水牛卵泡卵母细胞体外成熟及体外受精研究
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摘要
世界范围内，水牛的繁殖能力低下是阻碍水牛奶、肉生产性能提高的主要原因之一。尽管在环境恶劣的热带地区，水牛有黄牛和肉牛无法比拟的优点，水牛的发展仍然一度被忽视。近年来体外受精(in vitro fertilization，IVF)和胚胎移植技术(embryo transplant，ET)的发展，使来自优秀水牛的后代数量快速增加成为可能。但是，将牛体外受精和胚胎移植技术应用于水牛并取得成功的报道非常有限。鉴于此，本研究参照牛卵母细胞的体外受精技术，对水牛卵母细胞体外成熟和体外受精进行了较为详细、系统的研究，并首次对采用水牛的附睾尾精子进行体外受精的可行性进行了探讨，以期为进一步建立和完善水牛体外受精技术、加速我国水牛品种改良提供理论依据和实践参考。
试验研究表明，在体外成熟(in vitro manturation，IVM)培养液中含有3μg/ml促卵泡素(follicle-stimulating hormone，FSH)时，随着促黄体素(luteinizing hormone，LH)添加量的增加，第一极体(polar body Ⅰ，PBl)排放率和成熟率逐渐提高：添加30μg/ml LH水牛卵母细胞的第一极体排放率(26.67％ vs 2.86％，7.50％)和体外成熟率(40.00％ vs14.29％，17.50％)均显著(p＜0.05)提高。在含有30μg/ml LH的IVM液中进一步将FSH增加为30μg/ml，水牛卵母细胞的第一极体排放率(24.24％)和体外成熟率(45.45％)没有提高；在含有激素的成熟培养液中添加10％的卵泡液(bovine follicular fluid，BFF)，水牛卵母细胞的第一极体排放率(52.33％)和成熟率(66.28％)均进一步显著(p＜O.05)提高；添加发情当天的牛血清(oestrous cow scru，OCS)，水牛卵母细胞的第一极体排放率和成熟率(48.1％，59.04％)均高于胎牛血清(fetal calf serum，FCS)(37.39％，52.87％)，但无统计学差异；颗粒细胞单层共培养对水牛卵母细胞的第一极体排放率(49.21％)和成熟率(65.08％)有提高，但差异不显著。在本实验室条件下，综合以上成熟培养条件可获得60.61％的第一极体率和72.73％的成熟率。
本试验采用添加肝素的BO液和TALP液分别对水牛精子进行获能和受精处理。用BO液和TALP液处理，卵裂率分别为51.85％和55.04％；就发育率而言，相对于培养卵数发育率分别为27.19％和26.36％；相对于卵裂数达48.21％和47.89％。两种处理，水牛精子体外受精效果差异不显著，说明BO液和TALP液均可用于水牛体外受精。
本试验首次对采用水牛的附睾尾精子进行体外受精的可行性进行了探讨。水牛附睾尾精子的受精率为60.71％，卵裂率为50.39％，相对于培养卵数发育率达24.41％，相对于卵裂数发育率达48.44％；与来自广西品种改良站的细管冷冻精液(64.00％，54.31％，26.72％，49.21％)相比，它们的受精能力差异不显著。分别用BO液和TALP液处理水牛附睾尾精子，受精后的卵裂率分别为46.67％和53.73％；发育率分别为21.67％和26.87％；受精效果差异也不显著，与冻精的相应指标相比差异也不显著。
附睾尾精子的活率与采集附睾的方法有关，用保温干储的方法可获得活率好、存活
水牛妞榕吸母纫屉洋办屈撤戾赵丹笋菏研穷
时间长的附睾尾精子：形态学观察及染色结果表明：水牛附宰尾精子解高，原生质滴
率也高。附睾尾精子受精能力可能与原生质滴率有关。试验结果表明，将水牛附睾尾精
子用于水牛体外受精研究和体外受精胚的生产是可行的。
The inadequate reproductive capability of domestic buffalo is one of the major impediments to the improvement of buffalo milk and beef production wordwild. Domestic buffalo development is neglected all the time despite its superiority over cow and beef cattle in the harsh environment of the tropical regions. Recently, with the development of IVF and embryo transplant (ET), it becomes possible to obtain large numbers of offsprings from high-quality buffalos rapidly. But there are few reports on applying these techniques to buffalo IW successfully. Therefore, we systematically studied the detailed processes of external maturation and fertilization of the bufflo oocyte, with reference of IVF of yellow cattle. And for the first time, investigated the possibility of external fertilization using sperm from epididymis of bufflo, in order to establish an optimized system for buffalo IVF, and thus to provide the theoretic basis and practical references for speeding up the improvement of buffalo in china.
