Isolation characterization and standardization of culture of goat spermatogonial stem cellisolation characterization and standardization of culture of goat spermatogonial stem cell

Abstract

The current study was carried out to i) examine the morpho-biometric evaluations of testes newlineand comparative analysis between pre and post pubertal spermatogonial stem cell (SSC) newlineculture colonies ii) examine the effect of digestion method on isolation of SSC iii) study the newlineeffect of lectin coating on SSCs and feeder layer of sertoli cells iv) analyze the effect of newlinesupplementation of growth factor on proliferation of SSC colonies v) analysis the effect of newlinefetal bovine serum (FBS) on SSC colonies in different concentration of FBS with respect to newlinetotal number of colonies and number of passages in SSC culture vi) standardization of SSC newlineculture in different types of culture media vii) analysis of SSC culture in breeding and newlinenonbreeding season with respect to proliferation of SSC colonies due to seasonal effect. SSCs newlinewere isolated from pre and post pubertal slaughtered goat testes by double enzymatic newlinetreatment and through mechanical method. The isolated cells were enriched by filtration newlinemethod (80 µm and then 60 µm net filters), differential adherence selection method and newlinepercoll density gradient centrifugation method. SSC cells were cultured on sertoli cell feeder newlinelayer culture conditions were optimized by observing the effect of age of buck, digestion newlinemethods, coating, growth factors, culture medium, FBS with respect to total number of newlinecolonies and number of passages in SSC culture. SSC colonies were further characterized by newlineexamining the expression of alkaline phosphate, immunofluorescence characterization by newlinepluripotent markers NANOG and OCT4 and SOX2 and SSC specific marker PGP9.5 and newlinePLZF. The results of the present study suggest that i) all the biometrical parameters except newlinedensity were significantly higher (plt0.05) in the post pubertal testes as compared with pre newlinepubertal testes but isolated cells and proliferation of SSC colonies was significantly higher in newlineculture of pre pubertal SSC culture ii) enzymatic digestion method showed significantly newlinehigher number of cells from mechanical method i