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.

Assessment of quantitative dual-parameter flow cytometric analysis for the evaluation of testicular toxicity using cyclophosphamide- and ethinylestradiol-treated rats.

“…The absolute count of cells was measured using the procedure described by Takizawa et al (1995) based on Toppari et al (1986). A constant volume of Immuno-check (standard fluorescent beads solution, EPICS, Hialeah, Florida) was added to each sample as an internal volume standard.…”

Evaluation of Mitochondrial Function and Membrane Integrity by Dual Fluorescent Staining for Assessment of Sperm Status in Rats

“…Spermatozoa could be classified into three different populations: one stained with CAM alone, another stained with EthD-1 alone, and still another stained with both CAM and EthD-1. Both eosin and propidium iodide, widely used for viability assay of mammalian spermatozoa, are indicators for membrane integrity (Dougherty et al, 1975;Garner and Johnson, 1995;Takizawa et al, 1995;Yamamoto et al, 1998), and their mechanism of staining dead sperm is similar to that of EthD-1 (MacCoubrey et al, 1990;Moore et al, 1990;Kaneshiro et al, 1993). When spermatozoa are dead, membrane integrity is lost and affinity for dyes becomes greater (Blom, 1950;Noda, 1992), and energy metabolism, which is determined by mitochondrial function, is also lost.…”

“…On the other hand, red fluorescence produced by EthD-1 is also shown in the nucleus (MacCoubrey et al, 1990;Moore et al, 1990;Kaneshiro et al, 1993). The mechanism of producing fluorescence of EthD-1 indicated by the membranebreached cell and conjugate with DNA is similar to that of other dyes currently used, such as eosin and pro-pidium iodide (Dougherty et al, 1975;Garner and Johnson, 1995;Takizawa et al, 1995;Yamamoto et al, 1998).…”

“…Some DNA dyes, such as eosin or propidium iodide, have been widely used for viability assay on spermatozoa in humans (Dougherty et al, 1975;Kramer et al, 1993), in bulls, boars, rams, rabbits, and mice (Garner and Johnson, 1995), and in rats (Takizawa et al, 1995;Yamamoto et al, 1998;Vetter et al, 1998). However, in rat spermatozoa, it is difficult to discriminate between viable and dead spermatozoa by eosin, because, as John (1970) reported, both living and dead spermatozoa display a strong affinity for eosin.…”