【作者】 冯金华；

【导师】 易建明；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 本论文内容包括三部分:第一部分对武汉市经屠宰所获卵巢的质量进行了调查研究。主要收集了卵巢形态特征,黄体形态特征,不同大小卵泡数量三个方面的数据,并对数据间的关系进行了分析,旨在摸清武汉当地经屠宰所获得的卵巢质量的情况,提高卵母细胞收集效率,为卵母细胞的体外培养方案的制定提供参考。第二部分为体外受精体系的优化试验。旨在通过研究种公牛、胚胎培养密度、颗粒细胞共培养三个方面对体外受精效率的影响,进一步优化本实验室早前建立的牛体外受精体系。第三部分为培养液对牛体外受精胚胎发育的影响研究。着重研究了三种牛体外胚胎培养常用培养液(KSOM,CR1,SOF)对胚胎发育的影响。通过对胚胎囊胚率,细胞总数及抗冻性进行比较,以获得最适合牛体外受精胚胎体外培养的培养液。1第一部分结果1.1本次调查分别在5月份,9月份,12月份收集到了95对(190枚)卵巢。以文献报道作为参考,从卵巢形态学上进行评定,在所收集的卵巢中,质量一般的卵巢(NO-NO,CL+DF-NO)占到了总数的46.31%,质量好的卵巢(CL-NO)只占总数的32.63%,质量较差的卵巢(DF-NO)占到了5.26%,此外还含有10.53%的因出现囊肿而不能利用的卵巢;从卵巢形态学上进行评定,在所收集的卵巢中,50%以上的卵巢不含黄体;从卵泡直径大小对所收集的卵巢的质量进行评定的话,绝大部分卵泡为2-4mm,占到总卵泡的85%,而6-8mm的卵泡所占比例却不到6%。1.2重量为5-10g组的卵巢,所含有的小卵泡的数量极显著地高于重量小于5g组的卵巢(P＜0.01),也显著地高于重量大于10g组的卵巢(P＜0.05)。在中卵泡数量上,5-10g组显著地高于大于10g组(P＜0.05)。在大卵泡数量上,三个组之间差异均不显著(P＞0.05)。三组不同的卵巢重量与小卵泡数量之间均存在正相关,而当卵巢重量超过10g后,卵巢重量与大卵泡数量之间不存在相关性。1.3卵巢形态对不同大小的卵泡数量存在影响。在小卵泡数和总卵泡数上,CL-NO组和NO-NO组要显著地高于DF-NO组和CY-NO组(P＜0.05);在中卵泡的数量上,各组差异均不显著(P＞0.05);在大卵泡的数量上,CY-NO组显著地低于其他五组,差异显著(P＜0.05)。2第二部分结果2.1选取了四头西门塔尔公牛的冻精进行实验,编号分别为6303,6305,6306,6307,研究种公牛冻精对牛体外受精胚胎发育的影响。结果发现,在卵裂率上编号为6303的公牛显著高于其他三头公牛(P＜0.05)。而在囊胚率上,编号为6303的公牛与编号为6305的公牛差异不显著(P＞0.05),但都显著高于其他两头公牛(P＜0.05)。说明公牛因素对体外受精效率存在非常大的影响,6303号公牛效果最好。2.2将假定受精卵随机按25μL/个、5μL/个、1μL/个三种培养密度进行培养,每个微滴中放入20～25个假定受精卵,研究培养密度对牛体外受精胚胎发育的影响。结果表明:三个处理间无论是在卵裂率、囊胚率,还是在孵化囊胚率上均不存在差异(P＞0.05)。说明三种培养密度均能用于牛体外受精胚胎的体外培养。2.3将假定受精卵随机放入含有GCM和不含GCM的微滴中进行培养,研究颗粒细胞共培养对牛体外受精胚胎发育的影响。结果发现,对比于非共培养组(对照组),共培养组在卵裂率,囊胚率和孵化率上差异均不显著(P＞0.05)。在本实验中,说明使用颗粒细胞共培养有利于胚胎发育的效应并不明显。3第三部分结果体外受精后,将假定受精卵随机放入三种培养液中,研究培养液对牛体外受精胚胎发育的影响。受精当天记为0 d,受精后第6～8 d统计囊胚率。将获得的囊胚一部分用Hoechst 33342进行染色,统计其细胞总数。余下的采用OPS法进行冷冻,解冻。解冻后分别在12 h,24 h,36 h观察胚胎复活情况。结果发现,在卵裂率和总囊胚率上,KSOM组显著高于CR1组(P＜0.05)。在第7 d的囊胚形成率上,KSOM组显著高于其他两组(P＜0.05);在细胞总数方面,KSOM组细胞数最多,显著高于其他两组(P＜0.05);在胚胎抗冻性上,各处理间差异不显著(P＞0.05)。说明KSOM更加适合牛体外受精胚胎的发育,能获得质量更好的胚胎。更多还原

【Abstract】 There were three parts in this investigation.In Part 1,we collected and analyzed the data of ovary weight,ovary morphology,corpus luteum morphology and follicle size. The objective was to investigate ovary quality which derived from local abattoirs.In Part 2,the objective was to investigate the effect of bulls,culture density, co-culture with or without granulose cell on the efficiency of IVP bovine embryos,in order to optimize bovine embryos IVC system.In Part 3,we focused on the effect of culture medium on the developmental competence of IVP Early Bovine Embryos.For selecting the best culture medium, blastocyst rates,cell number and cryosurvival were assesed.The results were as follows:1 In this investigation,we collected 95 pairs(190) bovine ovaries in May, September and December.Assessed for the ovary morphology by the standard from Varisanga,there were 32.63%good ovaries(CL-NO),46.31%fair ovaries(NO-NO, CL+DF-NO),and 5.26%poor ovaries(DF-NO).Apart from this,there were 10.53% useless ovaries which had cysts;For follicle size,85%of follicles were small follicles (2～4mm),the large follicles(6～8mm) were less than 6%.2 Small follicles in 5～10g group were significantly higher than≤5g