羔羊JIVET相关技术及影响其卵母细胞发育功能基因的研究
发布时间：2018-10-17 10:14
【摘要】：幼畜体外胚胎生产和移植技术(juvenile in vitro embryo transfer,JIVET)是集成了幼龄母畜超数排卵技术、卵母细胞体外成熟技术、体外受精技术、胚胎体外培养技术和胚胎移植技术等一系列繁殖技术为一体的生物技术体系。JIVET技术将幼龄母畜卵巢上大量的卵母细胞为卵源在体外培养生产胚胎,有利于缩短家畜繁殖的世代间隔,充分发掘优良遗传性状母畜的繁殖潜能,提高畜群的遗传改良速度与生产效率,为家畜育种提供丰富的遗传资源。体外成熟是JIVET技术的一个重要环节,通过调节激素浓度等手段来提高体外成熟培养效果以达到细胞质和细胞核共同成熟的目标,能显著提高后期胚胎生长发育的潜力,极大地提高JIVET技术的生产效率。本研究以海安县当地的长江三角洲白山羊羔羊为主要研究对象,经羔羊屠宰场取卵后进行体外成熟培养,然后对培养成熟的卵母细胞进行体外受精。在成熟培养液里添加不同浓度的FSH+LH(2.5、5.0、7.5、10.0、12.5μg/mL),以研究FSH+LH浓度对卵母细胞成熟率的影响,旨在不断完善培养体系,并且对比羔羊与成年母羊的早期胚胎发育能力,来探究卵母细胞成熟的调控机制。本试验运用单细胞转录组测序技术筛选影响羔羊卵母细胞成熟的关键基因,并通过实时荧光定量技术对筛选出来的五个基因(MOS、RPS6KA1、CPEB1、 ANAPC13和CDK1)进行定量验证,从分子水平研究卵母细胞成熟的调控机制,以期提高羔羊卵母细胞发育到后期胚胎的潜力。本次研究结果如下：1、在成熟液里添加FSH+LH浓度分别为2.5、5.0、7.5、10.0、12.5μg/mL进行分组成熟培养后,其结果显示成熟液中添加不同浓度FSH+LH对卵母细胞成熟率有明显的影响。其中5.0μg/mLFSH+LH组的羔羊卵母细胞成熟率明显高于其他四组；10.0μg/mLFSH+LH组的成年母羊卵母细胞成熟率明显高于其他四组；而且羔羊和成年母羊卵母细胞在相同浓度FSH+LH的成熟液中培养效果无显著差异(P0.05)。2、在相同条件下体外受精后,对羔羊和成年母羊卵母细胞的卵裂率、4细胞率、8细胞率、16细胞率、囊胚率进行统计分析,其结果显示羔羊和成年母羊卵母细胞的卵裂率、4细胞率无显著差异(P0.05),羔羊卵母细胞的8细胞率显著低于成年母羊(P0.05),羔羊卵母细胞的囊胚率极显著低于成年母羊(P0.01)。3、采用单细胞测序技术对羔羊和成年母羊成熟的卵母细胞进行转录组差异分析,筛选出MOS、RPS6KA1、CPEB1、ANAPC13和CDK1这5个重要的表达差异基因,并通过实时荧光定量技术验证了转录组测序数据的准确性。通过总结与这五个基因有关的研究表明,MOS、RPS6KA1、CPEB1、ANAPC13和CDK1基因可能是羔羊卵母细胞成熟的关键基因。
[Abstract]:In vitro embryo production and transfer (juvenile in vitro embryo transfer,JIVET) is an integrated technique of superovulation, in vitro maturation of oocytes and in vitro fertilization. A series of reproductive techniques such as embryo culture in vitro and embryo transfer are integrated. JIVET technique takes a large number of oocytes from young female ovaries as oocytes to produce embryos in vitro. It is beneficial to shorten the intergenerational interval of livestock breeding, fully explore the reproductive potential of excellent hereditary female animals, improve the speed of genetic improvement and production efficiency of livestock, and provide abundant genetic resources for livestock breeding. In vitro maturation is an important part of JIVET technology. By regulating hormone concentration to improve the effect of in vitro maturation to achieve the goal of co-maturation of cytoplasm and nucleus, it can significantly improve the potential of late embryo growth and development. Greatly improve the production efficiency of JIVET technology. In this study, the local Yangtze River Delta white goat lamb in Haian County was selected as the main research object. The eggs were harvested from the slaughterhouse of the lamb and cultured in vitro, and then the matured oocytes were fertilized in vitro. In order to study the effect of FSH LH concentration on the maturation rate of oocytes, the effect of different concentrations of FSH LH (2.55.07.50.0.0.0 渭 g/mL on the maturation rate of oocytes was studied in order to improve the culture system, and to compare the ability of early embryo development between lamb and adult ewe, in order to study the effect of FSH LH concentration on the maturation rate of oocytes. To explore the regulatory mechanisms of oocyte maturation. In this study, single cell transcriptome sequencing was used to screen the key genes affecting oocyte maturation in lambs, and the five genes (MOS,RPS6KA1,CPEB1, ANAPC13 and CDK1) were quantitatively verified by real-time fluorescence quantitative analysis. The regulation mechanism of oocyte maturation was studied at the molecular level in order to improve the potential of oocyte development to anaphase embryo in lambs. The results were as follows: 1. The results showed that the addition of different concentrations of FSH LH to the maturation fluid had a significant effect on the maturation rate of oocytes. The maturation rate of oocytes in 5.0 渭 g/mLFSH LH group was significantly higher than that in the other four groups, and the maturation rate of adult ewe oocytes in 10.0 渭 g/mLFSH LH group was significantly higher than that in the other four groups. Moreover, there was no significant difference between lamb and adult ewe oocytes cultured in mature liquid of the same concentration of FSH LH (P0.05). 2. After in vitro fertilization, the cleavage rate, 4 cell rate, 8 cell rate, 16 cell rate of oocytes of lambs and adult ewe were 4 cells, 8 cells and 16 cells, respectively. The blastocyst rate was analyzed statistically. The results showed that the cleavage rate of oocytes in lambs and adult ewe had no significant difference (P0.05). The oocyte 8 cell rate of lambs was significantly lower than that of adult ewe (P0.05), and the blastocyst rate of lamb oocytes was significantly lower than that of adult ewe (P0.01), and the oocyte blastocyst rate of lambs was significantly lower than that of adult ewe (P0.01). Single cell sequencing technique was used to analyze the transcriptional difference between mature oocytes of lamb and adult ewe. Five important differentially expressed genes, MOS,RPS6KA1,CPEB1,ANAPC13 and CDK1, were screened, and the accuracy of transcriptome sequencing data was verified by real-time fluorescence quantitative technique. MOS,RPS6KA1,CPEB1,ANAPC13 and CDK1 genes may be the key genes for oocyte maturation in lambs.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
