USE OF A SIMPLE STAINING TECHNIQUE TO DISTINGUISH ACROSOMAL CHANGES IN THE LIVE SPERM SUBPOPULATION

In order to determine acrosomal status in live spermatozoa, the two common staining techniques, nigrosin-eosin (NE) stain and the Giemsa stain, were combined. There was no significant difference between proportions of live boar spermatozoa determined by the NE technique and the nigrosin-eosin-Giemsa (NEG) technique. The proportion of live spermatozoa determined by the NEG technique was also compared with the results of fluorescent viability stains, carboxyfluorescein diacetate and propidium iodide. The difference, although significant (P<0.01) was small, 87% (NEG) vs. 80% (CFDA/PI). With ram semen, samples were compared for proportions of live cells and the NEG technique gave six percentage points lower than the NE technique (P<0.01). Acrosomal alterations, determined by differential interference contrast microscopy and by the NEG technique did not differ significantly for boar or ram spermatozoa. This novel staining technique is capable of determining four categories of spermatozoa: live acrosome-intact, live acrosome-reacted or damaged, dead acrosome-intact and dead acrosome-reacted or damaged. The method is simple to perform, requires only basic equipment and appears extremely reliable.