【作者】 桑国俊；

【导师】 余四九；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2008， 博士

【摘要】 牛胚胎体外生产技术包括卵母细胞体外成熟、体外受精、体外培养及冷冻保存等环节。本试验对牛卵母细胞及体外胚培养技术进行了研究,比较系统地掌握了牛卵母细胞的操作技术,为该技术的深入研究和应用铺垫了良好基础。1、卵母细胞体外成熟技术(1)COCs级别对卵母细胞成熟的影响。选择A、B、C和D级COCs进行成熟培养。A级成熟率为63.81%,B级为47.81%,C级为2.01%,D级为0.00%,各级成熟率之间差异显著(p<0.01),A、B级远高于C、D级。C级仅有个别发育成熟,D级未见成熟。因此,A、B级COCs是卵母细胞体外培养的主要资源,宜选择A、B级COCs进行成熟培养。(2)卵泡直径对卵母细胞成熟的影响。将卵泡分为3级:大卵泡(φ>8mm)、中卵泡(2mm≤φ≤8mm)和小卵泡(φ<2mm)。COCs成熟率分别为大卵泡组17.07%、中卵泡组61.82%、小卵泡组19.06%,中卵泡组显著高于大卵泡组和小卵泡组(p<0.01),大卵泡组和小卵泡组差异不显著(p>0.05)。卵母细胞的成熟率与采集COCs时的卵泡直径关系密切,在卵母细胞体外培养过程中,宜选择卵巢发育正常的卵泡采集,不宜选择发育过大或过小的边际卵泡。2、牛卵母细胞体外受精技术(1)直接离心法对获能效果和卵母细胞受精率的影响。①二次离心沉淀液:对第一次离心的沉淀定容后继续离心后获得的沉淀进行活力、获能效果和受精率测定。洗涤精子最终活力为0.44;获能效果以10min时最高,为0.41,15min时为0.32,超活化以15min时最好;受精率为40.19%。②二次离心上清液:对第一次离心的上清液定容后继续离心后获得的沉淀进行测定。洗涤精子最终活力为0.56;获能效果以10min时最高,为0.59,15min时较低,为0.585,超活化也以15min时最好;受精率为43.21%。二次离心上清液的各项指标显著高于沉淀液(p<0.01)。从试验结果看,宜选用上清液15min组的获能精子进行体外受精。本试验突破了精子离心法的传统方法,避开了单纯利用沉淀液进行精子获能的思路,而直接对上清液进行离心处理,获得了理想的精子洗涤和获能效果。(2)不同温度对精子获能和卵母细胞受精的影响。将精子分别在20～24℃室温和38.5℃恒温(CO2培养箱)上浮60min、获能10～20min。①室温上浮法:超净工作台上浮60min,精子活力以45min时最高,达到0.68;对上浮45min的精子获能15min后进行体外受精,平均受精率41.40%。②恒温上浮法:CO2培养箱上浮60min,精子活力30min时最高,达到0.697;对上浮30min的精子获能15min后进行体外受精,平均受精率41.51%。精子获能后的超活化现象与室温法一致。二种不同上浮方法的精子活力和受精率之间差异不显著(p>0.05),恒温上浮法的效果略高于室温组。从可控角度考虑,还以恒温上浮法为宜。(3)精子浓度与受精的关系。使用不同浓度(低1.0×106、中1.5×106和高3.0×106个/ml)精子与卵子共同孵育。中浓度组平均受精率为47.34%,显著高于低浓度组的42.38%(p<0.05)和高浓度组的33.96%(p<0.01);低浓度组的平均受精率显著高于高浓度组平均受精率(p<0.01)。本试验表明精子适宜浓度宜控制在(1.0～1.5)×106个/ml。3、牛体外受精胚胎培养技术(1)卵丘细胞、卵丘细胞+bFF共培养系统对胚胎发育的影响。卵丘细胞组、卵丘细胞+bFF组和对照组的桑椹胚率分别为29.07%、34.99%和26.67%;囊胚率分别为8.51%、10.63%和7.24%。卵丘细胞+bFF组的桑囊率显著高于卵丘细胞组和对照组(p<0.01)。本试验通过添加卵丘细胞、卵丘细胞+bFF共培养物质,较好地克服了2C发育阻滞。(2)培养液体积对胚胎发育的影响。分别用50μl和500μl培养液培养胚胎。50μl液滴的桑椹胚率为33.14%,囊胚率为9.07%,500μl液滴依次为36.23%和10.23%。大体积培养比微滴培养有优势,桑椹胚和囊胚率均较高(p<0.01)。本项试验中500μl的效果好于50μl。4、卵母细胞冷冻保存技术及解冻培养(1)程序化一步法冷冻时10%GL和10%EG对COCs冷冻效果的影响。COCs在液氮保存1周后解冻成熟培养。EG组成熟率为34.46%,桑椹胚率为11.18%,囊胚率为4.18%;GL依次为33.44%、10.56%和3.86%。EG组的成熟率比GL组高3.05%(p>0.05),桑椹胚率高5.87%(p>0.05),囊胚率高8.29%(p>0.05)。冷冻保护剂GL和EG均可用于COCs的冷冻保存,但EG的效果比较好。(2)玻璃化冷冻时麦管和OPS管对COCs冷冻效果的影响。以麦管和OPS管作为冷冻载体,二步法玻璃化冷冻COCs。对冷冻1周后的COCs解冻培养,麦管法成熟率为29.84%,桑椹胚率为9.34%,囊胚率为3.47%;OPS法依次为31.89%、9.66%和3.80%。OPS法的成熟率比麦管法高6.87%(p>0.05),桑椹胚率高3.43%(p>0.05),囊胚率高9.51%(p>0.05)。OPS管玻璃化冷冻的效果优于麦管法,但无统计差异,而麦管法宜于操作和储存,宜在实际操作中选用麦管法。(3)COCs冷冻方法的比较。COCs的4种处理方法在成熟培养、桑椹胚发育和囊胚发育方面的趋势基本一致,整体效果的优势序列为EG法>GL法>OPS管法>麦管法。玻璃化冷冻效果低于程序化一步法冷冻法。由于玻璃化冷冻的解冻处理程序复杂,建议实际应用时选用麦管程序化一步法冷冻法。5、小结通过牛卵母细胞体外培养技术试验的实施,比较系统地掌握了牛卵母细胞成熟培养、精子获能、体外受精和体外培养的技术环节和操作程序,利用共培养系统突破了2C发育阻滞,在精子获能方面建立了新的洗涤和获能方法。本研究的技术指标在同类研究中属于较高水平,在将后研究中将进一步探索和提高。更多还原

【Abstract】 It is the development that the bovine oocyte can be researched to produce embryos in vitro for studying and demonstration. Bovine embryo production includes oocyte in vitro maturation, in vitro fertilization, in vitro culture etc. The article had reported how to carry out the process of oocyte in vitro and had got more dates and materials for the future studies.1 Bovine Oocyte in Vitro MaturationThe Effect of in Vitro Maturation with Oocyte GradeThere were four COCs grade in the study. The maturated oocyte rate were 63.81% for grade A,47.81% for grade B, 2.01% for grade C and 0.00% for grade D. The difference among four grades were very significantly(p<0.01)each other. Grade A and B of COCs were significantly higher than grade C and D. The grade A and B of COCs would be chose to maturate in following system.The Effect of in Vitro Maturation with Different Diameter