Developing Efficient Methods of Sperm Cryopreservation for Three Fish Species ( L., , ).

Sperm cryopreservation is helpful for maintaining the genetic diversity of fish species. This study was aimed at developing efficient methods to cryopreserve the sperm of three fish species, including koi carp ( L.), Ya fish (), and . Firstly, based on the analysis of sperm viability, the cryomedium, dilution ratio, volume, and cooling procedure were assessed and optimized in koi carp. The results showed that the highest sperm viability was up to 63.23 ± 1.36% after a 14-day cryopreservation using the optimal method, briefly, sperm frozen with a volume of 50 μL (Vol.sperm:Vol.cryomedium = 1:9) of cryomedium containing 10% DMSO and 3% sucrose in D17 through ultrarapid cooling. Secondly, both the mitochondrial membrane potential and the DNA fragmentation index of sperm were examined and found to be significantly damaged after the cryopreservation. Intriguingly, the fertilization rate of sperm after 14-day cryopreservation is up to 63.03 ± 1.36% and the elongation of cryopreservation time (210 days) just slightly affected the fertilization rate (55.09 ± 4.70%) in koi carp. Thirdly, the optimal cryopreservation method was applied to cryopreserve sperm; the cell viability was 45.39 ± 4.70%. And then this method, after a minor modification (3% sucrose of cryomedium replaced with 3% SMP) was adopted to cryopreserve Ya fish sperm, the cell viability was up to 70.45 ± 2.23%. Lastly, the ultrastructure and morphology of sperm was observed by SEM, and it was found that the cryopreservation prominently caused sperm head swelling and tail shortening in three fish species. In conclusion, this study established effective methods for cryopreserving sperm in three fish species and elaborated the injuries on sperm caused by cryopreservation. And the findings facilitate developing more protocols with practical value to cryopreserve sperm in different fish species.