Sperm quality is evaluated for the calculation of sperm dosage in artificial reproductive programs. The most common parameter used is motility, but morphology has a higher potential as a predictor of genetic quality. Morphometry calculations from CASA-Morph technology improve morphological evaluation and allow mathematical approaches to the problem. Semen from 28 Holstein bulls was collected by artificial vagina, and several ejaculates were studied. After general evaluation, samples were diluted, packaged in 0.25 ml straws, and stored in liquid nitrogen. Two straws per sample were thawed, and slides were processed and stained with Diff-Quik. Samples were analyzed by a CASA-Morph system for eight morphometric parameters. In addition to the “classical” statistical approach, based on variance analysis (revealing differences between animals, ejaculates, and straws), principal component (PC) analysis showed that the variables were grouped into PC1, related to size, and PC2 to shape. Subpopulation structure analysis showed four groups, namely, big, small, short, and narrow from their dominant characteristics, representing 31.0%, 27.3%, 24.1%, and 17.7% of the total population, respectively. The distributions varied between animals and ejaculates, but between straws, there were no differences in only four animals. This modern approach of considering an ejaculate sperm population as divided into subpopulations reflecting quantifiable parameters generated by CASA-Morph systems technology opens a new view on sperm function. This is the first study applying this approach to evaluate different ejaculates and straws from the same individual. More work must be done to improve seminal dose calculations in assisted reproductive programs.
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.
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