A SIMPLE STAINING METHOD FOR EVALUATING ACROSOMAL STATUS OF CAT SPERMATOZOA

Abstract

The purpose of this study was to develop a simple staining procedure for determining the acrosomal status of felid spermatozoa. Previously, only a modified triple-stain technique suc cessfully differentiated the acrosomal reaction; however the complexity of the technique limited its usefulness. The single-step staining technique uses a solution of 1% fast green FCF, 1% rose bengal, and 40% ethyl alcohol in 0.1 M citric acid-0.2 M disodium phosphate buffer. Semen was diluted with 2.9% sodium citrate, and an equal volume of staining solution was added and incubated for 70 sec at room temperature. Ten microliters of the mixture then was pipetted onto a microslide, smeared with anther slide, and air dried at 37?C. Acrosomal status was evaluated using bright field microscopy ( x 1,000). An intact acrosome was indicated by the purplish-blue staining over the anterior portion of the sperm head. If acrosomal loss had occurred, the anterior caput was almost colorless to light pink. The postacrosomal region of the head was pale pink in spermatozoa with and without intact acrosomes. There was no difference in percentage of intact acrosomes in a comparison of three media used for semen dilution (Experiment la) or in a comparison of fresh versus premixed (stored) staining solution (Experiment lb). The percentage of intact acrosomes as determined by the single stain was higher (P < 0.001) than that determined by the modified triple stain in all four cat species studied (Experiment II). This technique can be used to evaluate the daily rate of acrosome loss in domestic cat semen during storage at 4?C (Experiment III). The single-step acrosome staining technique as described is useful for quantifying acrosome loss in felid spermatozoa