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Refining Methods for Using Opal Blue Stain in Evaluating Ram Semen¹

United States Department of Agriculture^,3

Abstract

For ram's semen the optimum stain mixture for opal blue Breslau and eosin was found to be: 12.5 percent opal blue and 0.5 percent eosin in pH 7.15 phosphate buffer. It was necessary to stain for 30 seconds before smearing. The dry smears were most suitable for observation. Balsam or oil decreases the color contrast. No suitable method was found to incorporate opal blue and eosin into a diluting fluid for use in the haemocytometer.

Under normal conditions of examination motile sperm took no stain, and unstained sperm were therefore regarded as the best estimate of the live sperm.

Prediction of percent live normal spermatozoa (on smear) from a combination (EMC) of the motility score and estimated percent motile had significant reliability.

The opal blue staining method was found to be simpler and more rapid than previous methods, allowing immediate examination for abnormalities of the sperm during routine examination of semen. The slides and the stain solution exhibited no noticeable depreciation after four years.

¹ This work was done in 1942 just previous to the entries of the authors into the armed services.

² The assistance and advice of Dr. C. E. Terrill in the problem are gratefully acknowledged.

³ Western Sheep Breeding Laboratory, Dubois, Idaho.