| Nuclear transfer technology has made much progress, but the efficiency is stilllow. The detailed technology approaches in the process of nuclear transfer mightaffect the efficiency. In the present study, some details ignored easily were studied ingoat nuclear transfer in order to optimize the procedure. The ovaries were collectedduring breeding season and non-breeding season respectively, and then the oocyteswere collected by surface cutting and then matured in vitro. The results showed that2.82 and 3.02 cumulus-oocyte complexed (COCs) per ovary were harvestedrespectively, and the maturation rate was 57% and 60% respectively. There was nosignificant difference in the collection efficiency and maturation rate (P>0.05).During the non-breeding season, the eligible COCs were collected by the surfacecutting and syringe aspiration, respectively, and then matured in vitro. Every ovaryproduced 3.02 and 1.10 COCs respectively (P<0.05) while the maturation rate was60% and 81% respectively (P<0.05). The goat ovaries were transported for about 8 hat 15-19℃, 20-24℃ and 25-30℃, respectively. The results showed that theoocyte maturation rate was 52%, 68% and 60% respectively. Maturation rate of COCstransported at 20-24℃ was significantly higher than that of the others (P<0.05).When the COCs were matured in vitro in LH/FSH or HCG produced by differentdomestic manufactures, there were no significant differences between the maturationrates among them (P>0.05). When goat parthenogenetic oocytes were cultured inmedium CR1aa, M199 supplemented 10% FCS, and HTF+P1 without cumulusmonolayes, the percentage of cleavage was 71%, 44% and 76%, respectively. Thecleavage rate in M199 was significantly lower than that of the others (P<0.05), but noembryos in any media developed to the blastulas. When parthenogenetic oocytes wereco-cultured in CR1aa, SOFaa and HTF+P1 with cumulus monolayer, the cleavage ratewas 75.68%, 67.33% and 76.27%, respectively, and blastulas rates was 21%, 10% and7%, respectively. The cleavage rates and blastula rates were significantly differentwith each other (P<0.05). When the reconstructed oocytes were electrofused, 250v/mm and one DC (direct current) pulse were used, and differetn pulse durations (10μs, 20 μs and 40 μs) was used, respectively. The results showed that after theelectrofusion, the livability of reconstructed oocytes was 52%, 44% and 37%,respectively (significantly different between any of them, P<0.05). The cleavage rateof the 40 μs electrofusion (65%) was significantly higher than that of the others(P<0.05). The results also showed that the livability of reconstructed oocytes with oneDC pulse electrofusion (52%) was significantly higher than that of two DC pulses(21%, P<0.05), one DC pulse electrofusion produced higher obtainable embryo ratethan two DC pulses (26% vs 15%, P<0.05), while lower cleavage rate (50% vs 69%,P<0.05). The reconstructed oocytes were cultured for 30 min, 90 min, 3-4 h and 9-10 h, respectively, and then electrofusion was undertaken by the optimized procedure.The results showed that the mortality of reconstructed embryos after 9-10 h (27%)and 3-4 h incubation (31%) was significantly lower than that of 30 min (49%) and 90min (56%, P<0.05), and that the cleavage rate of 9-10 h (68%) was significantlyhigher than 30 min (53%) and 90 min (56%, P<0.05), and the same tendency wasfound in the obtainable embryo rates (49% vs 28% vs 24%, P<0.05). Thereconstructed embryos were activated by ionomycin and then by 6-DMAP for 0 h, 2h,3h, 4h, 5 h, 8 h and 10 h, respectively. After activation, the reconstructed embryoswere cultured in vitro. The results showed that the cleavage rate was not significantlydifferent during 2-10 h activation in 6-DMAP (P>0.05), but the cleavage rate ofreconstructed embryos activated for 2-10 h were significantly higher than that ofembryos without activation (0 h, P<0.05). Ear-derived fibroblast cells of Boer goatand cumulus cells of Huanghai white goat were used as the donors for nuclear transferrespectively. The results showed that the fusion rates of reconstructe...
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