| There were many factors influenced the success rate of mammal nuclear transfer from preparation of donor cells and oocytes, reconstruction combination of embryos, culture of reconstructed embryos, pregnancy to birth of foetus. In vitro culture of donor cell is one important factor of them. In our studies, the method of separation and culture were investigated on bovine skin fibroblast cell, granulose cell and oviduct epithelia cell in order to establish a steady method of donor cell culture in vitro, and the same time the biological characteristics of somatic cells were studied and discussed. The results as follows:1. DMEM and 1640 are more appropriate to the culture of bovine ear skin in vitro than M199.2. Hot digestion, cold digestion and cold digestion combined hot digestion all can gain many bovine ear skin fibroblast cells, and cold digestion combined hot digestion bring more fibroblast cells and high yield of viable proliferating fibroblast cells than hot digestion and cold digestion.3. Effect of three different culture methods on growth of subculture fibroblast were studied. Digested tissue culture method was better than cell culture method and tissue culture method on gaining higher touch rate of cells, more alive cells and shorter culture time.4. In 37℃, 0. 25%trypsin+0. 02% EDTA was an appropriate digested medium for subculture of bovine granulose cells.5. The rate of chromosome numerical aberration of skin fibroblast cells (passage 0-2), granulose cells (passage 0-4) and oviduct epithelia cells (passage 0-3) were low (less than 10%) in vitro, and those cells can be used as donor cell in nuclear transfer.6. The volume of culture medium has more important effect on the touch rate of primary granulose cell than culture area. 1. 33mL of culture medium in 6-well culture plate was more appropriate to the development of cells than other groups, and there. were no significant difference in touch rate of primary granulose cell between different culture area.
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