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 400 X g for 10min at 37 degrees C and osmotic pressure was determined using an osmometer In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37 degrees C for 30s) semen samples using a phase-contrast microscope (X400). Plasma membrane integrity (916) was determined by mixing 50 mu l each of fresh and frozen-thawed semen with 500 mu l of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37 degrees C for 1 h. Viability (916) 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 (X400). In the first experiment, the mean +/- s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.8 +/- 1.17 and 256.0 +/- 1.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P < 0.05) in frozen-thawed semen samples as compared with fresh semen (60.1 +/- 1.34 v. 81 +/- 1.57, respectively). The plasma membrane integrity No) 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.