【作者】 郑小敏；

【导师】 史远刚；

【作者基本信息】 宁夏大学 ， 临床兽医学， 2005， 硕士

【摘要】 体外受精技术即体外胚胎生产技术一直是辅助繁育新技术的研究热点。但目前绵羊的体外受精技术体系还不是很成熟,本实验使用屠宰场获得的绵羊卵巢旨在研究绵羊体外成熟、体外受精(IVM/IVF)及早期胚胎培养中的影响因素,以期筛选出较为合理的绵羊卵母细胞体外成熟、体外受精和早期胚胎发育培养体系。 结果表明: 1 切剖法从屠宰绵羊卵巢表面卵泡采集得到的可用卵母细胞数显著多于抽吸法(4.47枚/卵巢VS2.51枚/卵巢),但耗时较长(55秒vs51秒)。 2 添加不同剂量的FSH均能刺激卵母细胞的体外成熟,且各组间卵母细胞的成熟率没有显著差异(P>0.05);但是,添加1μg/ml和5μg/mlFSH的成熟培养液得到的卵裂率都显著大于添加10μg/ml FSH的成熟培养液(P<0.05)。 3 表明绵羊成熟培养液中添加一定剂量的EGF能够显著提高卵母细胞的成熟率,且100ng/ml的EGF添加浓度与0ng/mlEGF及50ng/mlEGF添加浓度相比较还能显著提高受精后绵羊卵母细胞的卵裂率,建议为合适添加浓度。 4 培养液中一定浓度范围内颗粒细胞(浓度为2-6×10~6个/ml)的存在对卵母细胞体外成熟率没有明显的影响,但能显著的提高卵母细胞受精后的卵裂率(55.6%vs53.8%)和桑囊率(28.9%vs19.1%)(P<0.05)。 5 公羊个体对体外受精结果的影响不容忽视。实验结果表明,来自三只不同公羊的精液经相同的获能处理,进行卵母细胞体外受精,结果在卵裂率(54.7%,41.3%,43.3%)和桑堪胚/囊胚率(28.2%,19.7%,22.6%)上都有显著差异(P<0.05) 6 绵羊受精16小时的卵裂率、桑囊率显著高于受精8小时、12小时和20小时。 7 四种培养液Ⅰ液:TCM199+GCM+15%ESS+EGF;Ⅱ液:TCM199+15%ESS+EGF;Ⅲ液:SOF液+15%ESS+10μg/ml NEAA+20μg/mlEAA+EGF;Ⅳ液:SOF液+BSA+10μg/mlNEAA+20μg/mlEAA+EGF与受精后的胚胎共培养,结果表明,Ⅰ、Ⅱ、Ⅲ液卵裂率显著大于Ⅳ组培养液(P<0.05),Ⅰ液显著大于Ⅱ液(P<0.05),Ⅲ液与Ⅰ、Ⅱ液间差异不显著。四组胚胎发育培养液之间所得的桑囊率在Ⅰ液中较高但与其它三组差异不显著(P>0.05)。 8 绵羊卵母细胞体外成熟、体外受精及早期胚胎培养的TCM199体外受精渐变培养体系与目前沿用的常规体外受精体系相比较,虽然成熟率差异不显著(81.3%vs80.4%),但卵母细胞体外受精后的卵裂率(72.1%vs50.8)和桑囊率(94.8%vs24.5%)差异极显著(P<0.01)。更多还原

【Abstract】 It is well known that artificial insemination (AI). embryo transfer (ET) and in vitro fertilization (IVF) are among the most efficient techniques. While IVF technology in sheep is still unskilled,and it need more study before practical use.Ovaries obtained from the slaughterhouse and matured in vitro were used to reseach the factors which reported influence the results of IVF and early embryo culture in order to optimized the technology of IVF in sheep. The major results obtained are as follows:1 The recovery of COCs from slaughtered sheep ovary by surface — follicles cutting is efficient compared with the aspiration and more COCs could be recovered by surface-follicles cutting (4.47per ovary vs 2.51 per ovary). But the time used is a little longger(55s per ovary vs51s per ovary)2 Addition with different dose of FSH can stimulate the maturation of oocytes in vitro and the percent of maturation did not differ between 1 μ g/mk 5 μ g/ml and 10 μ g/ml.However both the 1 μg/ml and 5 μ g/ml treatments result in cleavage rate increase compared with 10 μ g/ml treatments3 Oocytes was incubated in maturation media with addition of EGF as both the 50 ng/ml and 100 ng/ml treatments displayed a significant increase (P < 0.05) in the maturation rate , but only in the 100 ng/ml EGF treatment, which displayed a significant (P < 0.05) increase in cleavage rate.4 Oocytes were co-cultured with granulosa cells of certain concentration ( 2-6×10~6 /ml)had no obvious effect on IVM compared with oocytes co-cultured without granulosa cells, but the former method can greatly increased the percentage of cleavage ( 55.6% vs 53.8% ) and morulae/blastocyst.(28.9% vs 19.1%) (P<0.05).5 The effect of individual rams cannot be neglect. The result of our experiment showed that when sperms coming from three different rams treated with the same method and used for IVF /There was significant difference in rates of cleavage ( 54. 7%, 41.3%, 43.3%) and morulae/blastocyst (28.2%, 19. 7%, 22. 6%) among different individual rams(P<0. 05).6 When oocytes and sperm were incubated for 8h, 12h, 16h 20h respectively. The highest rates of cleavage and morulae/blastocysts were obtained from 16h.7 When sheep’ embryos were co-cultured with different meduim I: TCM199+GCM+15%ESS+EGF ; II: TCM199 +15%ESS+EGF; III: SOF fluid+15%ESS+10 μg/ml NEAA+20 μ g/mlEAA+ EGF; IV: SOFfluid+BSA+10 μ g/mlNEAA+20 μ g/mlEAA+ EGF TCM199, The result showed that the cleavage rates of team Ⅰ、Ⅱ、Ⅲ was significant higher than team Ⅳ(P < 0.05),and team I was higher than team Ⅱ ,result of team Ⅲ resembled Ⅰ and Ⅱ; The results also showed that team I got the higher percentage of morulae/blastocysts ,but there is no significant difference when compared with other mediums8 The Gradual Changed TCM199 Culture System for IVF on Sheep can significant enhanced the rates of cleavage and morulae/blastocysts compared with the regular method (P < 0.01).This is an effective way to progress sheep’s Embryo in vitro fertilization technology .更多还原