Use of Fluorescent Dyes for Observing Bovine Spermatozoa in Opaque Media

A method for staining sperm with fluorescent dyes and examination with ultraviolet light is presented.By using this method with semen diluted with opaque media, the spermatozoa were made clearly visible with no interference from fat globules or other extraneous material£ and evaluation of sperm motility was readily made.These dyes in concentrations of 1:10,000 to 1:40,000 were nontoxic to the spermatozoa during the 15-rain.incubation at 37 ° C. and caused only a slight reduction in motility after storage at 5 ° C. for 24 hr.The staining of sperm so that they fluoresce in ultravioIet light was found to make the sperm clearly visible for evaluation of motility in whole milk, yolk-phosphate, or other diluents in which white light illmnination does not allow good discrimination between sperm and media.Other possible uses of this procedure also are discussed.The observation and evaluation of bovine spermatozoa in diluents such as whole milk and yolk-phosphate have always presented a problem, because of the opacity of the media due to the presence of large numbers of interfering fat globules (4, 7).In an attempt to make the sperm more visible in such media, a study was conducted on the practicality of the routine staining of sperm with dyes which would cause the spermatozoa to fluoresce in ultraviolet light.Fluorescent dyes for staining of spermatozoa have been utilized by a number of authors in the study of sperm morphology (1, 2, 3, 5).Bishop and Austin (2) reported that a spectacular polychromatic fluorescence, which clearly defined the aerosome and nucleus of the sperm, was obtained by staining fresh, living rodent spermatozoa with acridine orange, but less successful defi~fition of sperm structures was obtained with rabbit and bull spermatozoa.Bishop (I) was able to differentiate between live and dead spermatozoa by fluorescence microscopy after staining with prinmlin and rhodamin G. Stoekinger (5) obtained good fluorescence of human sperm after staining with acridine orange, but stated that this method could not be used for practical examilaation of sperm livability.On the other hand, Strugger and Rosenberger (6) claimed that the motility of goat sperm was undisturbed by staining with acridine orange.The latter also' report that 21 of 27 goats artificially inseminated with spermatozoa stained with fluorochromes became pregnant and gave birth to normal young.5~ore recently, workers in this laboratory have used the technique of staining with fluorescent dyes t~ make spermatozoa more visible when using visual methods (9), and to eliminate counting of fat particles and other extraneous material in milk diluents when using electronic methods of evaluating sperm motility (8).