Cryopreservation of spermatogonia from Yesso scallop, Mizuhopecten yessoensis.

The Yesso scallop is a commercially important species in the northern coastal regions of East Asia; however, its aquaculture production has recently become increasingly unstable due to global climate change. Cryopreservation of germ cells and surrogate broodstock technologies would be fundamental approaches for safeguarding the genetic resources of local populations of commercial bivalve species. Successful germ cell transplantation in bivalves requires the proper preparation of donor spermatogonia. In the present study, we established and optimized protocols for the enzymatic dissociation and cryopreservation of testicular tissue. Dissociation using 0.025% Liberase TM at 25 °C for 2 h yielded the highest number of viable cells among the conditions tested. Evaluation of cryoprotectants and their concentrations showed that 5-10% dimethyl sulfoxide (DMSO) was the most effective among the cryoprotectants tested. Further evaluation of cooling rates and thawing temperatures at these concentrations demonstrated that rapid freezing followed by thawing at 25 °C using 10% DMSO resulted in the highest cell viability. Immunohistochemical and immunocytochemical analyses confirmed that the resulting cell suspensions contained spermatogonia, indicating that the established methods are suitable for preparing testicular cell suspensions for germ cell transplantation.