本文選題：牛 + 未成熟卵母細(xì)胞　； 參考：《河南科技大學(xué)》2015年碩士論文

【摘要】：哺乳動(dòng)物卵母細(xì)胞的冷凍保存是生命科學(xué)領(lǐng)域的重要研究?jī)?nèi)容之一,近年來(lái),盡管人們對(duì)此進(jìn)行了大量的研究,但卵母細(xì)胞冷凍保存的效果仍不理想,尤其是未成熟卵母細(xì)胞的冷凍效果更差。影響卵母細(xì)胞玻璃化冷凍效果的因素很多,其中降溫速率被認(rèn)為是最重要的影響因素。目前,卵母細(xì)胞玻璃化冷凍主要采用液氮(-196℃)為冷源,而液氦的溫度可以達(dá)到-269℃,本課題擬在液氮OPS玻璃化冷凍的基礎(chǔ)上,嘗試以溫度更低的液氦為冷源,通過(guò)加快降溫速率來(lái)改進(jìn)牛未成熟卵母細(xì)胞的冷凍效果,建立并優(yōu)化牛未成熟卵母細(xì)胞的液氦玻璃化冷凍方法。1.牛未成熟卵母細(xì)胞液氮OPS玻璃化冷凍方法的建立試驗(yàn)1:OPS管拉制方法研究。分別以水浴鍋、普通酒精燈和自制小酒精燈為熱源,探究OPS管的拉制方法。結(jié)果表明,水浴鍋和普通酒精燈均無(wú)法拉制出符合要求的OPS管,只有自制小酒精燈可以拉制出內(nèi)徑和管壁厚度分別為0.8mm和0.07 mm左右的OPS管。試驗(yàn)2:細(xì)管法和OPS玻璃化冷凍法的對(duì)比。將采集到的COCs隨機(jī)分為3組,對(duì)照組、細(xì)管組和OPS組。對(duì)照組不經(jīng)冷凍直接進(jìn)行IVM、IVF和早期胚胎IVC。兩個(gè)冷凍組分別用細(xì)管法和OPS法進(jìn)行玻璃化冷凍,解凍后分別進(jìn)行IVM、IVF和早期胚胎IVC。結(jié)果顯示:與對(duì)照組卵母細(xì)胞的形態(tài)正常率、成熟率、卵裂率和囊胚率(100%、86.1%、69.7%和38.2%)相比,兩冷凍組的各項(xiàng)指標(biāo)均顯著降低(P0.05)。在兩個(gè)冷凍組中,細(xì)管組和OPS組卵母細(xì)胞的形態(tài)正常率分別為65.2%和77.8%,成熟率分別為31.0%和41.6%,卵裂率分別為19.9%和29.8%,囊胚率分別為0和7.0%。OPS組上述指標(biāo)均明顯高于細(xì)管組(P0.05)。結(jié)論:用自制小酒精燈更易于拉制出適于玻璃化冷凍的OPS管;液氮OPS法玻璃化冷凍牛未成熟卵母細(xì)胞的效果優(yōu)于細(xì)管法。2.牛未成熟卵母細(xì)胞液氦OPS玻璃化冷凍方法的探索將采集到的COCs隨機(jī)分為3組,對(duì)照組、液氮組和液氦組。對(duì)照組不經(jīng)冷凍直接進(jìn)行IVM、IVF和早期胚胎IVC。兩個(gè)冷凍組分別以液氮和液氦為冷源,以O(shè)PS管為載體對(duì)牛未成熟卵母細(xì)胞進(jìn)行玻璃化冷凍,解凍后分別進(jìn)行IVM、IVF和早期胚胎IVC。結(jié)果顯示:與對(duì)照組卵母細(xì)胞的形態(tài)正常率、成熟率、卵裂率和囊胚率(100%、87.0%、69.5%和42.0%)相比,兩冷凍組的各項(xiàng)指標(biāo)均顯著降低(P0.05)。在兩個(gè)冷凍組中,液氦組解凍后卵母細(xì)胞的形態(tài)正常率(84.7%)顯著高于液氮組(78.5%;P0.05)。經(jīng)過(guò)體外成熟后,液氦組卵母細(xì)胞的成熟率(43.9%)顯著高于液氮組(39.2%;P0.05)。經(jīng)過(guò)體外受精和早期胚胎體外培養(yǎng)后,液氦組的卵裂率和囊胚率(35.4%和6.4%)顯著高于液氮組(30.0%和4.1%;P0.05)。結(jié)論:液氦OPS玻璃化冷凍法的效果優(yōu)于液氮法;液氦作為冷源冷凍保存牛未成熟卵母細(xì)胞是可行的。3.液氦OPS玻璃化冷凍方法的優(yōu)化試驗(yàn)試驗(yàn)1:最佳冷凍保護(hù)液的篩選。將采集到的COCs隨機(jī)分為6組,其中一組不經(jīng)冷凍,作為對(duì)照組,其余五組分別使用五種濃度的冷凍保護(hù)液(EDS30、EDS35、EDS40、EDS45和EDS50)進(jìn)行液氦OPS法玻璃化冷凍,解凍后分別進(jìn)行IVM、IVF和早期胚胎IVC。結(jié)果顯示:與對(duì)照卵母細(xì)胞的形態(tài)正常率、成熟率、卵裂率和囊胚率(100%、84.5%、69.9%和41.1%)相比,所有冷凍組的各項(xiàng)指標(biāo)均顯著降低(P0.05)。在五個(gè)冷凍組中,EDS35組卵母細(xì)胞(90.3%、51.9%、41.8%和10.0%)冷凍-解凍后的冷凍效果均顯著高于其他四組(P0.05)。EDS50組(75.1%、36.0%、27.2%和2.6%)的冷凍效果最差,顯著低于其他四組(P0.05)。EDS30(83.0%、42.0%、33.8%和6.5%)、EDS40(84.9%、43.9%、34.9%和6.4%)、EDS45(82.6%、42.4%、33.2%和5.1%)組之間的差異均不顯著(P㧐0.05)。試驗(yàn)2:液氦玻璃化冷凍中兩種操作溫度的對(duì)比。將采集到的COCs隨機(jī)分為三組,對(duì)照組、39℃組和25℃組。對(duì)照組不經(jīng)冷凍直接進(jìn)行IVM、IVF和早期胚胎IVC。兩個(gè)液氦OPS法玻璃化冷凍組除操作溫度不同外,其他操作均相同,解凍后分別進(jìn)行IVM、IVF和早期胚胎IVC。結(jié)果表明:與對(duì)照組卵母細(xì)胞的形態(tài)正常率、成熟率、卵裂率和囊胚率(100%、84.6%、71.5%和40.6%)相比,兩個(gè)冷凍組的各項(xiàng)指標(biāo)均顯著降低(P0.05)。兩個(gè)冷凍組中,39℃組和25℃組中的形態(tài)正常率分別為89.2%和85.8%,差異不顯著(P㧐0.05)。39℃組卵母細(xì)胞的成熟率、卵裂率、囊胚率分別是51.4%、41.2%和10.0%,均顯著高于25℃組(45.7%、34.1%和6.3%;P0.05)。試驗(yàn)3:最佳預(yù)處理時(shí)間的篩選。將采集到的COCs隨機(jī)分為四組,對(duì)照組、1 min預(yù)處理組、3 min預(yù)處理組和5 min預(yù)處理組。對(duì)照組不經(jīng)冷凍直接進(jìn)行IVM、IVF和早期胚胎IVC。