Development of an in vitro culture system for immature oocytes of red swamp crayfish (Procambarus clarkii)

Abstract

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.