引用本文:	韩龙江,刘清华,于道德,官曙光,纪利芹,王文琪,刘名,温海深,李军.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.海洋与湖沼,2014,45(4):789-797.

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*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*
韩龙江^1,2, 刘清华¹, 于道德³, 官曙光³, 纪利芹², 王文琪⁴, 刘名³, 温海深², 李军¹
1.中国科学院海洋研究所青岛 266071;2.中国海洋大学水产学院青岛 266003;3.山东省海水养殖研究所青岛 266002;4.青岛农业大学海洋科学与工程学院青岛 266109

摘要:

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(P < 0.05);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(P < 0.05)。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(P < 0.05)。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。

关键词: 太平洋鳕(Gadus macrocephalus) 精子超低温保存抗冻剂添加剂超微结构

DOI：10.11693/hyhz20130700086

分类号:

基金项目:国家自然科学基金项目,31072212号,41076100号;国家高技术研究发展计划(863计划),2012AA10A402号;水产种质资源平台运行服务项目;海洋经济创新发展区域示范项目,12PYY001SF08。

附件

CRYOPRESERVATION AND ULTRASTRUCTURE OF GADUS MACROCEP HALUS SPERM

HAN Long-Jiang^1,2, LIU Qing-Hua¹, YU Dao-De³, GUAN Shu-Guang³, JI Li-Qin², WANG Wen-Qi⁴, LIU Ming³, WEN Hai-Shen², LI Jun¹

1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Fisheries College, Ocean University of China, Qingdao 266003, China;3.Mariculture Institute of Shandong Province, Qingdao 266002, China;4.College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China

Abstract:

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)μm/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.

Key words: Gadus macrocephalus sperm cryopreservation anti-freeze agent additive ultrastructure