【作者】 肖开田；

【导师】 李跃民；

【作者基本信息】 西南农业大学 ， 临床兽医学， 2004， 硕士

【摘要】 水牛繁殖存在许多问题，如性成熟较晚、发情不明显、发情后期长、受胎率低等。与黄牛相比，水牛胚胎生物技术相关研究较为滞后，文献报道也相对较少。有关水牛胚胎的程序化冷冻有过成功的报道并产犊，但未见程序化冷冻水牛卵母细胞的报道。本实验旨在探索水牛生发泡(GV)期卵母细胞程序化冷冻保存的条件，希望能提高其保护效果，为水牛的IVM、IVF、核移植等生物技术提供实验材料。 本实验采用程序化冷冻方法，对不同植冰温度(-5.5℃、-6℃、-6.5℃、-7℃)下几种冷冻液[PBSm(PBS+FBS)、PEGF(PBSm+EG+GL)、PEF(PBSm+EG)、PGF(PBSm+GL)]的结晶变化进行了探讨，进而探讨了不同植冰温度(-6℃、-6.5℃、-7℃)、不同冷冻保护液[PBSm(对照)、PEGF、PEF、PGF]对水牛GV期卵母细胞的冷冻保护效果。 1．实验过程中我们将水牛卵巢采卵情况与一同采回的黄牛卵巢采卵情况作了比较：从单个水牛卵巢回收的卵母细胞数平均为4.85个，从单个黄牛卵巢回收的卵母细胞数平均为13.4个，平均每个卵巢回收卵母细胞数水牛明显低于黄牛，外观形态正常、可用的卵母细胞比率也极显著低于黄牛(水牛66.3％，黄牛87.9％，P＜0.01)。 2．在-5.5℃、-6℃、-6.5℃、-7℃四种温度下植冰，以植冰后平衡10min结晶为适宜的保护指标，用PBSm作对照，观察PEGF、PEF、PGF在植冰后不同时问(2，5，8，10，15min)的结晶情况。结果显示：PBSm在四种植冰温度植冰后都很快完全结晶(2min)；植冰温度为-5.5℃时，植冰后10min PEGF、PEF、PGF未完全结晶，15min时PGF仍未完全结晶；植冰温度为-6℃，植冰后10min PEGF、PEF、PGF完全结晶；植冰温度为-6.5℃，植冰后8min PEGF、PEF、PGF完全结晶；植冰温度为-7℃，植冰后5min PEGF、PEF、PGF完全结晶。说明保护液中单独添加GL比单独添加EG有较低结晶速率，PEGF和PEF的结晶效果明显好于PGF和PBSm。在四种植冰温度下表现出温度越低结晶效果越好的趋势。 3．-6℃、-6.5℃、-7℃三种植冰温度下植冰，程序化冷冻解冻水牛GV期卵母细胞的形态正常率为：PEGF(84％、88.37％、90.9％)＞PEF(73.7％、78％、76.7％)＞PGF(58％、59％、57.5％)＞PBSm(50.7％、47.2％、47.4％)。同种植冰下，PEGF与PGF间差异显著(p＜0.05)，PEF与PBSm间差异显著(p＜0.05)，PEGF与PBSm间差异极显著(p＜0.01)。同种冷冻保护液，-6℃、-6.5℃、-7℃植冰，效果差异不显著。 4．-6℃、-6.5℃、-7℃植冰，程序化冷冻解冻后水牛GV期卵母细胞成活率(0.8％台盼兰染色判断)为：PEGF(76.9％、87.5％、83.3％)＞PEF(64.0％、70.8％、66.7％)＞PGF(47.4％、55.0％、55.6％)＞PBSm(16.7％、37.5％、35.3％)。植冰温度为-6℃时的成活率低于-6.5℃和-7℃的成活率。同种植冰温度下，PEGF与PEF间、PEF与PGF间、PGF与PBSm间染色成活率差异不显著，但PEGF与PGF间、PEF与PBSm间差异极显著(P＜0.01)，说明在-6.5℃或-7℃植冰温度下使用PEGF冷冻保护液比较好。西南农业大学硕士学位论文中文摘要旦旦口组且口里口级旦口.口口.. 5一6℃、芍.5℃、一℃三种植冰温度植冰，程序化冷冻解冻后水牛GV期卵母细胞成熟率为:PEGF(1 8.9%、20.5%、20.0%户PEF(9.7%、18.2%、15.6%卜PGF(6.9%、7.7%、8.3%卜PBSm(0、0、0).峨5℃植冰，PEGF作冷冻保护液有2枚卵母细胞发生卵裂，PEF作冷冻保护液有1枚卵母细胞发生卵裂;一℃植冰，PEGF作冷冻保护液有1枚卵母细胞发生卵裂.说明EG和GL混合使用，保护效果好于EG或GL单独使用，据此得出，在双5℃或一7℃植冰，用PEGF作冷冻保护液保存水牛GV期卵母细胞效果比较好。 6.通过将冷冻处理与未冷冻处理的水牛GV期卵母细胞的IVM效果比较，差异极显著 (P<0川)，说明冷冻对水牛GV期卵母细胞的损伤严重. 结果表明:用PEGF作冷冻保护液，装管前用2步法平衡，植冰温度用戒5℃或一℃，植冰后平衡l伽min，以0.3℃lmin降至一5℃投入液氮，解冻采用快速解冻、2步脱出保护剂，对水牛CV期卵母细胞冷冻效果好。更多还原

