【摘要】：幼畜體外胚胎生產(chǎn)和移植技術(shù)(juvenile in vitro embryo transfer,JIVET)是集成了幼齡母畜超數(shù)排卵技術(shù)、卵母細(xì)胞體外成熟技術(shù)、體外受精技術(shù)、胚胎體外培養(yǎng)技術(shù)和胚胎移植技術(shù)等一系列繁殖技術(shù)為一體的生物技術(shù)體系。JIVET技術(shù)將幼齡母畜卵巢上大量的卵母細(xì)胞為卵源在體外培養(yǎng)生產(chǎn)胚胎,有利于縮短家畜繁殖的世代間隔,充分發(fā)掘優(yōu)良遺傳性狀母畜的繁殖潛能,提高畜群的遺傳改良速度與生產(chǎn)效率,為家畜育種提供豐富的遺傳資源。體外成熟是JIVET技術(shù)的一個(gè)重要環(huán)節(jié),通過調(diào)節(jié)激素濃度等手段來提高體外成熟培養(yǎng)效果以達(dá)到細(xì)胞質(zhì)和細(xì)胞核共同成熟的目標(biāo),能顯著提高后期胚胎生長(zhǎng)發(fā)育的潛力,極大地提高JIVET技術(shù)的生產(chǎn)效率。本研究以海安縣當(dāng)?shù)氐拈L(zhǎng)江三角洲白山羊羔羊?yàn)橹饕芯繉?duì)象,經(jīng)羔羊屠宰場(chǎng)取卵后進(jìn)行體外成熟培養(yǎng),然后對(duì)培養(yǎng)成熟的卵母細(xì)胞進(jìn)行體外受精。在成熟培養(yǎng)液里添加不同濃度的FSH+LH(2.5、5.0、7.5、10.0、12.5μg/mL),以研究FSH+LH濃度對(duì)卵母細(xì)胞成熟率的影響,旨在不斷完善培養(yǎng)體系,并且對(duì)比羔羊與成年母羊的早期胚胎發(fā)育能力,來探究卵母細(xì)胞成熟的調(diào)控機(jī)制。本試驗(yàn)運(yùn)用單細(xì)胞轉(zhuǎn)錄組測(cè)序技術(shù)篩選影響羔羊卵母細(xì)胞成熟的關(guān)鍵基因,并通過實(shí)時(shí)熒光定量技術(shù)對(duì)篩選出來的五個(gè)基因(MOS、RPS6KA1、CPEB1、 ANAPC13和CDK1)進(jìn)行定量驗(yàn)證,從分子水平研究卵母細(xì)胞成熟的調(diào)控機(jī)制,以期提高羔羊卵母細(xì)胞發(fā)育到后期胚胎的潛力。本次研究結(jié)果如下：1、在成熟液里添加FSH+LH濃度分別為2.5、5.0、7.5、10.0、12.5μg/mL進(jìn)行分組成熟培養(yǎng)后,其結(jié)果顯示成熟液中添加不同濃度FSH+LH對(duì)卵母細(xì)胞成熟率有明顯的影響。其中5.0μg/mLFSH+LH組的羔羊卵母細(xì)胞成熟率明顯高于其他四組；10.0μg/mLFSH+LH組的成年母羊卵母細(xì)胞成熟率明顯高于其他四組；而且羔羊和成年母羊卵母細(xì)胞在相同濃度FSH+LH的成熟液中培養(yǎng)效果無顯著差異(P0.05)。2、在相同條件下體外受精后,對(duì)羔羊和成年母羊卵母細(xì)胞的卵裂率、4細(xì)胞率、8細(xì)胞率、16細(xì)胞率、囊胚率進(jìn)行統(tǒng)計(jì)分析,其結(jié)果顯示羔羊和成年母羊卵母細(xì)胞的卵裂率、4細(xì)胞率無顯著差異(P0.05),羔羊卵母細(xì)胞的8細(xì)胞率顯著低于成年母羊(P0.05),羔羊卵母細(xì)胞的囊胚率極顯著低于成年母羊(P0.01)。3、采用單細(xì)胞測(cè)序技術(shù)對(duì)羔羊和成年母羊成熟的卵母細(xì)胞進(jìn)行轉(zhuǎn)錄組差異分析,篩選出MOS、RPS6KA1、CPEB1、ANAPC13和CDK1這5個(gè)重要的表達(dá)差異基因,并通過實(shí)時(shí)熒光定量技術(shù)驗(yàn)證了轉(zhuǎn)錄組測(cè)序數(shù)據(jù)的準(zhǔn)確性。通過總結(jié)與這五個(gè)基因有關(guān)的研究表明,MOS、RPS6KA1、CPEB1、ANAPC13和CDK1基因可能是羔羊卵母細(xì)胞成熟的關(guān)鍵基因。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 文路;湯富酬;;單細(xì)胞轉(zhuǎn)錄組高通量測(cè)序分析新進(jìn)展[J];遺傳;2014年11期

2 陳利;趙薇;占思遠(yuǎn);李丹;李利;仲濤;王林杰;張紅平;;山羊不同組織及不同發(fā)育時(shí)期骨骼肌內(nèi)參基因的表達(dá)穩(wěn)定性分析[J];畜牧獸醫(yī)學(xué)報(bào);2014年08期

3 李文娟;字向東;;羔羊超數(shù)排卵及體外胚胎生產(chǎn)技術(shù)的研究與應(yīng)用進(jìn)展[J];畜牧與獸醫(yī);2014年07期

4 周超平;李鑫輝;;單細(xì)胞轉(zhuǎn)錄組研究進(jìn)展[J];生物化學(xué)與生物物理進(jìn)展;2013年12期

5 尹峰;王利民;;幼齡羔羊超數(shù)排卵及體外生產(chǎn)胚胎技術(shù)[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2013年12期

6 段彪;杜海燕;張榮;;精子獲能時(shí)間及精卵共孵育時(shí)間對(duì)小鼠體外受精和胚胎發(fā)育的影響[J];中華臨床醫(yī)師雜志(電子版);2013年07期

7 陳林華;趙月娥;陳曉燕;高英;;兩種微量RNA擴(kuò)增方法的比較研究[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2011年04期

8 張濤;劉超;呂麗華;;LH和hCG對(duì)山羊卵母細(xì)胞體外成熟的影響[J];畜禽業(yè);2009年04期

9 佟桂芝;韓永勝;丁昕穎;;幼畜胚胎體外生產(chǎn)技術(shù)研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2009年03期

10 王榮祥;陳靜波;;羔羊卵母細(xì)胞體外生產(chǎn)胚胎技術(shù)研究進(jìn)展[J];黑龍江動(dòng)物繁殖;2009年02期

，

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