Goat-bovine Inter-species Nuclear Transplantation

The present study on goat-bovine inter-species nuclear transfer dealt with the following six experiments: 1.Isolation and in vitro cultivation of goat ear-skin cells as nuclear donors; 2.In vitro maturation and cultivation of the goat and the bovine oocytes; 3.Application of Spindle-view in enucleation of oocytes from domestic animals; 4.Influences of ages of donor-cell animals and donor cells and nuclear-injection methods on cloning efficiency; 5.In vitro development of cloned goat-bovina inter-species and goat-goat intra-species embryos; 6.Transfer of cloned embryos.1. Isolation and in vitro cultivation of goat ear-skin cells as nuclear donors.Donor cells were obtained from ear tissue of 4 groups of goat including the adult male Boer goat, the six-month-old male Boer goat, the adult female Mayang goat and the six-month-old female Boer goat after sampling, isolation and cultivation.The primary cell culture of goat somatic cell can well established through attachment of ear tissue fragments. The study showed that the cell isolation efficiency of the lamb and the adult goat ear tissue was about 60% and 40% respectively, that the donor cells isolated from adult goat ear skin grew regressively after 5 passages and the addition of uracil and pyruvate of sodium to medium could obviously improve the cells growth.2.In vitro maturation and culture of the goat oocytes and the bovine oocytes. Five media used for in vitro maturation and culture of bovine oocytes cover ① TCM-199 supplemented with 0.22 mg/mL sodium pyruvate, 10 mM/L Hepes, 0.10 IU HMG/mL, 1mg/mL 17?-E2, and 10% fetal bovine serum(FBS), ② SOF solution supplemented with 0.10 HMG IU/mL, 1 mg/mL 17?-E2 and 6 mg/mL BSA, ③ CR1 solution supplemented with 0.10 IU HMG/mL, 1 mg/mL17?-E2 and 6 mg/mL BSA, ④ Medium ① supplemented with ITS and EGF and ⑤ Medium ① supplemented with ITS, EGF and uracil .The visibility rates of PB1 after 22h of in vitro maturation and cultivation in these media were 62.5%(303/485) in medium ①,52.5%(156/297) in medium ②,63.8%(192/301) in medium ③,63.6%(930/1462) in medium ④ and 67.9%(292/430) in medium ⑤. And this rate of medium ② was obviously lower than those of other four media(0.05.05). Three media ①,④ and ⑤ were used for in vitro maturation and culture of goat oocytes and the visibility rates of PB1 after 22 h cultivation were respectively 39.8%(209/525), 71.8%(1129/1572) and 67.9%(285/420). The rate of medium ① was much significantly different from those of other two media(P<0.01) and there was no significant difference between media ④ and ⑤(P>0.05).3.Application of Spindle-view in enucleation of oocytes from domestic animals. The enucleation research in cattle, goat, swine, mice and rabbit indicated that ①there were no obvious changes in the spindle and the position of polar body in matured bovine oocytes after 20 to 26 h of in vitro maturation and culture, ②the oocytes qualities could be evaluated according to the visibility of their spindle images and, ③Spindle-view could be used to observe the miotic spindles in oocytes of rabbit, mice and goat.4.Influences of ages of donor-cell animals and donor cells and nucleus-injection methods on cloning efficiency. Oocytes were enucleated and reconstructed with donor cells of adult goat and 6-month-old lambs to produce embryos for transfer and there was no obvious difference in fusion rate and cleavage rate between the two ages of donor-cell animals (44.5% and 72.0% vs. 48.8% and 65.4%). The fusion rate, cleavage rate and blastocyst rate of reconstructed embryos with enucleated oocytes and donor cells at different passages(up to 5 passages and after 6 passages) of an identical donor-cell animal were respectively 61.1%,60.6% and 35.0%(up to 5 passages) vs. 38.7%,78.3% and 23.4%(after 5 passages). There was a much significant difference of fusion rate between the two ages of donor cells(P<0.01) but no significant differences of cleavage rate and blastocyst rate between them(all P>0.05). And there was n...