Establishment And Characterization Of Ear Skin Fibroblast Cell Lines Derived From Wandong Bulls

Currently,cell engineering technogy,including cell culture,has become a powerful auxiliary means for protection of livestock and poultry genetic resources.Found recently in Anhui province,Wandong cattle has been accepted as a newly breed approved and registered by national yellow cattle genetic resource commision.Wandong cattle has strong adaptability,good meat performance.However,the number of existing population is declining,so it is urgent to be protected.In this experiment,we used tissue explants adherent culture method to established the ear skin fibroblast cell lines of two Wandong bulls.We analyzed the cytobiology characteristies of the cell line which we have established,tried gradient centrifugation to separate the somatic cells from the frozen-thawed semen,which will lay solid foundation for banking of Wandong bull's cells.The main results as follows:(1)The cultured cells were typical long spindle shaped fibroblast morpha,which have good state.The growth curve was "S" type.(2)The average cell viability of Wandong bull 34115053(later referred to as A for short)(Passage 2,P2 for short)before and after the cryopreservation was 99.75%,91.82% respectively and Wandong bull 34114125(later referred to as B for short)(P2)was 97.88%,92.90% respectively.(3)Seventy cells of A(P5)and B(P11)were observed respectively,and the frequency of diploidy(2N=60)was 91.43%(64/70)and 82.86%(58/70).(4)The proliferation rate and number of cells cultured in 15% serum were better than that of 15% Sijiqing,but the rate of proliferation was similar to that of 10% Gibco serum.Overall,the 10% concentration of Gibco serum was the best choice for cell.(5)There was no significant difference in cell status and logarithmic growth phase among cells of different generations of A(P2,P5,P8)and B(P3,P6,P9),which need further study.In all,the bovine fibroblast cell lines of two Wandong bulls were successfully established,which could be used as a genetic material for protecting resources of Wandong bull,and laid a solid foundation for the establishment of the Wandong bull's cell bank.