4 min in TE at 37℃, and the digestion enzyme dissolved with D-PBS was suitable for digestion; DMEM + F12(1:1) supplemented with 10% NCS well supported the fibroblast growth. Addition of cGMP(0.2mM) and Insulin(20μg/ml) to the culture medium respectively promoted the division of the fibroblasts; Horse skin fibroblast had 81.3% normal chromosome content after at least twelve passages(2n=64); DMEM supplemented with 10% DMSO and 20% NCS was the most efficient cryopreserving solution(efficiency of attachment at 24h after thawing, 84.98%); The effects of cryo- preservation in programming-cryo-preservation were obviously better than those in manual cryo-preservation (86.32%,79.98%). The horse fibroblasts were used as donor of nuclear to reconstruct cloned embryos with enucleated bovine oocytes. The results of experimerts indicated that: Matured bovine oocytes were efficiently activated by 5mM Ionomycin,A23187 and 7% ethanol together with 6-DMAP, and developed to blastocysts. The percentage of cleavage and the rate of blastocyst development, which were activated by Ionomycin-6-DMAP, were significantly higher than those of activated by the others(p<0.05); When the actived oocytes were cultured in SOFaa added with 10% FBS and cumuluses, a higher rate of cleavage and blastocyst development were achieved(72.30%,14.91%); Co-culture of oocytes and cumuluses stimulated the development of the constructed cloned embryos; The cloned embryos reconstructed by sub zona pellucida injection associated with electrofusion. Under the condition of 20V start-voltage, 10-second duration, 0.2MHz frequency, 15V end-voltage, 2 pulses and 0.125 seconds internal, when the amplitude was 2.0KV/cm and pulse duration was 40μs, both the rates of fusion and the cleavage of reconstructed embryos reached peak values (52.27%, 71.74%); The reconstructed embryos progressed to the 8-cell stage.