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An Eosin-Nigrosin Stain for Differentiating Live and Dead Bovine Spermatozoa

University of Tennessee

Abstract

The application of a staining mixture containing eosin and nigrosin for the differential staining of live and dead bovine spermatozoa has been investigated. This staining mixture gave results comparable to those with an eosin-fast green mixture when it was mixed in isotonic sodium citrate solution. Mixing in distilled water resulted in abnormally high percentages of dead sperm. Variations in pH from 6.4 to 8.5 and varying the proportion of semen to stain from 1:1 to 1:20 did not affect the percentage of dead sperm. The concentration of dyes found most suitable was 1 percent eosin B and 5 percent nigrosin, although variations in these amounts from 50 to 200 percent did not alter the accuracy of the live-dead differential count. The nigrosin background stain mixes satisfactorily with egg yolk diluter, but fewer stained sperm are found from diluted than from pure semen. The nigrosin stain gives a very homogenous background and highlights clearly the morphological characteristics of the sperm.