三個(gè)液氦OPS法玻璃化冷凍組除在冷凍平衡液(VS1)中的預(yù)處理時(shí)間不同外,其他操作均相同。解凍后分別進(jìn)行IVM、IVF和早期胚胎IVC,結(jié)果顯示:與對(duì)照組卵母細(xì)胞的形態(tài)正常率、成熟率、卵裂率和囊胚率(100%、84.0%、70.6%和40.1%)相比,三個(gè)冷凍組的各項(xiàng)指標(biāo)均顯著降低(P0.05)。三個(gè)冷凍組中,1 min組、3 min組和5 min組解凍后卵母細(xì)胞的形態(tài)正常率分別為87.7%、87.2%和86.4%,彼此之間差異不顯著(P㧐0.05)。1min組解凍后卵母細(xì)胞的成熟率、卵裂率和囊胚率(49.6%、39.6%和10.4%)與3min組(47.4%、37.0%和8.8%)差異不顯著(P㧐0.05),但卻顯著高于5min組(41.0%、32.0%、6.1%;P0.05)。結(jié)論:液氦OPS玻璃化冷凍法的最佳冷凍保護(hù)液是EDS35,最佳操作溫度為39℃,最佳預(yù)處理時(shí)間為1min。
[Abstract]:The cryopreservation of mammalian oocytes is one of the important research contents in the field of life science. In recent years, although a lot of research has been done on this, the effect of cryopreservation of oocytes is still not ideal, especially the freezing effect of immature oocytes is worse. There are many factors affecting the vitrification effect of oocyte. The cooling rate is considered to be the most important factor. At present, the vitrification of oocyte mainly uses liquid nitrogen (-196 C) as the cold source, and the temperature of liquid helium can reach -269 C. On the basis of the cryopreservation of liquid nitrogen OPS vitrification, we try to use the liquid helium at a lower temperature as the cold source. The freezing effect of the mature oocyte, the establishment and optimization of the liquid helium vitrification of the immature oocytes of cattle.1. the establishment of the liquid nitrogen OPS vitrification method for the immature oocytes of cattle. A study on the method of drawing the 1:OPS tube. The method of drawing the OPS tube was investigated with water bath, ordinary alcohol lamp and self-made small alcohol lamp as the heat source. It shows that the water bath pot and the ordinary alcohol lamp can not make the OPS tubes that meet the requirements. Only the self-made small alcohol lamp can pull out the OPS tubes with the inner diameter and the thickness of the tube wall about 0.8mm and 0.07 mm respectively. The comparison of the 2: tube method and the OPS vitrification method. The collected COCs is divided into 3 groups, the control group, the tubule group and the OPS group. Two cryosurgery groups of IVM, IVF and early embryo IVC. were vitrified without freezing. The results of IVM, IVF and early embryo IVC. after thawing respectively showed that compared with the normal rate, maturation rate, cleavage rate and embryo rate (100%, 86.1%, 69.7% and 38.2%) of the control group, two freezing groups were compared. The morphological normal rates of the oocytes in the two frozen groups were 65.2% and 77.8%, the maturation rates were 31% and 41.6%, the cleavage rates were 19.9% and 29.8%, respectively, and the blastocyst rates were 0 and 7.0%.OPS respectively higher than those in the fine tube group (P0.05). Conclusion: the homemade small alcohol