Effects of antioxidants and antifreeze proteins on cryopreservation of blue catfish (Ictalurus furcatus) spermatogonia

Long-term storage of spermatogonial stem cells (SSCs) represents the next step for advancing aquaculture research by providing valuable genetic resources for genetic enhancement programs and xenogenesis technologies. Hybrid catfish, the cross between channel catfish, Ictalurus punctatus ♀ and blue catfish, I. furcatus ♂, are in high demand by the US aquaculture industry, but production can be limited by the availability of blue catfish gametes. Therefore, xenogeneic stem cell transplantation is a powerful method for generating blue catfish gametes within faster growing and maturing channel catfish hosts. These technologies could be facilitated by having cryopreserved SSCs from testicular tissue on-hand in gene banks. Cryopreservation protocols for blue catfish testicular tissues and for other fish species are still being optimized, and currently, data is lacking on the effects antioxidants and antifreeze proteins (AFPs) have on cellular post-thaw viability of SSCs. The objective of this study was to analyze the individual and combined effects of antioxidants (catalase, hypotaurine, and ascorbic acid) and AFPs (I and III) on post-thaw type A spermatogonia cell production and viability from testicular tissue. In this study, there were no improvements with individual antioxidant or AFP treatments. However, when the antioxidants hypotaurine and catalase were tested in combination with different AFPs, three treatments had higher type A spermatogonia cell production than the control, including hypotaurine 7 mM + AFPI 1 μg/mL, hypotaurine 3.5 mM + AFPI 0.1 μg/mL, and hypotaurine 7 mM + AFPIII 0.1 μg/mL. These treatments along with hypotaurine 7 mM + AFPI 0.1 μg/mL and catalase 100 IU + AFPI 0.1 μg/mL had higher viability than the control. From these results, we recommend adding these antioxidant-AFP combinations to future cryopreservation protocols. Overall, this is the first study showing that combining specific antioxidants and AFPs can improve spermatogonia cryopreservation for a fish species