乌克兰鳞鲤精子超低温冷冻保存方法研究

Cryopreservation of Sperm in Ukraine Scale Carp

为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4 ℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种“3步冷冻法”以及3种解冻温度(20、30、40 ℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12 ℃/min,解冻温度为30 ℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。

The motility was determined in both fresh and post-thawed sperm preserved at 4 ℃ in three extender solutions Hank′s,Cortland,and Freezefish at 3 dilution ratios (1∶1,1∶3,1∶5) and 3 cryoprotectants dimethyl sulphoxide DMSO,propylene glycol,glycerin at 10% volume fraction in Ukraine scale carp ( Cyprinus carpio ) in order to optimize the sperm cryopreservation,and the cryopreservation protocol for Ukrainian scale carp was subsequently tested with 3 “3-steps”cooling methods and 3 thawing temperature (20, 30 and 40 ℃) by using the screening cryoprotectant and dilution ratio. The optimal post-thawed motility (>68%) was observed in the sperm preserved at sperm to Hank′s ratio=1∶3 with 10% DMSO with mean 12 ℃/min cooling rate,and 30 ℃ water bath. The findings had significant advantages in germplasm protection,and provided reference for other fish sperm cryopreservation.