Study on Sperm Cryopreservation for Paternal Germplasm Repository of Selected Indigenous Fishes in Taiwan

This study aimed to investigate the biological characteristics and to establish the related cryopreservation protocol of sperm of selected indigenous freshwater fishes-Onychostoma barbatulus, Candidia barbata, Distoechodon tumirostris and Acrossocheilus paradoxus in Taiwan. The enhanced cryoprotectants were progressively studied using basic dimethyl sulfoxide (DMSO) with optional addition of 0.03, 0.06, 0.09 or 0.12 M sucrose or trehalose, and further addition of CAPS (3-Cyclohexylamino-1-propanesulfonic acid), TAPS (tris-hydroxymethyl methylamino-propane sulfonic acid) or milk. Milt mixture with various cryoprotectant combinations were frozen using freezing laboratory-designed protocols FW1, FW2, or FW3. Results indicated that 10 to 15% DMSO were favorable for four experimental fishes and trehalose was better than sucrose with overall optimal concentration of 0.06 M. Addition of TAPS was found favorable for milt of S. barbatulus and C. barbata while CAPS was suitable for D. tumirostris and A. paradoxus. There was obvious species-specific preference of enhanced cryoprotectant and freezing protocol in each species. The optimal combination of enhanced cryoprotectant and freezing protocol for respective species were further studied. Using the optimal enhanced cyoprotectant combinations and freezing protocol, 0.5-ml straws containing cryopreserved sperm of these fishes have been stored in liquid nitrogen at -196℃ as an example of paternal germplasm repository and related databank.