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

Abstract

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.

The effect of selected staining techniques on bull sperm morphometry

“…Within one ejaculate there are sperm with different shapes, mainly of the head, and of different sizes and forms. This has led researchers to attempt to identify and define normal sperm traits (Gago et al, 1998;Gage, 1998). …”

“…One solution in evaluation of sperm morphology is computer-assisted sperm analysis (CASA), which reduces the subjectivism of morphological analysis (Gago et al, 1998), but these systems are relatively expensive and also not free of errors resulting from automation of the analysis process. The concept of morphology is closely linked to sperm cell dimensions falling within the norms for a given species.…”

Computer assisted sperm morphometry in mammals: A review

“…Regarding the sperm head shape (morphology), the species may be classified into homomorphous and heteromorphous (Soler et al, 2014c). Homomorphous species, such as the ovine (Vicente-Fiel et al, 2013a,b), goat (Vicente-Fiel et al, 2013a,b), Cynomolgus monkey (Gago et al, 1998), mouse ), rabbit (Perez-Sanchez et al, 1998, or red deer (Soler et al, 2005a) have high homogeneous sperm morphology between and within individuals. Heteromorphous species, such as human (Moruzzi et al, 1988;Bellastella et al, 2010), Goeldi's monkey (Valle et al, 2013), dog (Dahlbom et al, 1997) or camelids (alpaca: (Buendia et al, 2002); llama: (Soler et al, 2014c) exhibit a high degree of sperm polymorphism both in the same ejaculate and in different individuals.…”

“…In consequence, the results of the different studies cannot be directly compared and there is a high dispersion in the morphometric traits of the sperm provided by different works for a given species. Gago et al (1998) Values are expressed as mean ± SD (when available). *H.…”

Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): Optimum sampling procedure and staining methods using Sperm-Class Analyzer®

“…The ASMA systems have been used to study the differences among males (Aziz et al, 1998;Dahlbom et al, 1997;Hirai et al, 2001;Sailer et al, 1996) and between species (Cummins Q3 and Woodall, 1985) and the relationship with fertility when samples are used (Gravance et al, 2009;Marco-Jiménez et al, 2010;Maree et al, 2010) as well as the effect of cryopreservation on sperm morphometry (Álvarez et al, 2008;Arruda et al, 2002;Esteso et al, 2003Esteso et al, , 2006aEsteso et al, ,b, 2009Gravance et al, 1998;Hidalgo et al, 2007;Thompson et al, 1994;Thurston et al, 2001). For greater accuracy of these systems, the standardization for each species of a number of analytical variables such as staining and sampling methods is required Foote, 2003;Gago et al, 1998;Gravance et al, 1995;Hidalgo et al, 2005Hidalgo et al, , 2006. The aim of the present study was to evaluate the influence of three different staining procedures and optimal number of sperm cells analyzed per sample to obtain accurate and repeatable sperm morphometric measurements in the Iberian ibex using SCA ® .…”