Abstract：Abstract Acrossocheilus fasciatus is a type of stream fish having great economic value in China. Recently, resource of A. fasciatus has declined rapidly due to overfishing and habitat destruction. Sperm cryopreservation is an effective way to protect germ cells; therefore, research in germplasm resource protection and germ cell conservation are in urgent need. In this study, the sperm was collected from cultured mature male fish and was stored in 0.25 mL straws for the study of sperm cryopreservation. Different conditions were evaluated for sperm cryopreservation, including extenders (Kurokura-1, Ringer, Cortland, D-14, D-15, D-16, D-17, D-19, D-20 and D-21), dilution ratios (1∶1, 1∶2, 1∶3, 1∶4, 1∶5, 1∶7 and 1∶9), cryoprotectants (dimethyl sulfoxide (DMSO), methyl alcohol (MeOH), ethylene glycol (EG), and propylene glycol (PG)), equilibrium times (0, 10, 20, 30, 40, 50 and 60 min), cooling heights (using two-step cooling procedures) (2, 3.5, 5, 6.5 and 8 cm), and thawing temperatures (27, 32, 37, 40 and 42 ℃). In addition, we detected the quality of fresh and corresponding frozen-thawed sperm through their enzyme activities. The experimental data were expressed as X±SD, using SPSS 17.0 statistical software. The statistically significant differences in sperm activation rate and enzyme activities of each group were detected by one-way ANOVA. P＜0.05 were considered significant. The fresh sperm density of Acrossocheilus fasciatus was (9.32±2.03)×109 cells/mL. The optimum result was obtained with the cryoprotectant comprising D-15 diluted with 10% DMSO, which was mixed with sperm at a ratio of 1∶5, equilibrated for 30 min, swung at 3.5 cm above nitrogen for 5 min, and finally stored in nitrogen. After thawing in a water bath at 37 ℃, the activation rate of the frozen-thawed sperm was (35.33±2.52)%; meanwhile, the rate of fresh sperms was (87.67±3.06)%. Enzyme activities were examined using reagent kits. Compared to the fresh sperm, the activities of the total ATPase, succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in optimized frozen-thawed sperm decreased from (9.31±0.17) U/mL, (30.33±5.69) U/mL, and (7 454.84±252.42) U/L to (7.23±1.08) U/mL, (17.67±6.03) U/mL, and (2 172.48±209.62) U/L, respectively. Thus, significant differences were observed in sperm activation rates and enzyme activities between the two groups. The decrease in enzyme activities indicated that cryopreservation caused damage to the sperm. With the given protocol, we got early success in sperm cryopreservation of A. fasciatus. This study provide a technical basis for the foundation of the cryobank for A. fasciatus; however, further studies on parametric optimization are warranted to improve the quality of frozen-thawed sperms.
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