The study indicated that, with 3ug/ml FSH external maturation medium, as LH increased, FBI exclusion and maturation rate of the buffalo oocyte increase as well. When the concentration of LH reaching 30ug/ml, FBI exclusion (26.67% vs 2.86%, 7.50%) and maturation ration (40.00% vs 14.29%, 17.50%) elevated significantly (p<0.05). In the medium with 30ug/ml LH, FSH rose to 30ug/ml, the FBI exclusion (24.24%) and maturation ratio (45.45%) did not increase further. When 10% BFF was added to the maturation medium, which contained gonadotropins, the FBI exclusion (52.33%) and maturation ration (66.28%) were increased further significantly (p<0.05). With the supplement OCS to medium, the FBI exclusion and maturation ratio were 48.19% and 59.04%, respectively, and 37.39% and 52.87% respectively in FCS medium. The former was superior to the latter, but no significant difference was found statistically significantly. Granulosa-cell monolayer co-culture system has improved the FBI exclusion (49.21%) and maturation ratio (65.08%), but there was no apparent difference. The 60.61% FBI exclusion and 72.73% maturation ratio had been obtained with the above cultured conditions.
We had adopted BO and TALP medium with heparin as sperm in vitro capacitation and
fertilization medium. The differences of the sperm capacitation and fertility between in BO and in TALP were investigated. The results indicate: the fertilized egg cleavage rates were 51.85% and 55.04% respectively; The development rates were 27.19% and 26.36% comparative to the number of cultured oocytes and 48.21% and 47.89% comparative to the number of the cleavage oocyte in BO and TALP respectively. The result showed no difference and the two media could be applied in buffalo IVF.
Furthermore, the feasibility to apply epididymal spermatozoa in buffalo IVF was first studied. The results demonstrate that the fertilization rate was 60.71%; egg cleavage ratio was 50.39%; development ratio was 24.41% comparative to the number of culture oocyte and 48.44% comparative to the number of cleavage oocyte. No significant differences between the epididymal spermatozoa and the thawed spermatozoa (64.00%, 54.31%, 26.72%, 49.21%) from Guangxi were found. The egg cleavage ratio and development ratio of the epididymal spermatozoa were 46.67%, 53.37% and 21.67%, 26.87% respectively. The effect of fertilization showed no apparent difference in the two media.
The spermatozoa stain and morphology indicated that the epididymal caudal sprmatozoa mobility is superior to thawed spermatozoa but have high percentage of protoplasm drop. The epididymal caudal sprmatozoa mobility was relevant to the collecting methods. The best method in the study was to conserve the epididymis in the conditions of constant temperature and anhydrous container. The results indicate the buffalo epididymal caudal sprmatozoa could be applied in buffalo IVF.
试验研究表明，在体外成熟(in vitro manturation，IVM)培养液中含有3μg/ml促卵泡素(follicle-stimulating hormone，FSH)时，随着促黄体素(luteinizing hormone，LH)添加量的增加，第一极体(polar body Ⅰ，PBl)排放率和成熟率逐渐提高：添加30μg/ml LH水牛卵母细胞的第一极体排放率(26.67％ vs 2.86％，7.50％)和体外成熟率(40.00％ vs14.29％，17.50％)均显著(p＜0.05)提高。在含有30μg/ml LH的IVM液中进一步将FSH增加为30μg/ml，水牛卵母细胞的第一极体排放率(24.24％)和体外成熟率(45.45％)没有提高；在含有激素的成熟培养液中添加10％的卵泡液(bovine follicular fluid，BFF)，水牛卵母细胞的第一极体排放率(52.33％)和成熟率(66.28％)均进一步显著(p＜O.05)提高；添加发情当天的牛血清(oestrous cow scru，OCS)，水牛卵母细胞的第一极体排放率和成熟率(48.1％，59.04％)均高于胎牛血清(fetal calf serum，FCS)(37.39％，52.87％)，但无统计学差异；颗粒细胞单层共培养对水牛卵母细胞的第一极体排放率(49.21％)和成熟率(65.08％)有提高，但差异不显著。在本实验室条件下，综合以上成熟培养条件可获得60.61％的第一极体率和72.73％的成熟率。
本试验采用添加肝素的BO液和TALP液分别对水牛精子进行获能和受精处理。用BO液和TALP液处理，卵裂率分别为51.85％和55.04％；就发育率而言，相对于培养卵数发育率分别为27.19％和26.36％；相对于卵裂数达48.21％和47.89％。两种处理，水牛精子体外受精效果差异不显著，说明BO液和TALP液均可用于水牛体外受精。
