The Sperm-Class Analyser was validated for assessing morphometric parameters of the head and midpiece of unwashed and washed human ejaculated spermatozoa from volunteers providing a wide range of semen quality. A higher proportion of sperm could be assessed (86% fresh semen and 75% washed sperm) if Hemacolor staining was used rather than DiffQuik (80 and 73%) or Papanicolaou (78 and 68%). Different stains employed different fixatives and the area, length, width and perimeter of the sperm head was significantly larger for washed sperm stained by Hemacolor and DiffQuik. Acrosomal area ranged from 48 to 51% of the sperm head area and this percentage was larger for washed sperm stained with DiffQuik. Sperm at the end of the slide, distant from the initial semen droplet, were larger in area and perimeter than those at that site or in the middle. The high precision and reproducibility of the equipment required assessing only 50 sperm on the slide. Far greater variation was found in head width, relative acrosomal area and midpiece width between different slides prepared from the same ejaculate, highlighting the inherent variability within the ejaculate and smear preparation, and requiring more than one slide to be assessed.

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 (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.

Early in his investigations, Leeuwenhoek (1670s)1 deduced that spermatozoa were alive and an integral part of semen, rather than artifacts or parasites. He eventually observed spermatozoa in the semen of men, dogs, horses, birds, fishes, amphibians, molluscs, and many insects, and concluded that they must be a universal feature of male reproduction. The huge differences in sperm form among species have been discussed in relation to evolutionary changes dictated by the egg and its investments.2 Spallanzani (1800s)1 was the first scientist to develop successful methods for artificial insemination, first with amphibians and later with dogs. With these experiments, he showed that physical contact between intact spermatozoa and ova was necessary to achieve the fertilization. Some years later (1820s), Prévost and Dumas1 performed the defining experiment to identify correctly the function of spermatozoa in reproduction.