Genetic Biotechnology to Improve Reproduction of North American Catfish for Aquaculture, Genetic Enhancement and Genetic Conservation

Abstract

Xenogenesis is an emerging technology for hybrid catfish production using primordial germ cells (PGCs), spermatogonial stem cells (SSCs), or oogonia stem cells (OSCs) transplanted to a sterile host species. The present study investigated the recovery of spermatogonial stem cells through short-term culture before transplantation using various incubation conditions. Stem cell research is a rapidly growing area that has the potential to generate therapeutic drugs to treat diseases as well as study disease progression from the beginning for humans. Many of the same techniques apply to human pluripotent stem cell culture as they do to normal mammalian cell culture. However, maintaining the undifferentiated state of human pluripotent stem cells (hPSCs) requires extra considerations to ensure that the cells keep their key traits of self-renewal and pluripotency. Such information is not available for blue catfish, Ictalurus furcatus, to enable catfish xenogenesis research. SSCs were extracted and isolated from the immature gonads of blue catfish. The maintenance of spermatogonial stem cells was investigated in this work used short-term culture prior to transplantation. The SSCs were incubated with and without 5% CO2 at 24-30°C temperature range and with 0-75mM or ROCK I. Spermatogonia produced in vitro discovered to be the best treatment at 30°C, with 50-75 mM ROCK I with 5% CO2 (p=0.001) for 72 hours. The timing of implantation and the number of transplanted cells were varied in order to optimize the selection of channel catfish (Ictalurus punctatus) fry and to evaluate the proliferation of donor cells into functional gonadal tissues. Xenogenic progeny were successfully produced in channel catfish when donor cells were implanted between 3 and 5 days post-hatch. Further experiments aimed to assess the efficiency of germ cell transplantation by introducing spermatogonial germ cells from blue catfish and channel catfish into sterile common carp fry, resulting in xenogenic common carp capable of producing channel or blue catfish gametes. The most effective injection window was determined to be between 15 and 25 days post-hatch. The effects of hormonal treatments on spawning success and reproductive performance in CRISPR/Cas9-generated melanocortin-4 receptor (mc4r) knockout channel catfish (Ictalurus punctatus) were investigated. Varying hormonal regimens, including luteinizing hormone-releasing hormone analog (LHRHa) and human chorionic gonadotropin (HCG), were evaluated for their impact on spawning rates, relative fecundity, hatch rates, and fry yield per kilogram of female body weight. Results demonstrated that HCG was crucial for successful spawning in mc4r mutants, with spawning failure observed in its absence, despite the presence of pronounced secondary sexual traits. The combination of HCG and LHRHa significantly enhanced reproductive outcomes, with mc4r x mc4r, pairings exhibiting fecundity and hatch rates comparable to wild-type controls under optimized hormonal protocols.