Abstract：Atrina pectinata and Crassostrea rivularis are important marine economic shellfish, but their output is declining sharply due to overfishing, disorderly use and environmental deterioration. The establishment of sperm cryobank and cryopreservation technology was significant to their germplasm resources preservation and genetic breeding. In this study, in order to establish a technology of sperm cryopreservation suitable for Atrina pectinata and Crassostrea rivularis, the series of selecting experiments for type and concentration of cryoprotectant, freezing procedure, balance time, sample volume, sperm dilution method and freezing time were designed and compared. The sperm survival rate was detected by both direct microscopic examination and eosin Y staining detection. The data indicated that the protection of dimethyl sulfoxide (DMSO) to sperm was obviously superior to glycerol (Gly). The sperm survival rates of both Atrina pectinata and Crassostrea rivularis were up to 60% with the protection of DMSO, while only 40% or so with the protection of Gly. The terminal concentration of DMSO was 8%, which was suitable for Atrina pectinata, and 10% suitable for Crassostrea rivularis. The cryoprotectant was divided into 3 parts and added to sperm by 3 times, interval 8 min every time. Using this dilution method, the sperm survival rate was enhanced about 30% than that using once-mix method. The precooling of samples for 15~25 min at 4 ℃ was necessary and could increase sperm survival rate by about 50% compared with direct freezing method. The optimum freezing procedure was firstly to prefreeze for 5 min at 15 cm above liquid nitrogen level, then to prefreeze for 10 min at 5 cm above liquid nitrogen level, and to freeze in liquid nitrogen at last. The sperm survival rate was more than 60% with this freezing procedure, while near to zero without any freezing procedure. Under this freezing procedure, the volume of sample between 1.0 to 1.4 mL could make the sperm survival rate of 60% or so, and less than 1.0 mL or more than 1.8 mL would reduce the sperm survival rate and were unsuitable for sperm cryopreservation of Atrina pectinata and Crassostrea rivularis. After cryopreservation, the sperm survival rate decreased sharply to 60% or so from 90% of fresh sperm. However, there was no significant difference in survival rate of sperm, which was frozen less than 180 d. The sperm survival rate was further decreased to about 56% after 180 d. Therefore, combined with the optimum condition above, we obtained the optimum sperm cryopreservation project for Atrina pectinata and Crassostrea rivularis. This study on the technology of sperm cryopreservation of Atrina pectinata and Crassostrea rivularis provides the theoretical and technical basis for germplasm resources preservation and genetic breeding in shellfish.
