Flow Cytometric Analysis for the Evaluation of the Rat Sperm Viability and Number in the Male Reproductive Toxicity Studies

Abstract We investigated the application of flow cytometric analysis to evaluate the rat sperm viability and number in the male reproductive toxicity studies. Flow cytometric procedure has been developed to evaluate sperm number and viability that uses fluorescent dye (propidium iodide, PI) to distinguish between viable (negative staining) and dead (positive staining) sperm. Sperm samples were collected from the caudal epididymides of SD rats (13–21 weeks old). PI staining patterns/viabilities were compared among several ranges of sperm concentrations, and several kinds of sperm viability. Viabilities determined by flow cytometry (FC) were also compared with motility by direct microscopical observation in several kinds of sperm viability. No notable changes in the PI staining patterns/viabilities were observed in the range from 1 × 10⁵ to 6 × 10⁶ sperm/ml. Essentially similar results were obtained from both FC and microscopical analyses for three degrees of viability sperm: live sperm (general preparation as a control), weakly viable sperm (mixed by vortex mixer for 30 seconds), and dead sperm (treated with 90°C or Triton X-100).
Viabilities of normal rat samples were 95.0 ± 4.0% in FC and 93.7 ± 4.6% in microscopical observation, indicating good correlation in both analyses. Sperm numbers with FC analysis were approximately 0.8 × 10⁶ to 1.8 × 10⁶ sperm per mg indicating good correlation with those by microscopy.
It was concluded that the present flow cytometric procedure was objective, rapid and reliable, and that it was one of the useful methods for measuring the number and evaluating the viability of sperm collected from the caudal epididymides of rats in the male reproductive toxicity studies.