The Study Of Somatic Cells Cloning Technology In Luxi Cattle's Conservation

The paper studied the application of somatic cell cloning technique in protecting domestic animal's genetic resources. It is based on domestic animal protecting programming and used culture method to establish Luxi cattle-ear skin fibroblast and epithelial cell lines which are not established until 2004.The efficiency of nuclear transfer using 6 passage epithelial cells as nuclear provider and matured oocytes as nuclear suffer was studied. Recipient cytoplasts were prepared from cumulus-oocyte complexes(COCs) which was got from ovaries collected from slaughter house. The results as follows:1. Luxi cattle-ear skin fibroblast and epithelial cells were derived by fragment of tissue. The primary and earlier period culture cells were mixture of fibroblast and epithelial cells.2.The study demonstrated the repeated attachment method could derive and pure fibroblast and epithelial cell lines because when the mixture growing cells were handled with trypsin(0.25%) in 2~3min by 37℃,the major cells of floating cells were fibroblast cells.3. The Propagate regulation of Luxi cattle-ear skin fibroblast cells was researched by drawing growth curve. It showed that the 4 passage cells cultured by high-sugar DMEM were in lag phase in first two day, then entered log phase and persisted six days, and began to die at eight day because of contact repression, they stayed at platform phase for about three days, cells died gradually.4. Luxi cattle-ear skin fibroblast cells contained normal chromosome content at least the twelfth passage.5. DMSO was better than glycerin as protection in cell refrigerate test(p<0.05) and the higher serum density was better in cell refrigerate test.6. It was proved that It could be derived and cultured normal cells which nuclear type was same to fresh tissue from refrigerate tissue by succeeding getting cells from refrigerate Luxi cattle-ear skin. 7. In cell cloning, serum starvation and contact repression were studied as handle method of provider cells. The results showed that the ratio was different(p<0.05) and the blastocysts ratio wasn't different(p>0.05).8. Differential staining of the inner cell mass(ICM) and the trophectoderm(TE) cells of blastocysts(Day 7)was performed. ICM was 37(44.2% of total cells) and TE was 47.9. It was proved the cell cloning technique could used in animal protection when cloning Luxi cattle in the experiment. We got 66 blastocysts, then used 10 cattle as nuclear transfer suffer . .The ratio of pregnancy(60 days) was 20%(2/10).