group(P＜0.01),and it was significantly higher than≥10g group(P＜0.05).Middle follicles in 5～10g group were significantly higher than≥10g group too(P＜0.05).There were no significant differene in the large follicles among three groups.Positive correlation was found between ovaries weight and small follicles’ number,but when weight over 10g, there was no correlation.3 Ovary morphology had effect on follicle number of different size.Group of CL-NO and NO-NO had more small and totall follicles than group of DF-NO and CY-NO(P＜0.05).There was no difference in middle follicles among each groups(P＞0.05),but there was lower large follicles in group of CY-NO than other groups(P＜0.05).4 Following IVM,oocytes were inseminated with sperm from four Simmental bulls, which number was 6303,6305,6306 and 6307 respectively.Cleavage rates were assessed at 48 h post insemination(hpi) and the proportion of oocytes developing to the blastocyst stage was recorded on Day 7,Day 8 post insemination(pi).The results were that cleavage rates in 6303 were significantly higher than others(P＜0.05);There were no significant difference in the blastocyst rates between 6303 and 6305(P＞0.05),but both were significantly higher than 6306 and 6307(P＜0.05).The results displayed that the selection of bulls producing sperm with a high fertilizing capacity is an important factor in achieving successful and consistent IVP results.5 After IVM,IVF,presumptive zygotes were randomly allocated to three culture density(25μL/embryo,5μL/embryo and 1μL/embryo),groups of 20～25 presumptive zygotes were placed in every culture droplets.Cleavage rates were assessed at 48 h hpi and the proportion of oocytes developing to the blastocyst stage was recorded on Day 7, Day 8 pi.There were no significant difference in differenct treatments on the cleavage rates,blastocyst rates and hatched blastocyst rates(P＞0.05).6 After IVM,IVF,presumptive zygotes were randomly co-cultured with or without GCM(Granulosa Cell Monolayer).Cleavage rates were assessed at 48 h hpi and the proportion of oocytes developing to the blastocyst stage was recorded on Day 7,Day 8 pi. The results indicated that there were no significant differences in cleavage rates, blastocyst rates and hatched blastocyst rates(P＞0.05).It was indicated that co-culture with GCM was not benefit to early embryos development in this experiment.7 After IVM,IVF,presumptive zygotes were randomly placed to three kinds of culture medium(KSOM,CR1 and SOF).The day of fertilized was 0 day,the blastocysts were collected on Day 7,Day 8.Several of blastocysts were stained with Hoechst 33342, the rest were freeze-thawing by OPS(Open Pulled Straw).After thawing,to record embryo survival rates in 12h,24h and 36h respectively.There was a significantly higher cleavage rates and total blastocyst rates in KSOM than CR1(P＜0.05);On Day 7, blastocyst rates of KSOM was higher than CR1 and SOF(P＜0.05),but embryo survival rates were no significant difference among three treatments(P＞0.05).The result indicated that KSOM is the optimal medium in IVP bovine embryos.更多还原