Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function

Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.