Buffalo spermatogonial culture and differentiation in vitro

To explore a developmental potential of buffalo spermatogonial culture in vitro,3-5-month-old buffaloes were used to spermatogonial and sertoli cells co-culture in vitro.The germ cells were obtained by two-step enzymatic digestion and were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) for 30 d at 37℃ in a humidified atmosphere with 5% CO2.Growth and morphologic changes of cells were observed during culture and cultured cells at 4 weeks were examined by reverse transcription-polymerase chain reaction (RT-PCR) for protamine-2 (PRM-2) and transition protein-1 (TP-1) mRNA.The result showed that buffalo spermatogonial(10.0-12.5 μm) were round and adhered to sertoli cells at 24 h later.After 1 weeks of culture,spermatogonial were congregating growth.Clones were formed after 10 days and elongated spermatid-like cells(8.0-10.0 μm) were observed after 30 days of culture.The expression of the spermatid-specific marker gene,PRM-2,was confirmed after 4 weeks of culture by RT-PCR.In conclusion,the culture conditions could sustain the survival and allow the proliferation and differentiation of buffalo spermatogonial during long-term culture in vitro,and ultimately spermatid-like cells were formed.