Determination of an Optimal Membrane-permeable Cryoprotectant Addition and Dilution Protocol for Water Buffalo Spermatozoa

BACKGROUND: There is a possibility to reduce the toxicity of glycerol and osmotic stress of Me_2SO/DMSO by lowering their concentrations in freezing extenders. OBJECTIVE: To investigate the effect of glycerol and dimethyl sulfoxide (Me₂SO/DMSO) in tris-citric acid based extender on post- thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. MATERIALS AND METHODS: Semen was collected from five adult buffalo bulls with artificial vagina. Five aliquots of semen per bull were separated for dilution with the treatment extenders. The first aliquot was diluted at 37°C with 6% glycerol (T1). The second aliquot was diluted at 37°C with extenders containing 4.5% glycerol and 1.5% DMSO (T2). The third aliquot was diluted with extenders containing 4.5% glycerol at 37°C and 1.5% DMSO at 4 °C (T3). The fourth aliquot was diluted with extenders containing 1.5% DMSO at 37°C and 4.5% glycerol at 4°C (T4). The fifth aliquot was diluted with extender containing 2.5% DMSO at 37 as well as at 4°C (T5). The final concentration of spermatozoa was 50×10⁶/ml in all the treatment groups. Semen was cooled from 37 to 4°C in 2 h and equilibration was done at 4°C for 4 h. Later on, packing of cooled semen was undertaken in 0.54 ml French straws and frozen in a programmable cell freezer. RESULTS: At post thawing, treatment groups T1 and T2 yielded significant (P<0.05) outcome for CASA parameters, longevity, acrosomal integrity, plasma membrane integrity, mitochondrial transmembrane potential and DNA integrity. CONCLUSION: We concluded that by decreasing glycerol concentration (4.5%) and combining it with Me₂SO/DMSO (1.5%) at 37°C (T2) in tris-citric acid based extender provided similar results to those observed when glycerol (6%) alone is used at 37°C (T1) for improving the post-thaw quality of buffalo bull spermatozoa.