CRYOPRESERVATION OF SPERM FROM TETRAPLOID EASTERN OYSTER Crassostrea virginica

Triploid oysters have been used for commercial aquaculture in several species because of their superior traits of growth especially in summer. All-triploid production are usually achieved by using sperm from tetraploids fertilizing with oocytes from normal diploids. Therefore, tetraploids are critical in triploid oyster aquaculture. Cryopreservation of sperm from tetraploids offers several benefits. It can extend the commercialization of triploid-tetraploid technology by the sale of frozen sperm from tetraploids, provide cost savings and security for maintaining tetraploids, and protect intellectual property. In addition, tetraploids are difficult to produce, and usually initial production only yields a few individuals. To build and maintain breeding populations, sperm cryopreservation from tetraploids is necessary. The goal of this study was to test the cryopreservation of sperm from tetraploid eastern oyster Crassostrea virginica by using the protocol for sperm cryopreservation of diploids. Two populations of tetraploids and one population of diploids were shipped over-night for processing (Table 1). For each population, individual oysters were opened after measuring the body size and whole body weight. Sex was identified by observing the gonad samples by microscope (100 x magnification). Testis from each male was dissected and suspended in Hanks' balanced salt solution at an osmolality of 700 mOsmol/kg, and sperm motility and plasma membrane integrity were estimated for selection of individuals to be pooled for cryopreservation. For tetraploids, four sperm pools were created with 5-6 males for each with the same amount of sperm contribution, and sperm samples were

packaged into 0.5-ml Fresh straws using fully automatic system, and then cryopreserved in large-scale digital freezer." . Fresh sperm motility ranged from 20-48%, and post-thaw motility ranged from 4-18%. Meanwhile, sperm from the individual males for pooling were cryopreserved as comparison. Fertilization data of the post-thaw samples is in the process of collection. Overall, this study demonstrated the feasibility of sperm cryopreservation for tetraploid eastern oysters. More investigation is needed to improve the post-thaw sperm viability.