The Influence of Developmental Stages and Different Kinds and Concentrations of Protective Additives in Cryopreservation of Surf Clam (Spisula Sachalinensis) Larvae

This study was performed to find out the optimal larval stage among trochophore, D-shaped and umbo stage larvae and the desirable protective additive such as fructose, glucose, sucrose and trehalose with cryoprotectant for cryopreservation of surf clam, Spisula sachalinensis larvae. Dimethyl sulfoxide and ethylene glycol were used as cryoprotectant and each cryoprotectant was made to 2.0 M with previous protective additives. The larvae were immersed in the preparations waited for 15 minutes to reach equilibration, and then frozen in a program freezer (-35$^{\circ}C$) and liquid nitrogen (-196$^{\circ}C$). The freezing rate of 1.0$^{\circ}C$ /min. was used for cryopreservation of trochophores before seeding temperature (-12$^{\circ}C$). The survival rate of frozen-thawed larvae increased as larval developing and that of umbo stage larvae was the highest as 96.1 ${\pm}$ 1.0%. The presence of lower concentration of disaccharides as sucrose or trehalose significantly enhanced survival rate when mixed with cryoprotectants (P<0.05). The results of our study indicate that desirable developmental stages of larvae and protective additive for cryopreservation are the umbo stage larvae and 0.2 M sucrose, respectively.