本文编号：2276330
[Abstract]:In vitro embryo production and transfer (juvenile in vitro embryo transfer,JIVET) is an integrated technique of superovulation, in vitro maturation of oocytes and in vitro fertilization. A series of reproductive techniques such as embryo culture in vitro and embryo transfer are integrated. JIVET technique takes a large number of oocytes from young female ovaries as oocytes to produce embryos in vitro. It is beneficial to shorten the intergenerational interval of livestock breeding, fully explore the reproductive potential of excellent hereditary female animals, improve the speed of genetic improvement and production efficiency of livestock, and provide abundant genetic resources for livestock breeding. In vitro maturation is an important part of JIVET technology. By regulating hormone concentration to improve the effect of in vitro maturation to achieve the goal of co-maturation of cytoplasm and nucleus, it can significantly improve the potential of late embryo growth and development. Greatly improve the production efficiency of JIVET technology. In this study, the local Yangtze River Delta white goat lamb in Haian County was selected as the main research object. The eggs were harvested from the slaughterhouse of the lamb and cultured in vitro, and then the matured oocytes were fertilized in vitro. In order to study the effect of FSH LH concentration on the maturation rate of oocytes, the effect of different concentrations of FSH LH (2.55.07.50.0.0.0 渭 g/mL on the maturation rate of oocytes was studied in order to improve the culture system, and to compare the ability of early embryo development between lamb and adult ewe, in order to study the effect of FSH LH concentration on the maturation rate of oocytes. To explore the regulatory mechanisms of oocyte maturation. In this study, single cell transcriptome sequencing was used to screen the key genes affecting oocyte maturation in lambs, and the five genes (MOS,RPS6KA1,CPEB1, ANAPC13 and CDK1) were quantitatively verified by real-time fluorescence quantitative analysis. The regulation mechanism of oocyte maturation was studied at the molecular level in order to improve the potential of oocyte development to anaphase embryo in lambs. The results were as follows: 1. The results showed that the addition of different concentrations of FSH LH to the maturation fluid had a significant effect on the maturation rate of oocytes. The maturation rate of oocytes in 5.0 渭 g/mLFSH LH group was significantly higher than that in the other four groups, and the maturation rate of adult ewe oocytes in 10.0 渭 g/mLFSH LH group was significantly higher than that in the other four groups. Moreover, there was no significant difference between lamb and adult ewe oocytes cultured in mature liquid of the same concentration of FSH LH (P0.05). 2. After in vitro fertilization, the cleavage rate, 4 cell rate, 8 cell rate, 16 cell rate of oocytes of lambs and adult ewe were 4 cells, 8 cells and 16 cells, respectively. The blastocyst rate was analyzed statistically. The results showed that the cleavage rate of oocytes in lambs and adult ewe had no significant difference (P0.05). The oocyte 8 cell rate of lambs was significantly lower than that of adult ewe (P0.05), and the blastocyst rate of lamb oocytes was significantly lower than that of adult ewe (P0.01), and the oocyte blastocyst rate of lambs was significantly lower than that of adult ewe (P0.01). Single cell sequencing technique was used to analyze the transcriptional difference between mature oocytes of lamb and adult ewe. Five important differentially expressed genes, MOS,RPS6KA1,CPEB1,ANAPC13 and CDK1, were screened, and the accuracy of transcriptome sequencing data was verified by real-time fluorescence quantitative technique. MOS,RPS6KA1,CPEB1,ANAPC13 and CDK1 genes may be the key genes for oocyte maturation in lambs.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
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