FolliclesThere were three kind of follicles: big follicle(sφ>8mm),middle follicle(s2mm≤φ≤8mm) and small follicles(φ<2mm). The maturated oocyte rate for different diameter follicles were 17.07% for big’s, 61.82% for middle’s and 19.06% for small’s. The middle’s was significantly higher than big’s and small’(sp<0.01).There were not significant difference between big’s and small’s(p>0.05). It had showed that the follicular diameter was very important for in vitro maturation of COCs. The best choice was to collect middle diameter follicles to do.2 Bovine Oocyte in Vitro FertilizationThe Effect of Sperm Capacitation and Oocyte Fertilization Rate by Direct Centrifuge to Select Sperm.Result for second centrifugal sperm for the deposit-liquid which was first centrifuged.After the second sperm centrifuged and capacitated, the sperm washed activation was 0.44, the high sperm capacitation was 0.41 at 10min and 0.32 at 15min, the super activation showed at 15min, and the fertilization rate of oocyte was 40.19%.Result for second centrifugal sperm for the up-liquid which was first centrifuged.After the second sperm centrifuged and capacitated, the sperm washed activation was 0.56,the high sperm capacitation was 0.59 at 10min and 0.585 at 15min, the super activation showed at 15min, and the fertilization rate of oocyte was 43.21%.It were made know that the sperm activation, super activation and oocyte fertilization rate of up-liquid of second centrifuged were significantly higher than the deposit-liquid of second centrifuge’s(p<0.01).The up-liquid’s were also observed that had clear field of microscope, had good diaphaneity, had not eyeable cryoprotectant and dilutent, had few died and abnormal sperms. The deposit-liquid’s were observed that had not clear field of microscope, had not clear diaphaneity, had much eyeable cryoprotectant and dilutent, had many died and abnormal sperms. It is a new method to wash and capacitate sperm with two step by centrifugating for the first up-liquid.The Effect of Sperm Capacitation and Oocyte Fertilization Rate under the Different Temperature by Swimming-upSwimming-up in room temperature(20～24℃).The thawing frozen sperm had been swum up 60min on the super clear desk in room temperature. It was indicated that the best sperm activation was 0.68 at 45min, the best sperm super capacitation was at 15min after treatment, and the fertilization rate was 41.40%.Swimming-up in constant temperature(38.5℃).The thawing frozen sperm had been swum up 60min in CO2 incubator in constant temperature at 38.5℃. It was indicated that the best sperm activation was 0.697 at 30 min, the best sperm super capacitation was at 15min after treatment, and the fertilization rate was 41.51%.The sperm activation and fertilization rate were not significantly difference between the two treatment. But the constant temperature group was better than the room temperature group. It was knew that the room temperature was not very stability and always changing along with the environment temperature. And the sperm activation and capacitation was also instability. Swimming-up in constant temperature would be the best way to select sperm because it was easy to control and stability for the sperm activation and capacitation.The Sperm Concentration for Oocyte Fertilization RateThe study was conducted to optimize the fertilization condition. Sperm concentration had great effect on the oocyte fertilization rate. It were showed that the oocyte fertilization