lamp was more than that of the small tube group (P0.05). It is easy to draw the OPS tube suitable for vitrification, and the effect of liquid nitrogen OPS method for vitrification of immature oocytes is better than that of liquid helium OPS vitrification of immature oocytes of.2. cattle. The collected COCs is randomly divided into 3 groups, the control group, the liquid nitrogen group and the liquid helium group. The control group is directly IVM, IVF and the control group without freezing. The early embryo IVC. two cryosurgery groups used liquid nitrogen and liquid helium as cold sources respectively, and OPS tube was used as the carrier to freeze the immature oocytes of cattle. After thawing, IVM, IVF and early embryo IVC. showed that compared with the control group, the normal rate, maturation rate, cleavage rate and blastocyst rate (100%, 87%, 69.5% and 42%) were compared with that of the control group. The indexes of the two freezing group were significantly decreased (P0.05). In the two frozen groups, the normal rate of oocyte in the liquid helium group (84.7%) was significantly higher than that in the liquid nitrogen group (78.5%; P0.05). The maturation rate of the oocyte in the liquid helium group (43.9%) was significantly higher than that in the liquid nitrogen group (39.2%; P0.05) after in vitro maturation. The cleavage rate and blastocyst rate (35.4% and 6.4%) in the liquid helium group (35.4% and 6.4%) were significantly higher than that in the liquid nitrogen group (30% and 4.1%; 4.1%; P0.05). Conclusion: the effect of liquid helium OPS vitrification is superior to the liquid nitrogen method; the cryopreservation of the immature oocyte by liquid helium as a cold source is a feasible optimization test of the.3. liquid helium cryopreservation method of liquid helium, the optimum freezing protection of 1: COCs randomly divided into 6 groups, one group without freezing, as the control group, the other five groups using five concentrations of cryopreservation solution (EDS30, EDS35, EDS40, EDS45 and EDS50) for liquid helium OPS method of vitrification, after thawing IVM, IVF and early embryo IVC. results showed: and the shape of the control oocyte Compared with the normal rate of state, maturity, cleavage rate and blastocyst rate (100%, 84.5%, 69.9% and 41.1%), all the indexes of all cryopreservation groups were significantly decreased (P0.05). In the five freezing groups, the freezing and thawing of group EDS35 oocytes (90.3%, 51.9%, 41.8% and 10%) were significantly higher than those of the other four groups (P0.05).EDS50 group (75.1%, 36%, 27.2% and 2.6%). The freezing effect is the worst, significantly lower than the other four groups (P0.05).EDS30 (83%, 42%, 33.8% and 6.5%), EDS40 (84.9%, 43.9%, 34.9% and 6.4%), EDS45 (82.6%, 42.4%, 33.2% and 