【作者】 李海峰；

【导师】 魏学良；

【作者基本信息】 西南大学 ， 临床兽医学， 2006， 硕士

【摘要】 本研究主要针对环境雌激素对雄性动物生殖机能造成的损害，并结合当前ICSI技术是否会对胚胎发育产生致畸作用尚存在争议，利用实验动物载体的优势条件，拟采用已证明具有环境雌激素效应的拟除虫菊酯类杀虫剂高效氯氰菊酯喷洒饲草后饲喂雄兔，进行人工致畸；用假阴道法采集致畸公兔精液，进行适当处理后，利用显微操作将畸形或活力较弱精子注入发育正常的成熟卵胞质内。选择捧出第二极体（PbⅡ）和具有两个原核的受精卵进行体外培养，研究弱、畸形精子ICSI受精卵获得较高受精率、分裂率和发育率的适宜条件，同时通过探讨精子获能与否、精子形态对ICSI受精卵的受精率、发育率的影响，为提高ICSI受精胚胎体外发育效果作一铺垫，进而提高ICSI成功率，并为男性不育症治疗、动物育种和濒危珍稀动物繁殖保种提供基础资料和技术参考。 实验主要从三个环节进行：第一，选取4月龄体重均匀的健康雄兔16只（体重为1.8+0.10kg），驱虫、隔离观察两周后，随机分成4组，采用稀释后的高效氯氰菊酯喷洒饲草后直接饲喂，使各组雄兔染高效氯氰菊酯量分别为0mg．kg^-1．d^-1、1.5mg．kg^-1．d^-1、3.0mg．kg^-1．d^-1和4.5mg．kg^-1．d^-1，持续56天后，假阴道法采集雄兔精液，对精液进行常规分析，检测该农药对家兔精液的致畸效果。第二，选用6月龄以上、体重2.0kg～3.5kg健康未孕母兔，采用PMSG+HCG法进行超排处理，并与正常雄兔同笼交配，19～20h左右手术法冲胚、清洗后分别放置到RPMI1640+10％FBS、RPMI1640+10％NBS、TCM199+10％NBS三个不同成份的培养液中，在相同的培养气相下（38.5℃、5％CO₂、饱和湿度）对家兔早期胚胎进行序贯培养，观察其胚胎体外发育状况，确定本实验中兔受精卵最佳的体外培养体系。第三，假阴道法获取被致畸雄兔的精液，稀释、离心洗涤处理（一部分精液用DM液获能处理，一部分仅用mPBS稀释、离心洗涤）；同时采取雌兔正常的成熟卵母细胞，清洗处理后在显微操作仪下吸取外观异常或活力较弱的精子注射到卵母细胞中，并在前一实验得到的最佳培养体系中进行体外培养，观察体外发育状况。实验还通过比较精子获能与否、精子不同形态对ICSI注射卵受精率、发育率的影响，为进一步提高ICSI注射卵体外发育效果奠定了基础。 实验结果发现，将高效氯氰菊酯通过分次低剂量饲喂雄性家兔56天后，3.0mg．kg-1．d-1剂量组雄兔不仅精子总数显著减少，而且精子活力和体外存活时间明显下降，畸形（卷尾、胖头、断尾）精子增加，尤其是卷尾精子明显增多（P＜0 05）；其睾丸组织的曲细精管中精母细胞数量明显减更多还原

【Abstract】 Recently, the harms of environmental estrogen to male anmimals and the ICSI technology’s effect of causing abnormal to embryo development are still disputed. This experiment was designed based on these disputations.Took advantages of experimental animal carrier, sprinkled β -cypermethrin,which a kind of pyrethrum ester pesticide that had been proved to have the environmental estrogen effect ,on the forage grass and feed them to male rabbits in order to induce abnormality artificially.Using artificial vagina collect the semen, after being treated properly, injected the abnomal sperm or inactive sperm into the cytoplast of mature oocyte by microoperation. Selected the zygotes which had vented the polar body Ⅱ or had two primary nucleus to proceed further incubation, research the best conditions to obtain the high rate of fertilization,cleavage and development. This research could be helpful to improve the ICSI zygotes’development, and the results could suggest whether the male infertile patients could have offsprings using the ICSI technology, so could provide basic information and technological reference for the treatment of male infertility, animal breeding and breeding protection of rarity animals.This experiment was mainly done in three aspects: firstly, collected four-month healthy male rabbits with symmetrical weight（1.8±0.10kg）,expelled parasite, isolated and observed two weeks, then divided them into four groups randomly, sprinkled the β-CP on the forgae grassa and feed them to the rabbits directly.let each group’s pesticide quantity as 0mg.kg^-1d^-1,1.5 mg.kg^-1.d^-1,3.0 mg.kg^-1.d^-1 and 4.5 mg.kg^-1.d^-1 56 days later, collected the semen by artificial vagina, proceed routine analysis with the semen to assay the pesticide’s effect of causing abnormality.Secondly, six-month healthy young female rabbits without pregnancy whose weight were above 2.0kg-3.5kg, dealed them with PMSG+HCG for superovulation and mated them with normal male rabbits in the same cage. After 19-20h, obtain the embryo by surgery, cleaning them, and then puting them in three different culture liquid as TCM199+ 10%NBS,RPMI1640+10%NBS,RPMI1640+10%FBS respectively. Under the same condition （38.5 ℃ ,5% CO₂,saturant humidity） cultured the pronulear embryos continually, observing the state of in vitro development,at last confirming the dominant culture system for rabbits’ zygotes. Thirdly, the first-group rabbits’semen were collected by artificial vagina.Diluted the semen and washing them by centrifugal effect,then storing them(some would be dealed with DM liquid to make sperm capacitation,some would更多还原