Effect of lactose, skim milk and Tris diluents on frozen buffalo spermatozoa

Abstract

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. This study was carried out to identify the suitable buffer for cryopreservation of buffalo semen. Sixteen split pooled ejaculates from two buffalo bulls possessing more than 70% visual sperm motility, were diluted at 370c either in lactose, skim milk or Tris extenders. The diluted semen was cooled to 40c within 2 hours, equilibrated at 40c for 4-6 hours following the addition of glycerol, filled in 0.5 ml French straws and frozen in a programmable cell freezer before plunging into liquid nitrogen. Semen was thawed at 370c for 30 seconds after 48 hours of storage inside liquid nitrogen. Post thaw visual sperm motility, plasma membrane integrity and acrosome morphology of each semen sample were assessed by warm plate microscopy at 370c, hypo-osmotic swelling test (HOST) and giemsa staining, respectively. Analysis of variance revelated that percentage of post thaw visual sperm motility (Mean± standard deviation) tended to be higher in Tris (50±3.6) than skim milk (44.5±2.5) and lower in lactose (24.4±10.5) extenders (P