本试验首次对采用水牛的附睾尾精子进行体外受精的可行性进行了探讨。水牛附睾尾精子的受精率为60.71％，卵裂率为50.39％，相对于培养卵数发育率达24.41％，相对于卵裂数发育率达48.44％；与来自广西品种改良站的细管冷冻精液(64.00％，54.31％，26.72％，49.21％)相比，它们的受精能力差异不显著。分别用BO液和TALP液处理水牛附睾尾精子，受精后的卵裂率分别为46.67％和53.73％；发育率分别为21.67％和26.87％；受精效果差异也不显著，与冻精的相应指标相比差异也不显著。
附睾尾精子的活率与采集附睾的方法有关，用保温干储的方法可获得活率好、存活
水牛妞榕吸母纫屉洋办屈撤戾赵丹笋菏研穷
时间长的附睾尾精子：形态学观察及染色结果表明：水牛附宰尾精子解高，原生质滴
率也高。附睾尾精子受精能力可能与原生质滴率有关。试验结果表明，将水牛附睾尾精
子用于水牛体外受精研究和体外受精胚的生产是可行的。
The inadequate reproductive capability of domestic buffalo is one of the major impediments to the improvement of buffalo milk and beef production wordwild. Domestic buffalo development is neglected all the time despite its superiority over cow and beef cattle in the harsh environment of the tropical regions. Recently, with the development of IVF and embryo transplant (ET), it becomes possible to obtain large numbers of offsprings from high-quality buffalos rapidly. But there are few reports on applying these techniques to buffalo IW successfully. Therefore, we systematically studied the detailed processes of external maturation and fertilization of the bufflo oocyte, with reference of IVF of yellow cattle. And for the first time, investigated the possibility of external fertilization using sperm from epididymis of bufflo, in order to establish an optimized system for buffalo IVF, and thus to provide the theoretic basis and practical references for speeding up the improvement of buffalo in china.
The study indicated that, with 3ug/ml FSH external maturation medium, as LH increased, FBI exclusion and maturation rate of the buffalo oocyte increase as well. When the concentration of LH reaching 30ug/ml, FBI exclusion (26.67% vs 2.86%, 7.50%) and maturation ration (40.00% vs 14.29%, 17.50%) elevated significantly (p<0.05). In the medium with 30ug/ml LH, FSH rose to 30ug/ml, the FBI exclusion (24.24%) and maturation ratio (45.45%) did not increase further. When 10% BFF was added to the maturation medium, which contained gonadotropins, the FBI exclusion (52.33%) and maturation ration (66.28%) were increased further significantly (p<0.05). With the supplement OCS to medium, the FBI exclusion and maturation ratio were 48.19% and 59.04%, respectively, and 37.39% and 52.87% respectively in FCS medium. The former was superior to the latter, but no significant difference was found statistically significantly. Granulosa-cell monolayer co-culture system has improved the FBI exclusion (49.21%) and maturation ratio (65.08%), but there was no apparent difference. The 60.61% FBI exclusion and 72.73% maturation ratio had been obtained with the above cultured conditions.
We had adopted BO and TALP medium with heparin as sperm in vitro capacitation and
fertilization medium. The differences of the sperm capacitation and fertility between in BO and in TALP were investigated. The results indicate: the fertilized egg cleavage rates were 51.85% and 55.04% respectively; The development rates were 27.19% and 26.36% comparative to the number of cultured oocytes and 48.21% and 47.89% comparative to the number of the cleavage oocyte in BO and TALP respectively. The result showed no difference and the two media could be applied in buffalo IVF.
Furthermore, the feasibility to apply epididymal spermatozoa in buffalo IVF was first studied. The results demonstrate that the fertilization rate was 60.71%; egg cleavage ratio was 50.39%; development ratio was 24.41% comparative to the number of culture oocyte and 48.44% comparative to the number of cleavage oocyte. No significant differences between the epididymal spermatozoa and the thawed spermatozoa (64.00%, 54.31%, 26.72%, 49.21%) from Guangxi were found. The egg cleavage ratio and development ratio of the epididymal spermatozoa were 46.67%, 53.37% and 21.67%, 26.87% respectively. The effect of fertilization showed no apparent difference in the two media.