5.1%) groups are not significant differences (P?). Test 2: liquid helium vitrification cold freezing operation temperature comparison. The collected COCs randomly divided into the group, the control group, the control group, The control group was not frozen directly for IVM, IVF and early embryo IVC. two liquid helium OPS vitrification group, except the operating temperature, the other operations were the same. After thawing IVM, IVF and early embryo IVC., respectively, showed that the normal rate, maturation rate, cleavage rate and blastocyst rate of the control group were 100%. (100% Compared with 84.6%, 71.5% and 40.6%, the indexes of two cryopreservation groups were significantly decreased (P0.05). In the two freezing groups, the normal rate in the 39 and 25 C groups was 89.2% and 85.8%, and the difference was not significant (P? 0.05), the maturation rate, cleavage rate and blastocyst rate were 51.4%, 41.2% and 10%, respectively. 4.1% and 6.3%; P0.05) screening the best pretreatment time for 3:. The collected COCs was randomly divided into four groups, the control group, the 1 min pretreatment group, the 3 min preconditioning group and the 5 min preconditioning group. The control group did not freeze the IVM, IVF and early embryos IVC. three liquid helium OPS method in the cryopreservation group (VS1). The other operations were the same. After thawing, IVM, IVF and early embryo IVC were carried out respectively. The results showed that compared with the normal rate, maturity, cleavage rate and blastocyst rate (100%, 84%, 70.6% and 40.1%) of the control group, the indexes of the three freezing groups were significantly reduced (P0.05). Three freezing groups, 1 min, 3 min and 5. The normal morphologic rates of oocyte in group min were 87.7%, 87.2% and 86.4%, respectively, and there was no significant difference between each other (P? 0.05).1min group after thawing, the maturation rate of oocytes after thawing, cleavage rate and blastocyst rate (49.6%, 39.6% and 10.4%) and 3min group (47.4%, 37% and 8.8%) were not significant (P? 0.05), but significantly higher than those of group 5min (41%, 32%, 6.1%; P0.05). Conclusion: the best cryopreservation solution for liquid helium OPS vitrification is EDS35, the best operating temperature is 39 C, and the best pretreatment time is 1min..

【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S823

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王彩玲;張華智;王利;彭三鳳;周學(xué)亮;陸陽(yáng)清;楊小淦;許惠艷;盧晟盛;盧克煥;;影響哺乳動(dòng)物卵母細(xì)胞玻璃化冷凍保存效果的因素[J];基因組學(xué)與應(yīng)用生物學(xué);2015年04期

相關(guān)碩士學(xué)位論文 前2條

1 史蕾;牛GV期卵母細(xì)胞玻璃化冷凍技術(shù)優(yōu)化及其作用機(jī)制研究[D];華中農(nóng)業(yè)大學(xué);2013年

2 陳君怡;液氦玻璃化冷凍對(duì)牛GV期卵母細(xì)胞發(fā)育及基因表達(dá)的影響[D];河南科技大學(xué);2014年

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本文編號(hào)：1806258