【作者】 刘晓辉；

【导师】 刘忠华；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 随着牛体外受精技术和胚胎移植技术在生产应用中的快速发展,以及牛体细胞核移植和转基因等胚胎工程技术的深入开展,如何稳定获得高质量的成熟卵母细胞已经成为该领域中的一个极其重要的问题。卵母细胞虽然可以通过超数排卵、活体采卵等方式获得,但其成本高昂,无法广泛开展。因此目前广泛被采用的方法主要是通过收集屠宰场废弃的卵巢来获得体外成熟卵母细胞。在某些特定的条件下,卵巢的收集和运输往往需要较长的时间,同时屠宰时间大多数都在夜间进行,给后续的工作带来许多不便。如果能够建立良好的卵巢保存体系,使其在一定时间内的储存不对卵母细胞的体外成熟和成熟后的发育能力造成不利影响,那么就会为相应的研究工作和生产应用带来极大的便利。本实验针对该问题,以卵母细胞体外成熟效果和成熟卵母细胞的后续发育能力为主要衡量指标,研究了“生物保鲜剂”、温度和储存时间等因素对牛卵巢保存效果的影响,同时以体外成熟的卵母细胞为核受体开展了初步的牛体细胞核移植研究。实验结果如下:1.低温4℃保存牛卵巢0h、3h、6h和8h,卵母细胞的成熟率分别为69.27%、67.95% 67.25%和47.98%,孤雌激活后卵裂率分别为36.99%、31.76%、36.77%和16.61%,囊胚率分别为14.16%、15.56%、14.43 %和0%,低温4℃保存0h、3h和6h三组卵母细胞的成熟率、孤雌激活后的卵裂率和囊胚率差异不显著(P>0.05),而低温4℃保存8h组卵母细胞的成熟率、卵裂率和囊胚率显著低于其它三组(P<0.01);2.低温4℃添加生物保鲜剂保存牛卵巢0h、3h、6h和8h,卵母细胞的成熟率分别为71.67%、69.57%、68.22%和67.14%,孤雌激活后卵裂率分别为36.99%、33.47%、32.29%和31.10%,囊胚率分别为14.63%、16.43%、13.89%和13.53%,四组之间各项指标差异不显著(P>0.05);低温4℃添加生物保鲜剂保存牛卵巢8h组与低温4℃保存牛卵巢8h组之间各项指标差异极显著(P<0.01);3.室温(15--20℃)添加生物保鲜剂保存牛卵巢0h、3h、6h、8h ,卵母细胞的成熟率分别为78.53%、71.32%,75.16%和77.98%,孤雌激活后胚胎卵裂率分别为38.63%、36.19%,33.87%和33.17%,囊胚率分别为18.28%、17.56%,16.85%和17.43%,四组之间各项指标差异不显著(P>0.05);室温(15--20℃)添加生物保鲜剂保存牛卵巢8h组与低温4℃添加生物保鲜剂保存牛卵8h组之间卵母细胞的成熟率差异显著(0.05<P<0.01),孤雌激活后卵裂率和囊胚率差异不显著(P>0.05);4.成熟培养液液中添加EGF 0ng/ml、10ng/ml、30ng/ml,卵母细胞的成熟率分别为64.86%、70.61%、72.27%,核移植胚胎激活后卵裂率分别为60.71%、80.61%、82.61%,囊胚率分别为11.88%、23.81%、25.00%.成熟培养液液中添加EGF 10ng/ml组与30ng/ml组之间各项指标无明显差异(P>0.05);与添加EGF 0ng/ml组之间各项指标差异极显著(P<0.01);结果表明添加生物保鲜剂可以延长牛卵巢的保存时间;室温(15--20℃)添加生物保鲜剂的保存效果优于低温4℃添加生物保鲜剂:成熟培养液中添加EGF10ng/ml可以有效地提高卵母细胞的体外成熟及核移植胚胎的发育。更多还原

【Abstract】 Bovine in vitro fertilization(IVF) and embryo transfer (ET) were getting progress greatly during the last two decades in China, the research of embryo biothechniques, such as, bovine somatic cell nuclear transfer(SCNT) , transgenic cattle, developed quickly during the last 10 years. Hence the requirments of bovine oocytes increased, it becomes a very important thing that how to consistently get high quality bovine oocytes for these application and research.Although the oocytes can derived from superovulated cattles,the cost is too expensive. Now main resource of matured bvine oocytes is in vitro matured oocytes derived from ovaries which were collected from slaughter house. In most cases, slaughtering often starts from midnight, and the collection and transport of ovarian often takes several hours, oocytes quality is very easily getting worse during this process and the ovaries collection schedule makes the followed work inconvenient. So in this experiment, addressed the temperature,“biology bao xian ji”, and the storage period as factors effecting on the oocytes quality during ovary storage, based on the in vitro maturation rate, parthenogenetic embryos in vitro development potential and the