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Abstract
A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.