Cryopreservation of spermatogonia of the giant freshwater prawn, Macrobrachium rosenbergii, using slow and ultra-rapid freezing

Abstract

The giant freshwater prawn, Macrobrachium rosenbergii, is an important species that is widely raised in tropical and subtropical regions. To develop techniques for preserving valuable traits in this species, we have devised protocols for cryopreserving spermatogonia-progenitor cells that give rise to spermatozoa through spermatogenesis-using slow freezing and ultra-rapid freezing techniques. Optimization of cryoprotectants and their concentrations revealed that 10 % dimethyl sulfoxide (ME2SO) is effective as a cryoprotectant in both methods. The optimal time for equilibration to the cryoprotectant before slow freezing was determined to be 15 min. Thawing temperature had a significant effect on cell recovery and viability, both being markedly higher when cells were thawed at 10 degrees C than at 27 degrees C. After long-term storage in liquid nitrogen, cells preserved by ultra-rapid freezing exhibited higher recovery and viability rates than those preserved using the slow freezing method, with recovery rates of up to 86.4 % +/- 5.9 % at 6 months. Therefore, using 10 % ME2SO with the ultra-rapid freezing method is recommended as the optimal protocol for long-term cryopreservation of M. rosenbergii spermatogonia.