【作者】 刘洋；

【导师】 孙中武； 刘玉堂；

【作者基本信息】 东北林业大学 ， 细胞生物学， 2007， 硕士

【摘要】 东北虎(Panthera tigris altaica)是国家一级保护动物，我国现存野生东北虎的数量很少，而人工圈养的东北虎又存在近亲繁殖的现象。对东北虎的精液进行超低温冷冻保存研究，为将来建立精液的种质资源库，通过人工授精在圈养虎和野生虎之间进行基因交流，避免近亲繁殖造成的种群退化，增加虎基因的多样性，对于更加有效的保护东北虎具有十分重要的意义。本研究对17只东北虎进行了23次电刺激采精，并对取得精液进行品质分析。结果表明，不同虎个体对电刺激采精的电压敏感度不同，在平均电压为4.78±1.3伏时射精。所采集精液量平均为4.02±1.18mL，pH值为7.05±0.26，精子密度为23.43±33.87×10~6个／mL，精子活率为62.9±19.96％，畸形率为45.06±14.96％。从总体上看12月份和1月份采精效果最好，有精率占全年的57.2％，10月和11月的采精效果最差，无精率占全年的44.4％，表明东北虎精液品质受季节影响。通过比较果糖和葡萄糖两种冷冻稀释液对精子的保护情况，发现果糖冷冻稀释液对虎精子的保护效果较好，冻融后精子活率达到58.85％，畸形率为58.88±11.88％，顶体完整率为82.43±4.04％；葡萄糖冷冻稀释液，解冻后精子活率为13.83％，畸形率为63.1±1.7％，顶体完整率为83.1±8.3％。用扫描电镜和透射电镜对东北虎精子形态和结构的观察表明，东北虎精子全长52.57±1.25μm，可分为头部和尾部。在头部和尾部颈段处及尾部中段前方常见原生质滴，内含线粒体。头部：正面呈圆形长约为3.98±0.58μm；宽约3.41±0.59μm。颈段：颈段是连接头部和尾部的结构，颈段较短0.66±0.106μm，内含中心粒和节柱。中段：长8.627±1.166μm，由线粒体包围，线粒体为31-34旋，轴丝为常见的“9+2”结构。主段和末段长39.31±1.25μm。冷冻对于精子损伤主要表现在质膜和线粒体，而这些变化会在不同程度上引起受精率的下降。精子在冻融过程中质膜易发生膨胀或断裂、顶体囊泡化或溃散，而质膜和顶体丢失现象较少。可见，冷冻保护液只能一定程度的保护精子，冷冻对于精子的损伤是不可避免的。更多还原

【Abstract】 Siberian tiger is first-grade state protected mammals in China and the total number of wild Siberian tiger is seldom today.The inbreeding phenomenon occurred often in Siberian tigers of artifical breeding.The study of cryopreserve sperma in Siberian tiger could allow to found long-range banking of gene pools in the future as well as promote gene flow between captive and wild population,hedging against losing more genetic variation and have more effect to protect it.An Electroejaculation technique was applied to the Siberian tiger for semen collection and characterization of their seminal traits,and the semen were recovered from 23 of 17 tigers.The results show different voltage was suitable for the electro of the different individuals.Ejaculation was happened on the mean of 4.78±1.3V.The mean values of ejaculation were:volume,4.02±1.18mL;pH,7.05±0.26;sperm concentration,23.43₃3.87x10⁶/mL;motility,62.9±19.96％;abnormal sperm percentage,45.06+14.96％.As a whole,the efficiency of eletroejaculation was best in December and January,accounting 57.2％of all the rate of producing seminal fluid containing spermatozoa in a year;it was worst in October and November,that accounting 44.4％of all the rate of aspermia in a year.That state seasonality has effect on semen strait.The effect on protecting of spermatozoal motility was compared by using these different cryoprotective diluent with fructose extender and glucose extender in this study.These results demonstrate that the fructose diluent is better than the glucose one in post-thaw motility of 58.85 and 13.83％,abnormal sperm of 58.88±11.88％and 63.1±1.7％,intact acrosome of 82.43±4.04％and 83.1±8.3％,respectively.Spermatozoal structure was observed by transmission electron microscope,scanning electron miceoscope and light microscope in the study.The spermatozoa total length is 53.424±0.7μm,and consist of the head and the tail.Cytoplasmic droplets can be seen in the distal or proximal position or the midpiece,including mitochondria of tiger spermatozoa.The head:rounded in shape and length 3.98±0.58μm,width 3.41±0.59μm.The nick:length 0.66±0.106μm,including of centriole and segmented columns.The middle piece of the tail is 8.627±1.166μm.Its axonemal complex have an usual "9+2" pattern.;its mitochondria makes 31-34 turns.The principal and terminal piece of the tail is 39.31±1.25μm.The cryodamage was observed in the cell membrane and mitochondria.All of these changes were found to contribute to a general decline in post-cryopreservation semen fertility.The plasma membrane was expanded extremely or broken and the acrosome was vesiculatcd or dispersed while plasma membrane and acrosome seldom lost.Thus,cryoprotectivc diluent could protected sperm in partly,and the sperm cryodamage could be not avoided.更多还原