in vivo development potential of SCNT embryos, the bovine ovary storage systems were systemly studied. The experimental results are as follows:1. Low temperature 4℃kept cattle ovarian 0h, 3h, 6h, and 8h, oocyte maturation rate was 69.27%, 67.95% 67.25% and 47.98%, parthenogenetic activation after the cleavage rate was 36.99%, 31.76%, 36.77% and 16.61%, blastocyst rates was 14.16%, 15.56%, 14.43% and 0%, low temperature 4℃preservation 0h,3h and 6h three groups oocyte maturation rate, parthenogenetic activation and the cleavage rate of blastocysts there was no significant differenc(eP>0.05),and low temperature 4℃8h preservation group oocyte maturation rate, cleavage and blastocyst rate was significantly lower than the other three groups(P<0.01);2. Low temperature 4℃add the preservation of biological preservative cattle ovarian 0 h, 3h, 6h, and 8h, oocyte maturation rate of 71.67%, respectively, 69.57%, 68.22% and 67.14% ,Parthenogenetic activation after the cleavage rate was 36.99%, 33.47%, 32.29% and 31.10%, respectively blastocyst 14.63%, 16.43%, 13.89% and 13.53%,Between the four groups was not significant indicators(P>0.05);low temperature 4℃add biological preservative cattle ovarian preservation group and 8h low temperature 4℃kept cattle ovarian 8h group differences between various indicators significantly(P<0.01);3. At room temperature (15 - 20℃) add the preservation of biological preservative cattle ovarian 0h, 3h, 6h, 8h, oocyte maturation rate was 78.53%, 71.32%, 75.16% and 77.98%,parthenogenetic activation after embryo cleavage rate was 38.63%, 36.19%, 33.87% and 33.17%, respectively blastocyst 18.28%, 17.56%, 16.85% and 17.43%,between the four groups was not significant indicators(P>0.05);At room temperature (15 - 20℃) add biology bao xian ji cattle ovarian preservation group and 8h low temperature 4℃add the biology bao xian ji cattle ovarian group 8h between the oocyte maturation rate significantly(0.05<P<0.01),parthenogenetic activation after cleavage and blastocyst rate was not significantly different(P>0.05);4. Maturation of liquid added EGF 0ng/ml, 10ng/ml, 30ng/ml, oocyte maturation rate of 64.86%, respectively, 70.61%, 72.27%,nuclear transfer embryos activated cleavage rate was 60.71%, 80.61%, 82.61%, respectively blastocyst rate 11.88%, 23.81%, 25.00%. maturation of liquid added EGF 10ng/ml group and 30 ng / ml among the indicators there was no significant difference(P>0.05);with the addition of EGF 0ng/ml group differences between various indicators significantly(P<0.01)The results showed that biology bao xian ji can extend the ovary storage period, especially at room temperature (15 - 20℃). 10ng/ml EGF can improve both the in vitro maturation rate and the SCNT embryos development rate.更多还原