Producing xenogenic channel catfish, Ictalurus punctatus with cryopreserved testes and ovarian tissues of blue catfish, I. furcatus

Abstract

Xenogenesis has been identified as an innovative hatchery technology for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂) embryo production. The xenogeneic process can be accomplished by transplanting primordial germ cells, spermatogonial stem cells (SSC), or oogonial stem cells (OSC), derived from a donor diploid fish into a sterile recipient, which then enables recipient fish to produce donor-derived gametes. Currently, freshly extracted stem cells are used to create xenogens, which is challenging, especially considering germ cell production is donor size and age specific as well as dependent on seasonal cycles. Thus, having frozen stem cells available in germplasm repositories will help to facilitate xenogenesis technology for hybrid catfish production. This study was conducted to assess the effectiveness of fresh and cryopreserved stem cells for germ cell transplantation to support xenogenesis. At 5 days post hatch (DPH), triploid channel catfish were injected with fresh or cryopreserved SSC or OSC labeled with PKH26 dye. At 45 and 90 DPH, body size (total length and weight) and survival of these recipient fish were evaluated. In addition, colonization of fresh and cryopreserved donor cells was evaluated in recipients using PKH26 dye florescence (for percentage of cell area and cluster area) and PCR from gonadal samples. No significant differences in body size characteristics were detected between the fresh and cryopreserved injection treatments at 45 and 90 DPH. Survival was significantly higher in the control treatment than the SSC and OSC injected treatments at both sampling days. Fluorescent imaging revealed that percentage of SSC cell area, SSC cluster area, OSC cell area, and OSC cluster area did not significantly differ between the fresh and cryopreserved treatments at 45 and 90 DPH. There was a significant increase in cell area and cluster area from 45 to 90 DPH for all treatments. According to PCR analyses, a high proportion of xenogens (at 45 and 90 DPH, respectively) were detected in recipient fish injected with fresh SSC (72.2 and 83.3%), cryopreserved SSC (61.1 and 66.6%), fresh OSC (66.7 and 61.1%), or cryopreserved OSC (61.1 and 61.1%). Taken together, our results show that cryopreserved donor stem cells can recover in recipient gonads and perform as well as their freshly extracted counterparts. Hence, cryopreserved stem cells can be used for future germ cell transplantation to support xenogenesis. These findings will enhance the efficiency of germ cell transplantation in xenogenesis for commercial scale hybrid catfish production.