USE OF A TRIPLE-STAIN TECHNIQUE TO DETECT VIABILITY AND ACROSOME REACTION IN DEER SPERMATOZOA

A procedure is described for simultaneously evaluating of sperm viabil ity and acrosomal status in fixed deer spermatozoa. This technique, wh ich is a modification of the triple-stain technique (TST) developed fo r human sperm, utilizes trypan blue to identify the percentage of live sperm in culture and subsequent staining of fixed sperm to differenti ate the acrosome. Four classes of deer sperm can be distinguished with the TST: (1) live unreacted sperm, (2) live acrosome-reacted sperm (t rue acrosome reaction), (3) dead unreacted sperm, and (4) dead acrosom e-reacted sperm (false acrosome reaction). The study shows that the ac rosome status can be detected either by TST stain or by phase-contrast microscopy (r = .971, p < .001) and that TST does not differ from 0.4 % trypan blue in its ability to identify live and dead sperm (r = .995 ,: p < .001). This staining procedure enables differentiation of sperm atozoa that have undergone a true acrosome reaction (TAR) from those t hat have undergone a false acrosome reaction (FAR). In this way, incub ation of deer spermatozoa for 30 min at 37 degrees C in the presence o f calcium ionophore A23187 increased the proportion of spermatozoa und ergoing a TAR. Sperm incubated in the absence of A23187 undergo the TA R at a much lower rate (p < .001) than those incubated in the presence of ionophore. This stain procedure was also used to study the time co urse of the true acrosome reaction of deer sperm in vitro. Incubation of deer spermatozoa for up 24 h at 37 degrees C resulted in a decrease in the percentage of live acrosome-intact spermatozoa and a simultane ous increase in the percentage of spermatozoa categorized as having un dergone a TAR and FAR.