STAINING SPERM FOR VIABILITY ASSESSMENT

Abstract

SummaryAssessing the functional capacity of sperm in vitro is of particular interest to the artificial insemination industry in order to select sires with the best fertilizing capacity. Early efforts to evaluate semen dating into the past century were based on microscopical observation of motility and abnormal morphology. Frequently, eosin stain was used to quantify live and dead sperm while giemsa stain was used for morphological evaluation. These methods along with visual inspection of the semen were useful but their repeatability was questionable. The advent of fluorescent staining improved microscopical evaluation of sperm, however, one of the major drawbacks remains, that is that one can only evaluate 100 or 200 sperm per sample. The advent of flow cytometry in the early 1970's and the development of new stains has led to more accurate means of assessing sperm viability. Fluorescein diacetate (FDA), carboxyl‐fluorescein diacetate (CFDA) in combination with propidium iodide (PI) were applied to flow cytometric assessment in the early 1980's. With flow cytometry several thousands of sperm can be analyzed quickly and without detriment. Staining sperm with FDA, CFDA and PI is useful but is time dependent leading to deterioration of the sample which can alter results. Hoechst stain, 33342 and 33258 were classed as vital stains in 1979. Hoechst 33342, a highly permeable fluorescent stain became prominent for use to differentiate X from Y sperm based on binding in a stoichiometric manner to DNA. Hoechst 33258, a useful exclusion dye, will only stain sperm with lysed membranes. More recently a new membrane permeant dye, SYBR‐14 has been used in combination with PI and is characterized by entering only those cells possessing a membrane potential. It shows a very high correlation with living sperm and a highly negative correlation with PI stained dead sperm. Initial studies show that the SYBR‐14/PI combination is time insensitive, which allows one to evaluate semen outside of a strict time regimen. The use of CTC and FITC‐PSA to ascertain the condition of the acrosome, using both the fluorescent microscopy and flow cytometry has proven to be a very useful tool. Finding one single test to evaluate the fertilizing potential of sperm continues to be elusive. However, using several tests, in combination, such as motility, acrosomal integrity, and a fluorescent assessment of membrane integrity would add significant credibility to estimating sperm function.