【作者】 穆春雨；

【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， 2007， 硕士

【摘要】 本试验的目的是通过研究卵巢保存温度、精子获能方法、胚胎体外培养体系等方面来探讨诸因素对牛卵母细胞体外成熟和体外受精的影响，进一步完善胚胎体外生产体系；在此基础上，把性别控制技术与体外受精技术相结合，把建立起来的胚胎体外生产体系应用于性控精液体外受精的初步研究，探讨用性控精液体外受精生产体外性控胚胎的可行性。主要研究结果如下：1．把卵巢随机分成两部分，一部分放到37℃左右的DPBS中保存做对照，另一部分则放到4℃左右的DPBS中，各保存8h后，收集卵母细胞进行体外培养，研究卵巢保存温度对卵母细胞体外成熟的影响。结果发现卵巢经4℃保存8h后卵母细胞的成熟率急剧下降，与对照组之间存在显著差异(12.12％vs 68.52％)(P＜0.05)，说明卵巢保存温度过低影响卵母细胞的体外成熟。2．将采集到的卵母细胞随机分成四组，分别用含不同浓度EGF(0、25、50、75 ng/mL)的成熟培养液成熟培养22h后检查卵母细胞成熟情况，研究EGF对卵母细胞体外成熟的影响。结果发现添加25ng／mLEGF组的卵母细胞的成熟率为64.74％，与对照组(61.81％)之间差异不显著(P＞0.05)；添加50ng／mL(74.39％)与75ng／mLEGF(75.86％)组间的成熟率无显著差异(P＞0.05)，但均显著高于对照组与25ng／mLEGF组(P＜0.05)。成熟培养液中加入EGF后均能提高卵母细胞的成熟率，随着EGF浓度的增加，卵母细胞的成熟率也随之提高；EGF在培养液中的适宜添加量为50ng/mL。3．从湖北省种公牛站购买了西门塔尔(4头)、夏洛来(1头)和利木赞(2头)3个品种中的7头种公牛的精液分别进行体外受精实验，以便从中筛选出适合用于体外受精的种公牛精液。结果发现不同个体的种公牛的精液对体外受精效果影响很大，用于体外受精的公牛精液在使用时需要进行仔细的选择。4．将采集到的卵母细胞成熟培养后随机分成四组，以BO和TALP为基础液配制受精获能液。精液解冻后分别用BO+上浮法、BO+直接离心法、TALP+上浮法和TALP+直接离心法四种方法获能处理后用于体外受精实验，筛选合适的精子体外获能体系。试验结果表明精子的四种处理方法(BO+上浮法、BO+直接离心法、TALP+上浮法和TALP+直接离心法)对体外受精效果无显著影响(P＞0.05)。5．卵母细胞体外受精后，将受精卵随机分成四组，以TCM199和SOF为胚胎基础培养液，分别用(1)TCM199、(2)TCM199+GCM、(3)mSOF和(4)mSOF+GCM四种培养体系进行体外培养，筛选合适的胚胎体外培养体系。研究结果显示各组之间卵裂率无显著差异(P＞0.05)，但囊胚率上出现了明显的差异(P＜0.05)。M199组的囊胚率(16.98％)最低，与M199+GCM组(29.49％)mSOF组(33.69％)、mSOF+GCM组(41.24％)间差异的显著(P＜0.05)；mSOF组的囊胚率(33.69％)与M199+GCM组(29.49％)在统计学上差异不显著(P＞0.05)，mSOF和mSOF+GCM组(41.24％)间囊胚率差异不显著(P＞0.05)。6．从屠宰场分别采集初情期前的小母牛和成年母牛的卵巢，保存在37℃的DPBS中运回实验室。采集卵母细胞后进行体外成熟培养和体外受精实验。不同年龄的牛(小母牛和成年母牛)的卵母细胞体外成熟效果无显著差异(73.75％vs75.80％)(P＞0.05)，来源于小母牛卵巢组的卵母细胞成熟培养后的卵裂率与成年母牛组间无显著差异(66.13％vs 68.48％)(P＞0.05)，而囊胚率存在明显差异(16.98％vs 37.72％)(P＜0.05)，且差异极显著(P＜0.01)。7．性控精液解冻后分别用上浮法和45％-90％Percoll梯度离心法获能处理后分别对成熟卵母细胞进行体外受精，随后观察胚胎的卵裂率和囊胚发育情况，研究性控精液(X-精子)不同处理方法对体外受精效果的影响，筛选出适合于性控精液体外获能的方法。结果发现性控精液经Percoll法获能处理后体外受精的卵裂率(55.57％)与上浮法(21.72％)存在着显著的差异(P＜0.05)，但在囊胚率上两者差异不显著(35.54％vs38.69％)(P＞0.05)。Percoll法是性控精液较好的体外获能处理方法。8．性控精液与常规精液解冻后均用Percoll法获能处理后分别对成熟卵母细胞进行体外受精，随后观察胚胎的卵裂率和囊胚发育情况，研究性控精液进行体外受精的可行性。性控精液体外受精后卵裂率(54.39％)低于常规精液(66.17％)(P＜0.05)，但囊胚率(36.06％vs 40.56％)无显著差异(P＞0.05)。性控精液用于体外受精体外生产性控胚胎是可行的。更多还原

