In this study,DMSO was used as cryoprotectants,and HBSS solution was used as extender.Two-step cooling for cryopreservation of Nibea albiflora spermatozoa in 0.25 mL straws.DNA damage,mitochondria and membrane damage of N.albiflora sperm in response to the cryopreservation process by SCGE and FCM were also investigated.The results demonstrated that there were no significant differences between fresh sperm and frozen-thawed sperm which was diluted with 5%-20% DMSO in vitality.The activation rate,moving time and lifespan of frozenthawed sperm were 85.25%±3.95%,(3.23±0.27) min and(3.83±0.33) min when 10% DMSO was used as cryoprotectant.However,a significant drop in sperm vitality was observed when DMSO concentration was increased to 25% and 30%.The SCGE showed that there were no significant differences between fresh sperm and frozen-thawed sperm which was diluted with 5%-15%DMSO in DNA fragments,but DNA fragments differed significantly with fresh sperm when DMSO concentration rose to 20%,25%,30%.In fact,there was a positive correlation between comet rate of frozenthawed sperm and concentration of DMSO in protocol.Also,we found that the integrity of mitochondria and membrane of frozen-thawed sperm obtained high proportion when using 5%-20% DMSO as cryoprotectant,and there were no significant differences between fresh sperm and frozen-thawed sperm.But,the mitochondrial and membrane integrity had a significant drop when using 25% and 30% DMSO as cryoprotectants.So we concluded that toxicity of cryoprotectant was the main factor of DNA damage,mitochondrial membrane damage in frozen-thawed sperm.