Abstract：In order to investigate the effect of cryopreservation on spermatozoal structures in Pseudosciaena crocea, we used dimethyl sulfoxide (DMSO) or ethylene glycol (EG) as cryoprotectant, cortland solutions as extender, and two-step cooling procedure to cryopreserve P. crocea sperm in 0.25 mL straws. And we detected the DNA damage of frozen sperm by single cell gel eletrophoresis(SCGE) and membranous damage by flow cytometry(FCM). The results showed that there were no significant differences between fresh sperm and frozen-thawed sperm in vitality when 5%~20% DMSO or 5%~20% EG were used. The activation rate, moving time and life-span of frozen-thawed sperm were (87.00±2.45) %, (8.99±0.24) min and (13.11±0.65) min at 10% DMSO, and (87.50±2.52) %, (8.45±0.48) min and (12.84±0.50) min at 10% EG. A significant drop in sperm vitality was observed at 25% DMSO or 30% EG. SCGE results showed that there were no significant differences in DNA fragmentation between fresh sperm and frozen-thawed sperm diluted with 5%~20% DMSO or 5%~20% EG, but the DNA damage to frozen-thawed sperm significantly increased at 25% DMSO or 30% EG. There was a positive correlation between the DNA damage extent of frozen-thawed sperm and concentrations of DMSO and EG. FCM analysis suggested that the integrity of mitochondria and membranous structures of frozen-thawed sperm were similar to that of fresh sperm at 5%~20% DMSO or 5%~20% EG, but the proportion of frozen-thawed sperm maintained the integrity of mitochondria and membranous structures was significantly declined at 25% or 30% DMSO or EG. So we conclud that the weakening of sperm vitality and the enhanced damage of DNA, mitochondria and membranous structures in frozen-thawed sperm are mainly induced by the high concentration of cryoprotectant. 10% DMSO or 10% EG can be considered as the best cryoprotectant to ensure the quality of frozen sperm.
