Study On Somatic Cell Nuclear Transfer In Boer Goat

The present study was conducted to investigate the somatic cell nuclear transfer in Boer goat, such as donor cell culture, recipient oocyte in vitro maturation, reconstructed oocyte electrofusion and activation, embryo in vitro culture, and embryo transfer. The experiment successfully primary-cultured and sub-cultured ear skin fibroblasts derived from a Boer goat by tissue culture. The different freezing protocols were investigated to cryopreserve the fibroblasts. First, the fibroblast cells were equilibrated at 4℃for 1 h, and stored at -20℃for 6 h, and then plunged liquid nitrogen. Second, the cells were were equilibrated at 4℃for 1 h, stored at -20℃for 6 h and -80℃overnight, and then plunged into liquid nitrogen. Third, the cells were equilibrated at 4℃for 1 h, kept at -80℃overnight before being plunged into liquid nitrogen. The result showed that the adherence rates were most significantly different between them after thawing and culture (2.38%, 22.81 and 57.33% respectively, P<0.01). When goat oocytes derived from abattoir ovaries were cultured in vitro, the effects of epidermal growth factor (EGF), in vitro maturation (IVM) time and bovine follicular fluid (bFF) were investigated. The results showed that the supplementation of 10-30 ng/ml EGF had no distinct effect on the extrusion rate of first polar body (PB1) (P>0.05). The extrusion rate of PB1 of goat oocytes was significantly higher when the oocytes were cultured 21-25 h than that of 17 h (P<0.05), but there were no statistical difference during 19-25 h. Adding 10% bFF had no significantly effect on the extrusion rate of PB1. After electrofusion, the reconstructed oocytes were chemically activated by ionomycin alone, by combination of ionomycin and 6-dimethylaminopurine (6-DMAP), or by 6-DMAP and cytochalasin (CB).The result showed that the cleavage rates were 34.38%, 69.85% (P<0.05), and 72.02% respectively. The cleavage rate and blastocyst rate had no significant difference for reconstructed embryos cultured in 6-DMAP compared with those in 6-DMAP and CB after activation with ionomycin (69.85% vs 72.02%, P>0.05; 8.70% vs 7.50%, P>0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development was 8.70% but no blastocyst formation were obtained without co-culture. The reconstructed embryos were cultured for 1-3 h, 3-6 h, 6-9 h respectively after fusion, and then activation was undertaken by using ionomycin and 6-DMAP. We found that the cleavage rate had no significant difference between 1-3 h group and 3-6 h group (72.58% vs 72.97%, P>0.05), but both the rates were significantly higher than 6-9 h group (64.40%) (P<0.05). A total of 491 nuclear transfer embryos were transferred into 37 recipients. One of them was pregnant and gave birth to a live female lamb after 153 d gestation. But unfortunately, the clone lamb died 8 h after birth. The anatomy and tissue staining told that the clone lamb developed well in viscera.