本文選題：牛 + 卵母細胞��； 參考：《河南科技大學》2017年碩士論文

【摘要】：哺乳動物的卵子冷凍技術在珍稀動物品種資源保存、人類輔助生殖領域及胚胎研究中一直是重要的研究內(nèi)容。卵母細胞玻璃化冷凍過程中,冷凍體積、冷凍保護劑濃度、冷凍降溫-解凍速率等因素是影響冷凍效果的關鍵,合適的冷凍載體可以減小冷凍體積,從而提高降溫-解凍的速率。近年來,雖有多種載體的報道研究,但不同研究者報道的制作方法和效果不盡相同。因此本研究旨在探索不同載體的研究方法,以卵母細胞冷凍后的發(fā)育能力為評定指標,通過4種冷凍載體的對比試驗,篩選出在我們現(xiàn)有實驗條件下的最佳玻璃化冷凍載體并探討冷凍不同發(fā)育時期卵母細胞的冷凍效果,從而為提高卵母細胞的冷凍保存效果奠定技術基礎。1.冷凍載體制備方法研究試驗1:OPS載體的優(yōu)化。將細管一分為二,分別以常規(guī)酒精燈和簡易酒精燈為熱源,對細管一端旋轉(zhuǎn)加熱,待前端待細管前端膨大時,迅速用小鑷子將膨大部分向另一端平穩(wěn)拉伸,經(jīng)冷卻固定后,剪去前端膨大部獲得OPS冷凍載體。結果表明,只有以簡易小酒精燈為熱源可對OPS載體成功優(yōu)化,且此方法不僅能增加細管的受熱均勻度,易于操作,而且也能減少材料損耗。試驗2:GMP載體制備方法研究。以卵針前端細部(內(nèi)徑約為1 mm,管壁厚度約為0.09 mm,長度約為6 cm)為原材料,分別以PC-10型拉針儀、常規(guī)酒精燈和簡易酒精燈為熱源,探究GMP載體的制備方法;以玻璃管(外徑0.8cm)為原材料,以酒精噴燈為熱源,探究GMP載體的制備方法。結果顯示:以酒精噴燈為熱源,以細玻璃管為原材料可以成功制備出GMP載體。試驗3:GMP冷凍載體的改進。以酒精噴燈和常規(guī)酒精燈為熱源,分別對外徑為0.8 cm、0.4 cm和0.3 cm玻璃管旋轉(zhuǎn)加熱。結果顯示,外徑為0.8 cm的玻璃管制備的GMP載體與外徑為0.5 cm的制備效果并無差異;外徑為0.4 cm和0.3 cm的細玻璃管以酒精噴燈為熱源,制備出的載體外徑在0.43~0.5 mm間,保留其后端的載桿經(jīng)測量發(fā)現(xiàn)距前端載體約4 cm可以作為GMP載體使用,但該方法材料損耗較大;以常規(guī)酒精燈為熱源時,制備的載體經(jīng)測量外徑為0.4~0.5 mm,管壁厚度約為0.03 mm,距后端載體約3 cm可作為GMP載體,并且在制備的過程中載體和載桿間會有一小玻璃氣泡產(chǎn)生,冷凍時對虹吸作用也有一定促進作用。結論:用常規(guī)酒精燈,外徑為0.3 cm和0.4 cm玻璃管制備的GMP載體效果最佳,將塑料細管一分為二,使其一端加熱可在原有OPS制備方法基礎上極大降低了損耗。2.不同載體對牛未成熟卵母細胞玻璃冷凍效果的比較將抽取的COCs隨機分為5組,OPS組、GMP組、冷凍葉片組、自制葉片組以及對照組。4組冷凍組細胞經(jīng)液氮冷凍解凍后分別進行IVM、IVF和IVC,新鮮對照組不做冷凍處理,直接進行IVM、IVF和IVC,統(tǒng)計成熟率、卵裂率和囊胚率。結果顯示:冷凍組的形態(tài)正常率、成熟率、卵裂率以及囊胚率與新鮮對照組各項指標(100%、78.1%±3.1%、64.4%±2.8%、34.7%±2.5%)均顯著差異(P㩳0.05),OPS組和GMP組形態(tài)正常率(74.3%±1.8%,72.5%±2.6%)無顯著差異(P㧐0.05),上述二組均顯著低于冷凍葉片組(82.1%±1.3%,P㩳0.05),但三組間成熟率、卵裂率和囊胚率均無顯著差異(P㧐0.05),自制葉片組則與其他冷凍組的各項發(fā)育指標差異均顯著(P㩳0.05)。結論:自制OPS、GMP冷凍載體均可成功地玻璃化冷凍牛未成熟卵母細胞。3.不同發(fā)育時期卵母細胞玻璃化冷凍效果的影響將抽取的COCs隨機分為4組,體外培養(yǎng)0 h、8 h、16 h、以及22 h,采用GMP冷凍法分別冷凍培養(yǎng)不同時間的卵母細胞,解凍后繼續(xù)依此培養(yǎng)24 h、16h、8 h、和2 h,之后分別進行IVM、IVF、IVC,統(tǒng)計各組的形態(tài)正常率、成熟率、卵裂率以及囊胚率。結果顯示:經(jīng)解凍后培養(yǎng)0 h、8 h、16 h和22 h的形態(tài)正常率分別為72.7%、75.3%、77.7%和78.0%,四組間差異均不顯著(P㧐0.05),培養(yǎng)0 h的卵母細胞同培養(yǎng)8h的各項發(fā)育指標差異均不顯著(P㧐0.05),但培養(yǎng)16 h的卵母細胞與前兩者各項發(fā)育指標差異均顯著(P㩳0.05),而培養(yǎng)22 h的卵母細胞各項發(fā)育指標均高于其他發(fā)育時期組。結論:卵母細胞的抗凍性隨其成熟程度增加而增強。
[Abstract]:The technology of mammalian oocyte cryopreservation is an important research content in the conservation of rare animal species resources, in the field of human assisted reproduction and in the study of embryos. In the process of vitrification of oocytes, the factors such as freezing volume, concentration of cryopreservation agent, freezing cooling rate and thawing rate are the key factors affecting the freezing effect and suitable freezing carrier. The freezing volume can be reduced to increase the rate of cooling and thawing. In recent years, although there have been reports of various carriers, the methods and effects of different researchers have been different. Therefore, this study aims to explore the research methods of different carriers. The development ability of oocyte after cryosurgery is evaluated by 4 kinds of cryosurgery. The optimal vitrification carrier under the existing experimental conditions was screened and the cryopreservation effect of the oocyte in different cryopreservation period was investigated, thus the technical basis for improving the cryopreservation of oocytes was established, and the optimization of the study on the preparation of the.1. cryopreservation method was established. The tubes were divided into two parts. Do not take the conventional alcohol lamp and the simple alcohol lamp as the heat source, and heat the end of the pipe. When the front end of the tube is expanded, the bulk is stretched quickly with the small tweezers to the other end. After the cooling is fixed, the OPS freezer is cut to the front end. The result shows that only the simple small alcohol lamp can be used as the heat source for the OPS carrier. Work optimization, and this method can not only increase the heat uniformity of the tube, easy to operate, but also reduce the loss of material. Study on the preparation method of 2:GMP carrier. The raw material is the front end of the egg needle (the inner diameter is about 1 mm, the thickness of the tube wall is about 0.09 mm, the length is about 6 cm) as the raw material, with the PC-10 type drawing