Isolation And Identification Of Muscovy Duck Spermatogonial Stem Cells,and Optimization Of Its Culture System

In this study,Muscovy duck as the research object,provide the basic information for long-term culture of SSCs conducted Muscovy duck and genetic manipulation is provided through the isolation and identification of Muscovy duck spermatogonial stem cells(SSCs),and the optimization of its culture system1.Muscovy duck embryo age to 28-30 as the experimental material.The testicular single cell suspension is identified and cultured by two-step enzymatic digestion method combined with differential adhesion method to testicular single cell suspension.The SSCs exist in the single cell suspension was proved by morphological identification,AKP staining and specific gene expression testing.2.SSCs is cultured in vitro by different feeder layers and different serum concentrations.Count the rate of proliferation and colony of SSCs.The proliferation rates of SSCs culturing in feeder layer free,fibroblast feeder layer and Sertoli cell feeder layer are 106.62%,125.14%and 126.87%.And the colony rates of SSCs are 3.30%,27.80%and 29.85%.The proliferation rates of SSCs culturing in culture medium containing 0%,5%,10%,15%of FBS are 26.29%,123.81%,126.73%and 119.12%.And the colony rates of SSCs are 0%,30.78%,28.08%,25.08%.Different culture system with feeder layer or serum was significantly better than the control group(P<0.01).SSCs cultured in a culture system which contain feeder layer,and adding a appropriate level of serum grow well and proliferate rapidly.3.SSCs is cultured in vitro in different culture systems add different concentrations of leukemia inhibitory factor(LIF),basic fibroblast growth factor(bFGF),stem cell factor(SCF)or glial cell line-derived neurotrophic factor(GDNF).Detect the effects of cytokine concentration on the proliferation of SSCs by MTT assay.Compare with the control group,adding cytokine can significantly improve the proliferation level of SSCs(P<0.01).LIF and SCF promote SSCs proliferation at low level,and inhibit SSCs proliferation at high level.The optimal working concentrations of LIF and bFGF are 10-20ng/ml and 20ng/ml.The effects of different concentrations of bFGF on the proliferation of SSCs are not significantly different.The optimal concentration of bFGF is 10-30ng/ml.And the optimal concentration of GDNF is 20-30ng/ml.In summary,the Muscovy duck SSCs can be obtained through the two-step enzymatic digestion combine with differential adhesion method.The proliferation of SSCs in vitro will be promoted by using embryonic fibroblast feeder layer cells or supporting cells.The culture system with appropriate serum or cytokines can also promote the proliferation of SSCs effectively.