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Objective:To display the effect of different concentrations of glycerol, as a cryopreservative, on the quality of the frozen-thawed buffalo semen extended in TRIS extender enriched with disaccharides (trehalose or sucrose).Methods:Semen samples were extended in Tris-Citric acid-Fructose-Egg yolk without addition of trehalose / sucrose and with 6.4 % glycerol as a control (TFEG-C) and with the addition of Trehalose / Sucrose and different concentrations of glycerol to ensure 60 million motile spermatozoa mL-1. Semen cooled slowly up to 5 and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws. The straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen (LN2) for 10 min then dipped in liquid LN2. Frozen straws were thawed at 37曟for 1 min. The parameters studied were sperm motility, sperm viability, sperm abnormality, sperm membrane integrity (HOST), percent of normal intact acrosome and DNA fragmentation.Results:The best sperm motility, sperm liveability, sperm abnormality, sperm cell membrane and DNA integrities appeared with TFES-G 5.5%(41.00% ± 2.08%, 70.40% ± 2.27%, 7.80% ± 1.19%, 68.10% ± 1.55%, 98.90% ± 0.50%, respectively ) and TFES-G 7.3% (41.50% ± 1.98%, 70.70% ± 2.03%, 10.80% ± 0.88%, 69.30% ± 1.85% and 96.40% ± 0.88%, respectively).Conclusion: From the present study, it can be concluded that addition of glycerol (5.5% or 7.3%) to Tris-Fructose-Egg yolk-Sucrose extender might help in improvement of the post-thawed characteristics of buffalo frozen semen.