Standardization of sampling and staining methods for the morphometric evaluation of sperm heads in the Cynomolgus monkey (Macaca fascicularis) using computer-assisted image analysis

Automated sperm morphology analysis (ASMA) technology has improved the assessment of sperm morphology, but the results depend on the use of adequate and standardized procedures. In this study the Sperm-Class Analyzer(R) (SCA) ASMA system was used to assess sperm head morphometry in the Cynomolgus monkey and to evaluate the influence of sample size, intraslide variation, and the use of three staining techniques on the accuracy of image processing and sperm head morphometry. Haematoxylin is the staining technique of choice for Cynomolgus spermatozoa, as optimum contrast of sperm heads with the surrounding background allows efficient segmentation, i.e. sperm head boundary detection, making the image analysis process more accurate. The analysis of 100 spermatozoa is recommended since a larger sample size did not result in more accurate sperm head morphometry. There were no differences in either the percentage of correctly binarized sperm heads or sperm head dimensions among samples obtained from different zones of the slides, although differences in stain intensity (grey level) were detected. The measurements made on Haematoxylin, Diff-Quik and Hemacolor-stained slides yielded different values for all of the sperm head parameters under consideration. This result demonstrates that the procedures of fixation and staining significantly affect the dimensions of sperm heads.