【作者】 崔凯；

【导师】 李和平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2004， 硕士

【摘要】 随着胚胎生物工程技术的深入研究和不断发展，在我国开展对鹿胚胎体外生产技术的研究显得越来越重要与紧迫。本研究以我国的马鹿为试验对象，对鹿胚胎体外生产体系中最关键的基础性环节——鹿卵母细胞的体外成熟和体外受精进行探索，旨在提出一套在实践中具有可操作性的鹿卵母细胞体外培养体系，进而为鹿体外受精研究奠定基础。试验研究结果表明： 1、采用递减法肌肉注射Folltropin-V（Canada）并结合PG对马鹿进行超数排卵处理可以有效提高母鹿卵巢卵母细胞的采集数量。马鹿在埋入CIDR后第8、9、10、11天内每日注射Folltropin-V（Canada）的剂量分别为：120～140mg、80～100mg、60mg、40mg；或将第四天剂量用PMSG（500IU／头）代替，均可以得到较多的卵母细胞。 2、采用先抽吸再切割的方法可以提高所获取鹿COCs的数量。切割法的补充使用所检得的卵母细胞数约占检取卵母细胞总数的1／2（43枚／84枚）。两种方法的操作时间不超过3min／对卵巢。 3、将所获卵泡液和切割液经过冲洗处理后再检卵与直接检卵相比：视野明了，蛋白质等物质不易发生凝集，卵的移取方便、快速。 4、检取的鹿卵母细胞可分为4级：A级为卵丘细胞致密，不扩散，细胞质均匀，形状规则，至少完全包裹有4层卵丘细胞；B级为卵丘细胞1～3层，基本上包裹卵母细胞；C级为半裸卵，卵母细胞外有部分卵丘细胞存在；D级为裸卵、变形卵，包括退化的卵母细胞。研究结果表明：A、B级卵母细胞体外培养成熟率较高。 5、马鹿的卵母细胞在体外成熟培养中采用37℃和38.5℃两种温度条件的比较结果是：38.5℃条件下的卵母细胞成熟率（78.9％）显著（p＜0.05）高于37.0℃时的卵母细胞成熟率（44.4％）。 6、以M199作为马鹿卵母细胞体外成熟培养的培养基，所配制的成熟液（1）：M199+10％FBS+0.2mM丙酮酸钠+1mM双抗+10mM Hepes+10mg／ml GnRH+1mg／mlE₂与成熟液（2）：M199+20％FBS+0.2mM丙酮酸钠+1mM双抗+10mg／ml GnRH，都适宜进行卵母细胞的体外成熟培养，体外成熟率可以分别达到78.9％、84.2％。 7、采用上浮法可有效地使鹿的精子获能。培养的条件为38.5℃、5％CO₂、饱和湿度；洗精时两次离心的速度分别为2200rpm、2000rpm为宜；TALP沈精／受精液（2）与BO洗精／受精液（2），处理精子的效果分别好于TALP沈精／受精液（1）与BO洗精／受精液（1）；BO洗精／受精液（2）比TALP沈精／受精液（2）处理马鹿精子的获能效果更佳，精子存活时间可长达24h，后者达到9h。 8、TALP受精液（2）与BO受精液（2）均可用一J几鹿卵母细胞的体外受精。虽然鹿精子在两种受精液中的存活时间不同，但都可以得到受精卵，受精率分别为66.7%、70.6%，两种受精液对受精率的影响差异不显著（p>0.05）。 9、鹿的受精卵在M199胚胎培养液中进行体外培养，发育至2一细胞的比例达50%以上。更多还原

【Abstract】 Along with the development of embryo engineering technique, it changes more and more urgent and essential to begin to study in vitro embryo production of deer in our country. We studied in vitro maturation and fertilization of Wapiti(Cervus elaphus) ovary oocytes. The aim of the study is to upbuild a set of perfect protocol of in vitro oocytes culture that is feasible to deer in the practice, so as to give theoretic basis, experience and methods to the deep study of deer IVF.The results of our studies are as followings:l.Wapiti(Cervus elaphus) superovulation with Folltropin-V from Canada can increase the number of ovary oocytes derived from abattoir material. The program is as follows: After being treated with CIDR device for 8 d, all hinds received a four-day twice-per-day injection of 120~140mg, 80~100mg, 60mg, 40mg, Folltropin-V, or a single injection of 500IU, PMSG in place of Folltropin-V at the fourth day.2.The number of COCs collected by first aspirated and then deeply cut from the abattoir-derived deer ovaries can be increased. The method of deeply cut can make up for the shortage of single aspiration and have made the number of COCs increase to twice of single aspiration. The total operating time is within 3minutes.3.Gently washing the follicular fluid, compared with no washing and direct checking oocytes, has more advantage: convenient, the protein difficultly coacervating,’fastly moving ooocytes and saving time.4.The COCs can be divided into four degrees:grade A is with fully cumulus cells and have four layers of comulus cells at least; grade B have 1-3 layers fully cumulus cells; grade C is hemi-nude egg with sectional cumulus cells; grade D are nude eggs> deformed oocytes and degenerative oocytes. The COCs of grade A and grade B have higher maturation rates.5.The in vitro maturation rate of oocytes is obviously higher under 38.5℃ than under 37.0’C(p<0.05).6.Medium 199 is the culture medium of in our experiments.. The following culture systems are all available and can make the in vitro maturation rate reach 78.9%, 84.2%: (l)M199+10%FBS+0.2mM Na-pyruvate+lmM antibiotic+10mg/ml GnRH +10mM Hepes +lmg/ml E2; (2)M199+20%FBS+0.2mM Na-pyruvate+lmM antibiotic+lOmg/ml GnRH.7.The method of swim-up is effective for Wapiti(Cervus elaphus) sperm washing and capitation in vitro. The two centrifugalizing speeds in washing sperm being 2200rpm, 2000 rpm is best. The effects of operating in TALP sperm washing or fertilization medium(2) and in BO sperm washing or fertilization medium(2) are all better than in TALP sperm washing or fertilization medium(l) and in BO sperm washing or fertilization medium(l). The time of sperm living in vitro can reach to 24 hour when sperm cultured in BO fertilization medium(2), and the time in TALP-fertilization medium(2) is only 9h.8.In vitro fertilization with BO or TALP can all make the ovum culturing to cleavage stage, and the fertilization rate are 66.7%, 70.6%. The effects of fertilizing in the two mediums are unobviously(p>0.05).9.The early embryo of Wapiti(Cerv玸 elaphus) developed in embryo culture madium M199. The rate of the cells that have developed to 2-cell can reach to 50%.更多还原