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Abstract In Egypt, buffaloes are considered the main dairy animals and have a vital economic interest, however, this species is mostly out of the attention and still until now obscure. Current econormc demand for buffalo milk and meat has dictated breeding strategies which necessitate the extension of modern biotech- neology of embryo transfer and in vitro fertilization to this species. The in vitro fertilization of buffalo oocytes represents one of the most important applicable techniques that help in the improvement of the buffalo’s genome. However, the in vitro oocyte maturation,fertilization and culture up to blastocyst stage are sub optimal and need further substantial improvement. The present work aimed to develop a reliable in vitro technique for buffalo oocyte maturation, fertilization and culture, that could regularly provide a large number of normally fertilized oocytes for further biotechnological application. The oocytes were harvested from the buffalo ovaries post slaughter of the buffaloes from all visible surface immature follicles ranged from 2-8mm in diameter. The oocytes were washed three times with TCM-199 medium supplemented with sodium pyruvate and gentamycin sulfate. Only oocytes that surrounded by compact multi layers of cumulus cells, with evenly granular ooplasm was selected for in vitro maturation. The good quality oocytes were matured in TCM-199 that supplemented with 50J.lg/ml sodium pyruvate, 1 % (v/v) antibiotic antimycotic solution and 10% FCS, 5J.lg/ml, FSH, 10j.lg/ml LH and 1 j.lg/ml estradiol -17B. The rnedium was sterilized by filtration through 0.22 Millipore filter membrane. The selected oocytes were covered by sterile paraffin oil and |