Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 degrees C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 degrees C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.

Automated summary

Portunus trituberculatus, an important marine economic species, has a rapidly developing aquaculture industry. However, wild capture and germplasm degradation have become a serious issue, calling for the development of artificial farming and germplasm resource protection. Sperm cryopreservation technology is an effective method, and this study compared three methods for obtaining free sperm, finding that mesh-rubbing is the best. Optimal cryopreservation conditions were determined, including sterile calcium-free artificial seawater as the medium, 20% glycerol as the cryoprotectant, and specific cooling and thawing procedures. However, the expression of sperm-related genes and enzymatic activities significantly decreased after cryopreservation, indicating damage to the sperm. Overall, this research improves the cryopreservation technology for P. trituberculatus sperm and provides a technical basis for establishing a cryopreservation library of crustaceans.