needle instrument, the conventional alcohol lamp and the simple alcohol lamp respectively. For the heat source, the preparation method of GMP carrier is explored. Using the glass tube (outer diameter 0.8cm) as the raw material and the alcohol spray lamp as the heat source, the preparation method of the GMP carrier is explored. The results show that the GMP carrier can be successfully prepared with the alcohol spray lamp as the heat source and the fine glass tube is used as the raw material. The improvement of the 3: GMP freezing carrier is tested. The alcohol spray lamp and the conventional alcohol lamp are used. For the heat source, the external diameter of 0.8 cm, 0.4 cm and 0.3 cm glass tubes was rotated. The results showed that the GMP carrier with the outer diameter of 0.8 cm was not different from the preparation effect of the outer diameter of 0.5 cm; the outer diameter of the thin glass tube with the diameter of 0.4 cm and 0.3 cm was the heat source of the alcohol spray lamp, and the outer diameter of the prepared carrier was between the 0.43~0.5 mm and the back end. It is found that about 4 cm from the front end carrier can be used as a GMP carrier, but the material loss is great. When the conventional alcohol lamp is used as the heat source, the measured outer diameter of the carrier is 0.4~0.5 mm, the thickness of the tube wall is about 0.03 mm and the back end carrier is about 3 cm as the GMP carrier, and there will be a carrier and the carrier in the process of preparation. A small glass bubble is produced and freezing has a certain effect on the siphon effect. Conclusion: the GMP carrier with 0.3 cm and 0.4 cm glass tubes with the conventional alcohol lamp is the best, and the plastic pipe is divided into two, and the heat can be heated on the basis of the original OPS preparation method, and the loss of.2. is greatly reduced to the immature cattle. The comparison of the effect of vitreous cryopreservation of oocyte was divided into 5 groups: OPS, GMP, frozen leaves, homemade leaves and.4 groups of the control group, IVM, IVF and IVC were frozen after freezing and thawing. IVM, IVF and IVC, statistical maturity, cleavage rate and blastocyst rate were carried out in the fresh control group without freezing. The results showed that the morphological normal rate, maturity, cleavage rate and blastocyst rate of the cryopreservation group were significantly different (100%, 78.1% + 3.1%, 64.4% + 2.8%, 34.7% + 2.5%) of the fresh control group (P? 0.05). There was no significant difference in the normal rate (74.3% + 1.8%, 72.5% + 2.6%) in group OPS and GMP group (P? 0.05), and all of these groups were significantly lower than those of the frozen leaves group. 1.3%, P? 0.05), but there was no significant difference between the three groups of maturation rates, cleavage rate and blastocyst rate (P? 0.05). The difference between the self-made leaf group and the other freezing groups was significant (P? 0.05). Conclusion: homemade OPS, GMP cryopreservation can be successfully vitrified oocytes of cold frozen bovine oocytes at different developmental stages of vitrification. The effect of frozen effect was randomly divided into 4 groups of COCs, which were cultured in vitro 0 h, 8 h, 16 h, and 22 h. The oocytes were frozen for different time by GMP freezing method. After thawing, 24 h, 16h, 8 h, and 2 h were continued, then IVM, IVF, and 2 h respectively. The results showed that the normal morphological rates of 0 h, 8 h, 16 h and 22 h after thawing were 72.7%, 75.3%, 77.7% and 78%, respectively, and there was no significant difference between the four groups (P? 0.05). The differences in the development indexes of the oocytes with the culture of 0 h were not significant (P? 0.05), but the differences in the development indices of the oocytes in the cultured 16 h were both significant (P? P). However, the developmental indexes of oocytes cultured at 22 h were higher than those of other developmental stages. Conclusion: the antifreeze ability of oocytes increases with the maturity of oocytes.
【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S823

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