【Abstract】 The objective of this study were to investigate the effect of storage temperature ofovaries, the method of capacitation, in vitro culture system and other factors on the IVFefficiency of bovine oocytes, in order to improve bovine embryo IVP system.Furthermore, the established system of bovine embryo IVP was applied to the IVF ofsex-sorted sperm to investigate the suitability of sex-sorted sperm for producingsex-controlled embryos in vitro.Bovine ovaries were collected from Wu-Feng abattoir of Hong Shan District, Wuhan.The results were as follows:1. Bovine ovaries were randomLy allocated to two treatment groups. Ovaries wereput into DPBS at 37℃(as control) and at 4℃for 8h, respectively. Cumulus-oocytecomplexes (COCs) were aspirated and cultured in vitro, investigating the effect of storagetemperature of ovaries on the IVM efficiency of bovine oocytes. The results displayedthat the in vitro maturation rate of bovine oocytes derived from ovaries stored at 4℃for8h was significantly lower than that of oocytes derived from ovaries stored at 37℃for8h (12.12% vs 68.52%) (P＜0.05).The low storage temperature of ovaries affected the rateof in vitro maturation of bovine oocytes.2. Bovine oocytes with intact cumulus cell layers were randomLy allocated to fourtreatment groups, and then they were cultured in 100μl droplets of maturation mediumsupplemented with the different concentration of EGF(0, 25, 50, 75 ng/mL, respectively).Oocytes were cultured in vitro for 22-24h for maturation. The results indicated that therate of in vitro maturaion of oocytes in maturation medium supplemented with 25ng/mLEGF(64.74%) was not different compared to the control group (0 ng/mL, 61.81%) (P＞0.05). There was no significant difference between the oocytes cultured in maturationmedium supplemented with 50ng/mL EGF and the rate of in vitro maturaion cultured inmaturation medium supplemented with 75ng/mL EGF(74.39%, 75.86±4.46%,respectively) (P＞0.05), but they were both significantly higher (P＜0.05) than that of theother two groups.The in vitro maturaion rate of oocytes was significantly correlated to theconcentration of EGF in the maturation media. The optimal concentration of EGF inmaturation medium is 50ng/mL.3. The semen of Simental, Charolais and Limousin were used for in vitro fertilizationof bovine oocytes in order to observe the effect of different bull semen on in vitro fertilization efficiency. The results indicated that there were significant differencebetweeen different bull semen. Bull semen used for in vitro fertilizations should bechosen carefully.4. Bovine oocytes with intact cumulus cell layers after IVM were randomly allocatedto four treatment groups for IVF with four treatment methods of sperm capacitation(BO+swimming-up, BO+direct centrifugation, TALP+swimming-up, TALP+directcentrifugation). The results displayed that there was no significant difference of differenttreatment methods on the cleavage, morula and blastocyst rates (P＞0.05).5. After IVF, presumptive zygotes were randomLy allocated to four different culturesystems: 1) M199 without co-culture, 2) M199+Granulosa Cell Monolayer (GCM) withco-culture, 3) mSOF without co-culture and 4) mSOF+GCM with co-culture. There wereno significant difference on the rate of cleavages of different in vitro culture system ofembryos (P＞0.05), but the rate of blastocysts was significantly different among the fourculture systems of embryo (P＜0.05). Compared with treatment 2 (29.49%), treatment 3(33.69%) and treatment 4 (41.24%), the blastocyst rate of embryos cultured in treatment1 (16.98%) was the lowest (P＜0.05). There was no significant difference on the rate ofblastocysts between treatment 2 and treatment 3, There was also no significant differenceon the rate of blastocysts between treatment 3 and treatment 4 (P＞0.05).6. Ovaries from prepubertal heifers and adult cows were recovered from a localslaughterhouse and transported to our laboratory at 37℃in DPBS, and then ooocyteswere collected for IVM and IVF. The results indicated that there were no significantdifference on the rate of IVM between heifer and adult cow (73.75% vs75.80%) andcleavage (66.13% vs 68.48%, respectively) (P＞0.05), but the rate of blastocysts ofembryos derived from adult cow was significantly higher than that of embryos derivedfrom heifers (37.72% vs16.98%) (P＜0.05).7. Motile frozen-thawed sex-sorted sperms were isolated with swimming-up and45%-90% Percoll gradient centrifuge. There were significant difference on the cleavagerate of oocytes fertilized with different treatment method of sex-sorted sperm (21.72% vs55.57% ) (P＜0.05), but there were no significant difference on the blastocyst developmentrate of oocytes fertilized with different treatment method of sex-sorted sperm (P＞0.05). These results indicated the method of Percoll gradient centrifuge was an suitablemethod to the in vitro capacitation of sex-sorted semen. 8. Frozen-thawed sex-sorted and unsorted semen was washed with a 45%-90%discontinuous Percoll gradient solution by centrifugation. And then the semen was used toIVF. The results indicated that there was significant difference on the cleavage rate ofoocytes fertilized with sex-sorted or unsorted sperm (54.39% vs 66.17% ) (P＜0.05), butthere was no significant difference on the blastocyst development rate of oocytesfertilized with sex-sorted or unsorted sperm (P＞0.05). These results proved thefeasibility of IVF using sex-sorted semen.更多还原