太平洋鳕(Gadus macrocephalus)精液超低温冷冻方法的建立及精子超微结构分析

采用分步降温法冷冻保存太平洋鳕(Gadus macrocephalus)精液,并用扫描电镜和透射电镜技术研究了精子的超微结构损伤。分析了添加剂(蛋黄)及五种抗冻剂(PG(丙二醇)、DMSO(二甲基亚砜)、EG(乙二醇)、GLY(甘油)、MeOH(甲醇))不同浓度(8%、10%、12%、14%、16%、18%)对太平洋鳕精子冷冻保存效果的影响。实验结果表明,蛋黄能够显著提高太平洋鳕冷冻保存效果(P0.05);12%PG中添加10%蛋黄保存的冻融精子运动率最高(85.87%±1.6%),显著高于其他冻存组(P0.05)。添加蛋黄的12%浓度的DMSO组解冻后精子运动速率较高,平均直线速度、平均曲线速度、平均路径速度分别达到了(120.39±20.78)μm/s、(120.39±20.78)μm/s、(155.64±12.02)μm/s,与其他各实验组差异显著(P0.05)。通过扫描电镜和透射电镜观察新鲜和冻融后的鳕鱼精子发现,鲜精中75.5%精子形态结构正常、24.5%精子形态结构异常;运动率最高的冻精中67%精子形态结构正常、33%精子形态结构异常。超低温保存对太平洋鳕精子结构产生了显著影响,细胞膜破裂、线粒体肿胀变形,鞭毛脱落、断裂。

CRYOPRESERVATION AND ULTRASTRUCTURE OF GADUS MACROCEPHALUS SPERM

We developed a cryopreservation method for sperm of Pacific cod Gadus macrocephalus and studied the ultrastucture of sperm with scanning electron microscope and transmission electron microscope. An additive (egg yolk) and five antifreeze agents including PG (propanediol glycol), DMSO (dimethyl sulfoxide), EG (ethylene glycol), MeOH (methanol), and GLY (glycerol) at six different concentrations (8%, 10%, 12%, 14%, 16%, and 18%) were studied to test the influence on cryopreservation of G. macrocephalus sperm. The results show that the egg yolk can improve the cryopreservation significantly (P<0.05). The group of 12% PG with yolk in solution had the highest sperm motility at 85.87%±1.6%, significantly better than those of other groups, while the group of 12% DMSO with yolk performed best in average linear velocity, average velocity, and average path velocity at (120.39±20.78), (120.39±20.78), and (155.64±12.02)pm/s, respectively. SEM and TEM observation on fresh and frozen-thawed sperm showed that the cryopreservation had a significant impact on the ultrastructure of sperm for causing membrane rupture, mitochondria swelling, and flagellar falling or fracture.