台湾泥鳅精子超低温冷冻保存技术研究

Cryopreservation Technology of Taiwan Loach Sperm

为建立台湾泥鳅精子超低温冷冻保存方法,采用显微镜观察精子活率,流式细胞术检测质膜完整性,研究4种稀释液[Hank′s平衡盐溶液(HBSS)、D-15、D-17、鱼用任氏液],5种抗冻剂(甲醇、乙二醇、丙二醇、二甲基亚砜、N,N-二甲基甲酰胺)在4个体积分数(2.5%、5.0%、7.5%、10.0%),不同平衡时间、熏蒸高度和时间、解冻温度和时间对台湾泥鳅精子超低温冷冻保存的影响。试验结果显示:以Hank′s平衡盐溶液为稀释液的精子活率最高,为(45.28±5.75)%;以5%甲醇作为抗冻剂的台湾泥鳅精子活率和质膜完整比例最高,分别为(51.25±5.03)%和(81.70±2.35)%;最佳平衡时间为30 min,最佳熏蒸高度和时间为9 cm和10 min,最后将冻精在37℃水浴锅中解冻10 s的解冻效果更佳。用冷冻复苏的台湾泥鳅精子进行人工授精,受精率达(68.94±6.22)%。综上,用Hank′s平衡盐溶液稀释台湾泥鳅鲜精,与甲醇1∶1混合至终体积分数为5%,放于4℃平衡30 min,而后装入麦管,在液氮面上方9 cm处熏蒸10 min后投入液氮保存可作为台湾泥鳅精子超低温冷冻的保存方法,解冻时在37℃水浴锅中解冻10 s,可获得更好的解冻效果。

To establish cryopreservation method of sperm for Taiwan loach Paramisgurnus dabryanus ssp.taiwan,effects of four extenders(HBSS,D-15,D-17,and Ringer),five cryoprotectants[methanol(MeOH),ethylene glycol(EG),propylene glycol(PG),dimethyl sulfoxide(DMSO),and N,N-dimethylformamide(DMF)]at four volume concentrations(2.5%,5.0%,7.5%,and 10.0%),different equilibrium time(0,10,20,30,40 and 50 min),fumigation height and time(5,7,9,11 cm and 5,10,15 min),and thawing temperature(17,27 and 37℃)and time(5,10 and 15 s)on the sperm viability of Taiwan loach was observed by microscopy and the plasma membrane integrity was measured by flow cytometry(FCM).The results revealed that the sperm with HBSS had the maximal motility(45.28±5.75)%,and the sperm with 5%methanol had the maximal motility of(81.70±2.35)%and the plasma membrane integrity of(51.25±5.03)%,with the best equilibrium time of 30 min,and the best fumigation height of 9 cm for 10 min.The better thawing effect was observed by thawing in 37℃water bath for 10 s,with the fertilization rate of(68.94±6.22)%at artificial insemination with thawed Taiwan loach sperm.In summary,the fresh sperm of Taiwan loach was diluted with HBSS and mixed with MeOH to a final concentration of 5%at 4℃for 30 min,then put it into straws,fumigated at 9 cm above the liquid nitrogen surface for 10 min,and then was stored in liquid nitrogen.When thawing,good results were observed when the fresh sperm of Taiwan loach was thawed in a 37℃water bath for 10 s.The findings provided data foundation with artificial breeding and germplasm resource protection of Taiwan loach.