为建立圆口铜鱼（）精子超低温冷冻保存方法，采用计算机辅助精子分析系统（CASA）评价了4种稀释液（D15、D20、L1、D1），3种抗冻保护剂[二甲基甲酰胺（DMF）、二甲基亚砜（DMSO）、甲醇（METH）]在3种体积浓度（7.5%、10%、12.5%，）下对圆口铜鱼精液的冷冻保存效果，并比较了150 mOsm/kg NaCl与超纯水作为激活剂对精子活力的影响。结果表明，D1组稀释液的精子活率（MOT）最高，为（74.64±13.17）%，与鲜精无显著差异（P>0.05），精子运动速度达最大，平均曲线运动速度（VCL）、平均直线运动速度（VSL）、VAP（平均路径运动速度）分别达到（50.28±12.46）μm/s、（35.06±10.82）μm/s、（39.44±12.46）μm/s，精子快速运动时间和寿命分别为（8.67±1.15）s、（33.33±5.00）s，显著低于鲜精（<0.05）；7.5%甲醇组和7.5%二甲基甲酰胺组的MOT次之，分别为（77.71±17.74）%、（76.42±12.49）%，抗冻剂二甲基亚砜组的MOT显著低于甲醇组、二甲基甲酰胺组（<0.05）。12.5%的3种抗冻保护剂中，几乎无精子存活；在不同种类不同浓度的抗冻剂保护下，10%甲醇组，相较于使用超纯水激活，用150 mOsm/kg NaCl激活后的精子活率和寿命更高，说明Na⁺有延长冻精寿命，提高精子活率的作用。研究表明，D1+10% METH（7.8 g/L NaCl+0.5 g/L KCl+15 g/L葡萄糖+10% METH）可用于圆口铜鱼精液超低温冷冻保存，为圆口铜鱼的繁育工作和种质资源保护奠定了基础。

The largemouth bronze gudgeon () is a dominant species and important economic fish in China, which is distributed in the upstream regions of the Yangtze River. However, it is currently classified as threatened due to a rapid decline in its population. Cryopreservation technology enables germplasm cells to be safely and effectively preserved for a long time, which will help with artificial reproduction (e.g., captive broodstock management and seedling cultivation). In addition, cryopreservation can assist in maintaining original genotypes for the conservation of threatened and endangered species. Many studies have suggested that due to the species specificity of fish sperm, the extender composition, cryoprotectant type, and cryoprotectant concentration are different during cryopreservation. More importantly, there is currently no research on sperm cryopreservation of the largemouth bronze gudgeon. In the present study, to establish a sperm cryopreservation method for the largemouth bronze gudgeon, a computer assisted sperm motion analysis system (CASA) was used to find a suitable sperm cryopreservation method, by comparing the effects of four extenders (D15, D20, L1, and D1) and three cryoprotectants, including dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and methanol (METH), at three concentrations (7.5%, 10%, and 12.5%; ) on sperm motility after thawing. Our procedures followed the two-step method of cooling the mixture and thawing the frozen semen in a 37℃ water bath for 12-15 s. The results showed that D1 and METH are suitable choices for the extender and cryoprotectant, respectively. The motility of the sperm reached (78.11±14.74)%, which showed no significant difference compared to fresh sperm (>0.05). In comparison with the other treatments, the 10% METH group performed better in terms of the average velocity of (50.28±12.46) μm/s, average linear velocity of (35.06±10.82) μm/s, and average path velocity of (39.44±12.46) μm/s, which are significantly different values compared to those of fresh sperm (<0.05). The fastest movement time and lifetime of the sperm were (8.67±1.15) s and (33.33±5.00) s, respectively. In summary, 10% METH as an antifreeze protective agent in D1 was the best cryoprotectant protocol for sperm and could be applied in practice. The results provide a feasible technique for sperm preservation of largemouth bronze gudgeon, which could assist in artificial breeding and species conservation of the species.

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现代农业产业技术体系专项资金资助项目（CARS-46）；中国长江三峡集团公司科研专项（JGJ0252013）.