【作者】 陈永昌；

【导师】 崔泰保； 陈学进；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 为了建立行之有效的猕猴体外受精—胚胎移植技术方案，对上海动物园的5只雄性猕猴进行了精液采集及其品质分析实验。自行设计了超数排卵方案，并利用国产兽用促性腺激素刺激15只猕猴超数排卵，手术法采集卵母细胞，然后选择高品质的精液，经过液化、优选、精子获能后对体内或体外成熟的卵母细胞进行体外受精，并将发育到8-细胞期的胚胎冷冻保存。2003年3月将其中3枚解冻后移植给1只处于月经周期第15d的受体猕猴。实验结果如下： 1．阴茎刺激时采精量显著高于直肠刺激(P＜0.01)，直肠灌肠刺激时，精子密度和活率与阴茎刺激时差异不显著(P＞0.05)，不灌肠时，精液量、精子密度和活率极显著降低，深度麻醉时，采精成功率明显降低。1％α-胰凝乳蛋白酶可使40％以上的猕猴精液凝块液化，加入稀释液后，液化率更高，但精液品质下降。 2．用四种方案超数排卵时，C、D方案均可获得20枚以上的卵母细胞；用1500 IU的HCG可使53.6％的卵母细胞成熟至MⅡ期；当采卵时间延迟到HCG后34h时，46％的卵母细胞成熟至MⅡ期。 3．上游法和Percoll法均能显著提高精子形态正常率(68±10至82±7和84±6)和精子活率(72±11至85±6和83±9)，但受精率差异不显著(P＞0.05)；体外受精实验中共用了136枚卵母细胞，其中30枚在精子获能实验中老化受损，其余106枚受精56枚，6枚进行固定，其余50枚用于体外发育培养，共获得28枚卵裂胚，11枚发育为8-细胞期胚胎。冷冻保存一段时间后解冻了其中3枚，存活率为100％，移植后没有妊娠。 结果表明： 1．灌肠可以明显提高直肠刺激的成功率；阴茎刺激效果优于直肠刺激；轻度麻醉更有利于精液采集；精液可以采用物理和化学方法处理使其部分液化。 2．C、D方案取得了较好的超排效果；HCG对卵母细胞的成熟至关重要；随着采卵时间的延迟，卵母细胞的成熟程度升高；1月份和2月份的超排效果最好。 3．上游法和percoll法都是有效的精子优选法，对受精率的影响基本无差异；咖啡因和dbcAMP在精子体外获能时缺一不可；理想的受精密度应在2×10~5精子／ml左右；在使超数排卵后采集的卵母细胞进一步成熟时，血清和激素的添加会取得更好结果；在胚胎的发育过程中，血清和氨基酸都是必要的能量物质。 4．胚胎冷冻保存后的解冻存活率很高，移植后没有受孕受多种因素的影响。更多还原

【Abstract】 In order to set up an effective route of in vitro fertilization and embryo transfer in rhesus monkeys, 5 male rhesus monkeys’ semen were electroejaculated and evaluated. The superovulation protocols were made using gonadotropin for animals made in China, 15 female rhesus monkeys were stimulated and oocytes were recovered using invasive surgical techniques. In vivo or in vitro maturated oocytes were inseminated with liquefied, separated and capacitated motile sperm. Zygocytes were cultured in vitro, and 8-cell embryos were cryopreserved, three of which were thawed and transferred to a recipient on the fifteenth day of its menstrual cycle. The whole results were as followings.1. More semen was obtained using penile electroejaculation. Sperm concentration and motility showed no difference between penile electroejaculation and rectal probe electroejaculation after being clystered. The quality and quantity of semen reduced significantly using rectal probe electroejaculation without clystering. 1 percent a -chymotrypsin liquefied over 40 percent semen clot of rhesus monkeys.Dilution made semen quality declined.2. Among the four stimulating protocols, more than 20 oocytes were obtained in C and D. When the dose of HCG was added to 1500IU, 53.6 percent of oocytes were in metaphase II (MII). As the interval extended to 34h after HCG administration, the percentage of MII oocytes was 46.3. The percentage of normal morpha (68±10 to 82±7 and 84±6) and motility of sperm(72±l 1 to 85±6 and 83±9) could be improved by using the two motile sperm separation methods, but there was no difference in fertility rate. 136 oocytes were used in the experiment of IVF, 30 of which were damaged to death during capacitation; the rest were inseminated and 56 of which fertilizated. 6 of fertilized oocytes were fixed, and 28 cleavaged. 11 of the 28 fertilizated oocytes developed to 8-cell embryos. Although the cryosurvival rate of three embryos was quite high after thawed (100%, 3/3), pregnancy rate was zero after being transferred.The conclusions resulted from above are as followings:1.Clyster made rectal probe electroejaculation more effective, and penile electroejaculation was more effective than rectal probe electroejaculation. Proper anaesthesia was more suitable for semen collection for rhesus monkeys. The ejaculates can be partly liquefied by physical or chemical means.2. Scheme C and D showed better superovulating results. HCG is very important for oocytes maturation, and the longer interval after using HCG can improve the degree of maturation. January and February seems to be the best seasons for superovulation in rhesus monkeys.3. There was no significant difference in fertilization rate between Percoll and swim up groups both methods were effective motile sperm separation methods. Both caffeine and dbcAMP are necessary for inducing sperm to capacitation, they have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction. The optimal sperm concentration for IVF is 2x105sperm/ml or so.Serum and hormone can improve the maturation rate of oocytes. And serum and amino acid are necessary for embryo development.4. The failure of pregnancy maybe resulted from multiple factors.更多还原