The present study assessed general strategies for the isolation and in vitro culture of immature ovary-derived cells (oocytes) in red swamp crayfish (Procambarus clarkii). In phase 1 of this study, the effects of different digestive enzymes on cell retrieval from developing ovarian tissues were examined to facilitate efficient oocyte isolation. Subsequently, the ovary-derived cells/oocytes were cultured under various conditions to evaluate the effects of basal media and supplement concentrations. Collagenase I treatment at 26 ℃ for 25 min was more effective (approx. 95 %) in oocytes isolation from ovarian tissues than other treatments. Furthermore, DMEM media with HEPES (DMEM-H) were more effective than L15 for oocyte culture (72.4 % survival rate), while oocyte survival improved to 90.6 % using a 10 % fetal bovine serum supplement. In phase 2, the top-performing media were used in immature oocyte culture to assess 17α-Hydroxyprogesterone (17-OHP) induction in vitro. An up-regulated expression of maturity-related genes, i.e., cyclin B, cdc2, ef1α, after 12 h of incubation was evident in qPCR results, demonstrating that the exogenous hormone 17-OHP could induce P. clarkii oocyte maturation in vitro. These results may provide a basis for developing an in vitro system for P. clarkii germline cell culture, which may ultimately lead to the genetic improvement of this species.
