Cryopreservation Of The Fibroblast In Black Bear And The Effect On Black Bear-Bovine Cloned Embryos

In this study, we adopted the method of primary culturing on the fibroblast of black bear to screen the most suitable kind of cryoprotectant and freezing method, and we also discuss the influence of the treated fibroblast to the development of black bear-bovine cloned embryo. Thus we can establish a suit of reconstructed embryo culture system which is systemic, stabile, high performance to improve the blastular developing rate.As a result, we can improve the efficiency of producing transgenosis animals, cloned animals, test tube animals, and so on, make the technic not only used in laboratory but also in practicing. It can apply a theory evidence to the practicing, too. The result is as follow:By means of Method and enzymic method culturing, obtained the black bear fibrolast system.Freezing the tissue cubes, compare to the unfreezing one, as a result the unfreezing tissue cube begun to appear free cells after culturing 5d, the rate of survival cell is 75%;and in the anabiosised freezing tissue cubes, the free cell appeared after culturing 6d, the cell survival ratio is 68.7%.2. Select and apply three kinds of cryoprotectant in different concentration:dimethyl sulfoxide (DMSO:5,7.5,10,12.5%), ethylene glycol(EG:10,15,20,25%), and glycerin (GC:5,10,15,20%), to freeze the black bear fibroblast, and the outcome is the DMSO in a concentration of 7.5% is most suitable.3. Freeze the black bear fibroblast in-70℃and in-196℃separately, for one week, one month, and one year, examine the cell survival ratio after anabiosis, cell viability tests found a higher survival rate of the week-70℃,-196℃week, month, three months, no difference in survival rate.4. Use the 3-6,7-11,and 10-12 generation fibroblast of the ear skin of black bear as honor nucleus, find that the 7-9 generation is the best, and also for the development of reconstructed embryo 5. Freezing 3-6,7-9,10-12 generation of the fibroblast of black bear ear skin in DMSO at a concentrate of 7.5% separately, then use them as donor nucleus, the index of cleavage rate, coenocyte rate, and blastula rate has no significant difference(P>0.5).,But the 7-8 generation group is better than the other two.6.Examine the cell activity of freeze and anabiosis in generation 3-6,7-9,and 10-12.The result is generation 3-6 has no difference, generation 7-9.10-12 has a significant difference.