【Abstract】 There are many problems with buffalo’s reproduction, such as delayed sexual maturation, unobvious estrus, long interim between estruses, low conception rate. The researches concerning embryo biotechnologies on buffalo are lagged behind that on cattle, and the reports about it are also relatively fewer. Although the success of program freezing of buffalo embryo had been reported and gave birth to children, there’s no report about program freezing of buffalo oocytes. This study was to explore program freezing conditions for buffalo GV oocytes, aiming at improving protective effect of program freezing, to provide experimental materials for buffalo’s IVM, IVF, nuclear transfer and other biotechnologies.Program freezing method was applied in this experiment, according to discussing crystal changes of several different freezing medium under different seeding temperatures, the freezing and protective effects of different cryopreservation medium on buffalo GV oocytes at different seeding temperatures were tested.1. During the experiments, the ovaries from cattle and buffalo were compared, the results indicated the number of oocytes from one cattle ovary is 13.4 on average, and that from one buffalo ovary was 4.85 on average. The latter was lower than the former, the diffrence was significant (p<0.01). The normal rate of oocyte configuration between cattle and buffalo was significantly different (p<0.01), respectively 87.9% and 66.3%.2. Seeding at -5.5℃,-6.5℃,-7℃ and using PBSm as control, the crystal status of PEGF, PEF, PGF at different time after seeding (2, 5, 8, 10, 15min) were observed.The results showed that PBSm completely rimmed in a short time after seeding (2min); when the seeding temperature came to -5.5℃, PEGF, PEF, PGF did not completely rime 10min after seeding, PGF did not completely rime 15min after seeding; When -6℃, PEGF, PEF, PGF did completely rime lOmin after seeding; When -6.5X:, PEGF, PEF, PGF completely rimed 8min after seeding; When -7℃, PEGF, PEF, PGF completely rimed 5min after seeding. And this demonstrated the crystal speed solely adding GL was lower than that solely adding EG, the Crystal effect of PEGF and PEF was better than that of GL and PBSm. At four seeding temperature, the crystal effects increased as seeding temperature decreased.3. Seeding at -6℃, -6.5℃, -7℃, the rate of oocytes with normal morpha after freezing and thaw-freezing was: PEGF (84%, 88.37%, 90.9%)>PEF (73.7%, 78%, 76.7%) > PGF (58%, 59%, 57.5%) > PBSm (50.7%, 47.2%, 47.4%). At the same seeding temperature, there was different between PEGF and PGF(p<0.05), between PEF and PBSm (p<0.05); and significance difference between PEGF and PBSm (p<0.01). When respectively seeding at -6℃, -6.5℃ and -7℃, thae effect of the same cryopreservation medium showed no difference.4. The survival rate of oocytes dyed by Trypan blue was: PEGF (76.9%, 87.5%, 83.3%) > PEF (64.0%, 70.8%, 66.7%) > PGF (47.4%, 55.0%, 55.6%) > PBSm (16.7%, 37.5%, 35.3%). The survival rate of oocytes was lower at -6℃ seeding than at -6.5℃ and -7℃ seeding. At these three seeding temperatures, the survival rate respectively between PEGF and PEF, PEF and PGF, PGF and PBSm was not different, but the survival rate respectively between PEGF and PGF, PEF and PBSm was significant different (p<0.01).5. Respectively seeding at -6℃,-6.5℃,-7℃, the maturation rate was : PEGF (18.9%, 20.5%, 20.0%) > PEF(9.7%, 18.2%, 15.6%) > PGF(6.9%, 7.7%, 8.3%) > PBSm(0, 0, 0). With regard to oocytes with normal morpha, after seeding at these three temperatures , PEGF and PEF, PEF and PGF were respectively not different ,but PEGF and PGF were different (p<0.05).after seeding at -6.5℃,two oocytes with PEGF as cryopreservation medium cleavaged ;one oocyte with PEF as cryopreservation medium cleavaged; one oocyte with PEGF as cryopreservation medium cleavaged after seeding at -7℃.In terms of these evidences ,the buffalo GV oocytes’ freezing effect using PEGF as cryopreservation medium was good when seeding at -6.5℃ or -7℃.6. For更多还原