| Zebrafish is an important model species and has been widely used in geneticbreeding, molecular biology and toxicology studies besides its value as ornamental fish.Sperm cryopreservation offers a venue to preserve its genetic resources and retain geneticdiversity for different strains and lines. This study addressed the optimization of zebrafishsperm cryopreservation.The method of sperm collection had an effect on sperm motility. Sperm collected bydissecting without crushing, dissecting with crushing and abdominal massage wereevaluated, and the highest motility (90±5％) was found in abdominal massage, followedby dissecting without crushing (89±3％), which were both higher than the method ofdissecting with crushing (68±13％). Because dissecting without crushing yielded 20times more sperm number than that of abdominal massage, thus was chosen forsubsequent studies.Factors such as osmolality, pH, temperature, and dilution ratio have been shown toaffect fish sperm motility. Effect of osmolality on zebrafish sperm motility was evaluatedat a wide range in this study, and the optimal osmolality for sperm inhibition was300mOsm/kg. Selection of extenders revealed that Hanks' balanced salt solution(HBSS) yielded the highest sperm motility aider activation. Studies of pH effectshowed the highest motility was obtained with extender of pH 7.5 (73±6％) in roomtemperature and pH 8.0 (90±5％) at 4℃. There were no significant difference amongextenders of pH 6.5, 7.0, 6.5 and 8.0 (P＞0.05). For samples stored at room temperatureand at 4℃, motility were not significant different between them during the first 4hstorage, but decreased dramatically at 8-10h for samples stored at room temperature (65±7％), while retained as high as 80％for samples stored at 4℃. Dilution ratio wasevaluated to maximize the sperm volume, and a ratio of testis to extender of 1:50 wasfound to retain motility of 87±6％for 2h at 4℃. Activation solutions of deionized water,3％NaCl and HBSS170 had no significant difference on sperm motility (P＞0.05),however, sperm retained motile for longer periods of time with samples activated bydeionized water than by others. To determine sperm concentration rapidly, aspectrophotomeric method was used, and wavelengths of 405, 450, 490, 630nm were compared. Positive correlation (r~2=0.823, P＜0.05) of sperm concentrations was foundbetween hemacytometer counts and absorbance at 405 nm, thus 405nm was selected forrapid estimation of sperm concentration for subsequent studies. Evaluation of fertilizationrevealed that sperm (fresh) to egg ratio of 2.64×10~6:1 yielded the highest percent hatchingrate (37.6±21.7％), so 2.64×106:1 was chose subsequent studies.Studies of cryoprotectant toxicity with fresh sperm showed lower motility of 10％methanol, dimethyl sulfoxide (DMSO), and propylene glycol than those of 5％(P＜0.01).Selection of optimal cryoprotectant revealed that the highest fertilization (31.3±13.7％)and hatch (60.5±0.9％) were obtained for samples cryopreserved with 8％N,N-dimethyl acetamide (DMA), though its post-thaw motility (21.70±7.70％) was lowerthan samples cryopreserved with 8％DMSO (26.7±11.6％). Evaluation of equilibrationtime showed no significant difference of post-thaw motility and fertilization among 10,30, and 60 min, therefore, 30 min equilibrium would satisfy the needs of samplepreparation while minimize the cryoprotectant toxicity. There were no significantdifference of percent fertilization for samples cooled at -0.5, -5, -15 and -25℃/min (P＞0.05), though post-thaw motility of samples cooled at -5℃/min were significant higherthan others (P＜0.01). In summary, fertilization (e.g., 34.3±12.8％), especially percenthatch (e.g., 41±13.2％) that comparable to controls with fresh sperm (fertilization: 74.60±5.54％, hatch: 27.1±0.8％) can be obtained with samples suspended in 8％DMA,cooled at -5℃C/min from 4℃to -30℃, followed by -45℃/min from -30℃to -80℃,and hold at -80℃for 5 min before plunging into liquid nitrogen (-196℃).
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