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Estimation of membrane integrity in spermatozoa using flow cytometry is often hampered by non-specific staining and the high autofluorescence of spermatozoa. The influence of the choice of immersion media on the estimation of sperm membrane integrity was investigated. Membrane integrity of spermatozoa from buffalo bulls was estimated using a lactate dehydrogenase (LDH) based assay and a fluorescent probe-based assay. Immunofluorescence staining using SDF (Synthetic Diethyl Fluorophosphate), a fluorescent dye used to assess sperm membrane integrity was also carried out. Immersion media, namely PBS (Phosphate buffered saline), glycerol and egg yolk-Tris (EYT) for the flow cytometry procedure and PBS, PBS-Tween and EYT for the immunofluorescence staining procedure were tested. Immersion media had a significant influence on the flow cytometric assay and the sperm membrane integrity estimates. The membrane integrity estimates using the LDH-based assay were unaffected. Immersion media can cause a significant effect on the estimation of membrane integrity using flow cytometry. The effect of immersion media was found to be higher in flow cytometric than in immunofluorescence based estimation of membrane integrity.Spread the love
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