Sperm cryopreservation of Sebastes schlegelii and application in large scale seed breeding production

Korean rockfish (Sebastes schlegelii) is a commercially important viviparous teleost widely distributed in China, Japan and Korea, and is characterized by internal fertilization and long-term sperm storage. However, artificial breeding of S. schlegelii has not been well established, and the optimal conditions for sperm preservation remain unclear. In the present study, an effective sperm cryopreservation protocol was successfully developed using 15-20% glycerol as the cryoprotectant combined with a programmable freezer. Post-thaw sperm kinematic parameters were evaluated using a computer-assisted sperm analysis system, with motility, VCL, and LIN values of 61.95 ± 6.74%, 66.48 ± 2.26 μm/s, and 48.61 ± 8.31%, respectively. Using the cryopreserved sperm, artificial intraovarian insemination was performed, and successful sperm localization within the ovarian cavity was further confirmed through HE staining and PKH26 fluorescence labeling. After artificial insemination, all females exhibited normal ovarian development until final maturation. The fertilization and parturition success rates reached approximately 98% and 94%, respectively, which were markedly higher than the natural mating success rate of approximately 70% under conventional hatchery culture conditions. No morphological deformities were observed in larvae or juveniles during subsequent seedling rearing. Morphometric measurements of larval body length and height over 60 days post-parturition showed no significant differences between the artificial insemination and natural insemination groups. Overall, this study established a reliable technical framework for sperm cryopreservation and artificial insemination in S. schlegelii, providing a solid methodological foundation for future pedigree construction, controlled selective breeding, and broodstock management in internally fertilizing fish species.