The spermatozoa stain and morphology indicated that the epididymal caudal sprmatozoa mobility is superior to thawed spermatozoa but have high percentage of protoplasm drop. The epididymal caudal sprmatozoa mobility was relevant to the collecting methods. The best method in the study was to conserve the epididymis in the conditions of constant temperature and anhydrous container. The results indicate the buffalo epididymal caudal sprmatozoa could be applied in buffalo IVF.
引文
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71．江金益，等．1991 猪卵母细胞体外成熟与冷冻附睾精子的体外受精．动物学报．，37(3)：313～316
72．李键．1996．IGF-I对山羊小腔卵泡体外生长发育的影响．博士学位论文
73．李青，阳年生，卢晟盛．1999．成熟培养时间对水牛卵母细胞体外成熟的影响．广西农业生物科学，18(2)
74．李荣凤，等．2000．牛卵母细胞皮质颗粒荧光染色法质成熟鉴定．畜牧兽医学报 31：(3)203-210
75．李英，侯健，郑云胜等 肝素和羊血清对绵羊附睾精于体外获能的影响 内蒙古农牧学院学报 1998，19(4)：20～24
76．刘东军等 1992．不同体外培养系统对牛体外受精胚胎冷冻解冻的孵化能力的影响．内蒙古大学学报(自科版)
77．刘建民，刘健，朱裕鼎，等．1989．牛卵泡卵体外成熟及其受精能力的研究．兽医大学学报，9(4)：356-362
78．卢晟盛，等．1994．不同血清种类及其深度对牛卵母细胞体外受精的影响．广西农业大学学报，
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80．邵华．2000．山羊卵泡卵母细胞核成熟抑制与恢复的研究．硕士学位论文
81．石德顺，等．1996．肝素及血小板激活因子对牛卵母细胞体外受精的影响．广西科学，3(3)：36-40
82．石德顺，卢克焕，等．1994．体细胞因子对牛卵母细胞体外受精的影响．广西农业大学学报，13(1)：37-41
83．石德顺，卢克焕．1994．牛卵泡液对牛卵母细胞体外成熟的影响．广西农业大学学报，13(1)：1-5
84．石德顺，卢克焕．1996．第二信使因子对牛卵母细胞体外成熟的影响．中国兽医学报，16(1)：80-84
85．孙青原，等．1995．性成熟前牛卵母细胞体外受精研究，中畜牧杂志，31(2)21-22
86．谭景和等1989体外培养前黄牛卵母细胞的分类、形态结构及成熟能力研究。中国农业科学 22：83-88
87．谭世俭，阳年生，石德顺，等．1998．现行牛IVF程序在水牛体外受精的应用研究．广西农业大学学报，17(4)312-317
88．王锋．1994．颗粒细胞生长发育及其在卵泡发生中的作用．国外兽医学—畜禽疾病，15(4)：8-12
89．王建辰，等，1998 动物生殖调控 安徽科技出版社
90．王祖昆，张益辉1994．水牛胚胎移植技术进展及其困扰问题探讨(上)．广东畜牧兽医科技，19(3)26-29
91．王祖昆，张益辉1994．水牛胚胎移植技术进展及其困扰问题探讨(下)．广东畜牧兽医科技，19(4)19-20
92．向(?)．1990．家畜生理学原理．北京：农业出版社出版．495-496
93．旭日干，张锁链，薛晓先．1989．屠宰母牛卵巢卵母细胞体外受精与发育的研究，畜牧兽医学报， 20(3)：193-198
94．杨膺白，等．1994．卵丘细胞对牛卵母细胞体外成热和体外受精的影响．广西农业大学学报， 13(1)：16-20
95．袁玉英等1998卵丘细胞及胞外基质诱发仓鼠精子顶体反应。解剖学报 29(4)：404-409
96．曾申明，朱士恩等1997．用屠宰场绵羊卵巢和附睾进行体外胚胎生产的可行性研究．中国养羊． 3：24-25
97．张锁链，刘东军．1996．通过体外受精技术缩短绒山羊育种周期的研究．内蒙古大学学报(自科版)
98．张涌，刘泽隆，李裕强．1999．山羊卵泡卵母细胞体外成熟的研究．西北农业大学学报，27(1)：14-18
99．张忠诚，2000。家畜繁殖学，农业出版社
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