The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

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Xenogenesis is an innovative tool for hybrid catfish (female Channel Catfish Ictalurus punctatus × male Blue Catfish I. furcatus) seed production, accomplished by transplanting undifferentiated germ cells derived from a donor diploid fish into a sterile recipient, which then enables recipient fish to produce donor‐derived gametes. There is potential to collect donor‐derived germ cells from mature fish during certain times of the year depending upon seasonal temperature and serum sex steroid hormonal fluctuations. The objective of this study was to evaluate seasonal variations in germ cell counts and serum sex steroid hormonal profiles in mature Blue Catfish. Mature fish were collected monthly over the full annual cycle to quantify the number of live germ cells (spermatogonia A, oogonia), viability of germ cells, and levels of serum sex steroid hormones, including testosterone, 11‐ketotestosterone, and 17β estradiol. Extracted spermatogonia A counts were highest from April to June, whereas a significant decline was detected from July to November. Extracted live oogonia counts were highest in April and gradually decreased to zero over the months of May to August. Seasonal variations in serum testosterone, 11‐ketotestosterone, and 17β estradiol followed a similar pattern as the live spermatogonia A and oogonia counts. Even though spermatogonia A counts were relatively lower in mature than in immature Blue Catfish males, extracting spermatogonia A from mature Blue Catfish males during April to June provides an added advantage to the process of artificial fertilization, as it is required to sacrifice these fish to collect sperm.