Co-culture of buffalo preantral follicles with different somatic cells

The effect of co-culture of buffalo preantral follicles (PFs) with different somatic cells, i.e, cumulus, granulosa, ovarian mesenchymal and oviductal epithelial cells was studied. Large PFs (250-450 microm) were isolated by microdissecting the trypsin (1%) digested ovarian cortical slices. Cumulus cells were isolated by repeated pipetting of oocytes, granulosa cells were isolated by aspirating from punctured PFs and ovarian mesenchymal cells were isolated from ovarian cortex by scraping the cortical slices and passing through 20 microm filter. Preantral follicles were cultured in standard culture medium without somatic cells or co-cultured with cumulus cells, granulosa cells, ovarian mesenchymal cells and oviductal epithelial cells for 80 days. The growth rate (microm/day) of the PFs was monitored by measuring follicular diameter on day 0, 30, 60 and 80 days of culture. The viability of PFs was evaluated by trypan blue staining. The results indicated that PFs co-cultured with cumulus, granulosa and ovarian mesenchymal cells had a better development and survivality compared with control and those co-culture with oviductal epithelial cells. Maximum growth and survivality of PFs were achieved when cultured with cumulus cells. It is concluded that inclusion of somatic cells in PF culture media had beneficial effect on the growth of PFs and cumulus cells supported maximum growth and survivality of PFs in vitro of all somatic cells tested.