rate of middle sperm concentration group(1.5×106/ml, 47.34%)was significantly higher than the high sperm concentration group(3.0×106/ml, 33.96%)and low sperm concentration group(1.0×106/ml, 42.38%)(p<0.01). And the low group’s was significantly higher than the high group’s(p<0.01). The sperm concentration would be applied at (1.0～1.5)×106/ml and had less abnormal sperm and increased the fertilization rate.3 Bovine Oocyte in Vitro CultureIn Vitro Culture with Co-culture SystemThere were 3 co-culture system groups in the experiment.All of them were cumulus, cumulus+bFF and control group. The morula rate of its were 29.07%、34.99% and 26.67% and the blastocyst rate of its were 8.51%、10.63% and 7.24%.The morula and blastocyst rate of cumulus+bFF group were significantly higher than the cumulus and control group(p<0.01).There was 2-cell block in the field for oocyte embryo development.Co-culture with cumulus+bFF played a important role for embryo development to blastocyst and had overcome the 2-cell block.Oocyte Embryos Development with Medium VolumeIt were treated that maturated oocyte were cultured with 50μl and/or 500μl medium volume in 4-hole culture plate. The morula and blastocyst rate of 50μl medium were 33.14% and 9.07%. The morula and blastocyst rate of 500μl medium volume were 36.23% and 10.23%. The two rates of 500μl medium volume were significantly higher than the 50μl medium’s(p<0.01).It was reported that the small medium volume would not maintain the minienvironment verywell because it was easy to change, but the large medium volume as 500μl would maintain the minienvironment well and had had embryo development.4 Freezing of COCsOocyte been Frozen with 10%GL and 10%EG of Cryoprotectant by One-way ProcessHigh grade COCs was frozen and stored following the designed process and thawed one week late. The maturation and morula and blastocyst rate of 10%EG group were 34.46%,11.18% and 4.18%, and 10%GL group’s were 33.44%、10.56% and 3.86%.The difference of all between the two groups was not significantly(p>0.05).The trial had evidenced 10%GL and 10%EG of cryoprotectant adapted to freeze and store COCs and had the same function.Oocyte been Frozen for Carrier by VitrificationHigh grade COCs was also frozen and stored following the designed process and thawed one week late. The maturation and morula and blastocyst rate of wheat straw group were 29.84%, 9.34% and 3.47%, and OPS group’s were 31.89%, 9.66% and 3.80%. The difference of all between the two groups was not significantly(p>0.05). The results had showed that wheat straw and OPS of carrier adapted to freeze and store COCs, and had the same function.Comparing with the Frozen WaysAccording to above exprements of COCs frozen, the effect of one-way process was better than the vitrification process. The predominance sequence of frozen effect could express as follow as 10%EG group>10% GL group>OPS group>wheat straw group.5 Brief Summary.It was carried out that the bovine oocyte in vitro production and freezing were studied. Through this series experiments it had been learned and applied how to maturate oocyte in vitro, to capacitate sperm, to fertilize oocyte in vitro, to freeze oocyte by one-way or vitrification process, and to culture thawing oocyte been frozen. The 2-cell block had been overcomed by co-culture system with cumulus or cumulus+bFF. One new centrifugal method for sperm capacitation had been tested and applied in this research and got better than before.更多还原