EFFECT OF DIFFERENT GLYCEROL CONCENTRATIONS ON MOTILITY BEFORE AND AFTER FREEZING, RECOVERY RATE, LONGEVITY AND PLASMA MEMBRANE INTEGRITY OF NILI-RAVI BUFFALO BULL SPERMATOZOA

A.Abbas and S.M.H. Andrabi

Abstract   

    The present study has designed to observe the effects of different concentrations of glycerol on sperm motility before after freezing and thawing, recovery rate, longevity and plasma membrane integrity in Nili- Ravi buffalo bull spermatozoa. Semen was collected and evaluated from four Nili-Ravi buffalo bulls in a standardized procedure. The ejaculates possessing more than 65% visual motility were selected and pooled at 37°C. Pooled samples were divided into eight aliquots of 0.5 ml each and extended (50 x 106 sperm/mi) in Tris-citric acid extenders (pH=7.0. OP=320 mOsmol/kg) at 37°C with different (2. 4. 5, 6. 7. 8. 10 or 12%) concentrations of glycerol. Semen was cooled to 4ºC in 2 hours, equilibrated for 4 hours, packed in 0.5 ml French straws and frozen from +4°C to -15°C at the rate of 3°C per minute and from -15°C to -80°C @ 10°C/ minute in a programmable cell freezer. Semen straws were thawed at 37° (for 30 seconds after 24 hours of storage in liquid nitrogen at -196°C.
The motility before freezing did not differ due to the treatments. The motility after freezing did not differ among the extenders containing glycerol (%) concentrations either 5, 6, or 7. This was higher than that in extender containing glycerol (%) concentrations either 4, 8 or 10. Post thaw motility in extenders having glycerol (%) concentrations either 2 or 12 compared to all other treatments was the lowest. The recovery rate of spermatozoa (%) after freezing and thawing did not differ among the extenders containing glycerol (%) concentrations either 4.5.6. 7 or 8. This was higher (P<0.05) than that in extenders containing glycerol (%) concentrations either 2 or 10 and was lowest (P<0.05) in extender containing 12% glycerol. The mean sperm longevity (37 ºC) at 2 hours was highest (32.6 ± 5.2, P<0.05) for 6. 7 or 8% glycerol, intermediate (13.9 ± 4.4, P<0.05) for 4.5 or 10% and lowest (3.3 ± 2.5 P<0.05) For 2 or 12%. Plasma membrane integrity (%) of spermatozoa frozen in 7% glycerol (42.0 ± 1.3) was superior (P<0.05) to 5.6. 8. 10 or 12% glycerol (29.6 ± 2.9). Plasma membrane integrity of Nili-Ravi buffalo bull spermatozoa was least protected (P<0.05) when frozen in 2 or 4% glycerol (10.4 ± 1.8). It is concluded that glycerol (%) concentrations of 6 or 7 in the extender may be suitable for crypreservation of Nili-Ravi buffalo bull spermatozoa.