Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa

In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili- Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 4003g for 10 min at 378C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (378C for 30 s) semen samples using a phase-contrast microscope (3400). Plasma membrane integrity (%) was determined by mixing 50 ml each of fresh and frozen-thawed semen with 500 ml of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose1sodium citrate) and incubating at 378C for 1 h. Viability (%) of fresh and frozen-thawed spermatozoa before and after challenging them to osmotic pressure (hypotonic treatments) was assessed using supravital stain under a phase-contrast microscope (3400). In the first experiment, the mean6s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.861.17 and 256.061.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P,0.05) in frozen-thawed semen samples as compared with fresh semen (60.161.34 v. 8161.57, respectively). The plasma membrane integrity (%) and magnitude of osmotic stress in fresh and frozen-thawed semen samples was higher (P,0.05) at 50, 100, 150 and 190 mOsm/l as compared with 250 mOsm/l. Loss of viability (%) in fresh and frozen-thawed semen samples was higher (P,0.05) at 50 mOsm/l (59% in fresh, 70% frozen thawed) as compared with other osmotic pressures, while it was lowest at 250 mOsm/l (4.1% for fresh, 9.7% frozen thawed). In conclusion, osmotic pressure of Nili-Ravi buffalo semen and seminal plasma is determined. Furthermore, variation in osmotic pressure below 250 mOsm/l is not favorable to fresh and frozen-thawed buffalo spermatozoa. Keywords: buffalo semen, fresh and frozen-thawed semen, osmotic pressure, plasma membrane