The functional integrity of spermatozoa in various domestic animals like horse, cattle and swine is evaluated by hypo osmotic swelling test. The hypo osmotic swelling test has not been tested in the fresh semen of Nili-Ravi buffalo bull. The aim of the present study was evaluation of membrane integrity by establishing the best hypo osmotic swelling solution. Ten solutions with different osmolarities were used: 70(HS1), 90(HS2), 100(HS3), 120(HS4), 140(HS5), 150(HS6), 190(HS7), 230(HS8), 280(HS9) and 300 (HS10). Twenty samples were collected (ten from each bull) with forty eight hours interval. The collected semen was placed in water bath at 37°C and was evaluated macroscopically and microscopically for color, volume, mass activity and motility. After evaluation 0.1 ml of semen was mixed with 1ml of hypo osmotic swelling solutions and incubated for one hour at 37°C. A total of two hundred sperms were counted in five fields and sperms were classified as swollen and strongly swollen. The percentages of coiled spermatozoa in ten hypo osmotic swelling solutions mentioned above were 18.3%, 23.5%, 37.3%, 41.4%, 50.2%, 57.5%, 38.6%, 26.0%, 13.9% and 6.2% respectively. The percentages of strongly swelling solutions were 9.2%, 12.0%, 21.6%, 32.8%, 31.0%, 39.1%, 29.3%, 21.7%, 10.0% and 5.7% for ten solutions respectively. HS6 was greater to HS1, HS2, HS7, HS8, HS9 and HS10 in increasing the ability of swelling (P<0.05). Similarly HS6 showed greater ability of swelling as compared to HS1, HS2, HS8, HS9 and HS10 (P<0.05).These results indicated that the solution having 150 mOsm/L was the best for functional membrane integrity of spermatozoa in fresh semen of Nili-Ravi buffalo bull semen.
