Effect of sample size and staining methods on stallion sperm morphometry by the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis has improved the assessment of sperm morphology, but the results depend on the use of adequate evaluation and staining procedures of spermatozoa from individual species. In this study, the morphological module of the Sperm Class Analyzer® was used for the morphometric analysis of stallion sperm heads and midpieces. Semen samples were obtained from six fertile stallions in order to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris’ Haematoxylin) on the accuracy of image processing and sperm morphometry, and the effect of the sample size on sperm morphometric measurements. Harris’ Haematoxylin was the staining technique of choice on the accuracy of the image processing with an optimum contrast of sperm cells with the surrounding background that allows an efficient boundary detection and segmentation which results in the highest proportion of sperm heads and midpieces assessed (80.47%). The results indicate that the staining methods affected significantly the sperm dimensions with increased values from Diff-Quik than Hemacolor and Harris’ Haematoxylin respectively (Diff-Quik > Hemacolor > Harris’ Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. In conclusion, to obtain objective and accurate sperm morphometric measurements by the Sperm Class Analyzer®system in the stallion, it’s recommended the analysis of 100 spermatozoa from slides which have been previously stained with Harris’ Haematoxylin.