Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS®

When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2 h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 °C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20 s at 37 °C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris' Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS®. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS®, and discriminates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals.