Summer flounder (Paralichthys dentatus) sperm cryopreservation and application in interspecific hybridization with olive flounder (P olivaceus)

The present study established an efficient technology for summer flounder (Paralichthys dentatus) sperm cryopreservation, and successfully applied the cryopreserved sperm into interspecific hybridization with olive flounder (P olivaceus). The best motility of postthaw sperm (78.00 ± 4.70% and 76.60 ± 7.90%), fertilization rates (95.70 ± 3.60% and 79.40 ± 5.20%), and hatching rates (93.10 ± 4.00% and 77.20 ± 2.90%) were achieved when using propylene glycol or DMSO as cryoprotectant. Furthermore, we have successfully improved the cryopreservation method from using 2-mL cryotube to 5-mL cryotube, and the dilution ratio has been increased to 4:1. By this method, the cryopreservation efficiency has been improved to 30 times of that with routine method. Computer-assisted sperm motion analysis showed the freezing-thawing process decreased the sperm speed but did not significantly change the sperm movement pattern, and the progressive linear motion still was the dominant movement pattern. The ultrastructural analysis showed 50% to 60% of the spermatozoa had normal morphology, 20% to 30% were slightly damaged, such as swelling or rupture of head, midpiece, and tail region, and 10% to 20% were severely damaged. In the artificial hybridization experiment, high fertilization rates and hatching rates were achieved when using 15% DMSO (95.7 ± 3.6% and 79.4 ± 5.2%, respectively) and 15% propylene glycol (93.1 ± 4.0% and 77.2 ± 2.9%, respectively), with no significant difference in comparison with control (94.7 ± 2.6% and 72.5 ± 6.5%, respectively). In addition, we found the embryos and larvae from postthaw sperm of P dentatus developed and grew normally. The results of the present study further validated the safety of the cryopreserved sperm in breeding production by assessing the fertilization capacity, embryo development, and larval growth.