| 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 andfertilization 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.
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