Regionalization of epididymal duct and epithelium in rats and mice by automatic computer-aided morphometric analysis.

AIM: To establish a rat and mouse epididymal map based on the use of the Epiquatre automatic software for histologic image analysis. METHODS: Epididymides from five adult rats and five adult mice were fixed in alcoholic Bouin's fixative and embedded in paraffin. Serial longitudinal sections through the medial aspect of the organ were cut at 10 microm and stained with hematoxylin and eosin. As determined from major connective tissue septa, nine subdivisions of the rat epididymis and seven for the mouse were determined, consisting of five sub-regions in the caput (rat and mouse), one (mouse) or three (rat) in the corpus and one in the cauda (rat and mouse). Using the Epiquatre software, several tubular, luminal and epithelial morphometric parameters were evaluated. RESULTS: Statistical comparison of the quantitative parameters revealed regional differences (2-5 in the rat, 3-6 in the mouse, dependent on parameters) with caput regions 1 and 2 being largely distinguishable from the similar remaining caput and corpus, which were in turn recognizable from the cauda regions in both species. CONCLUSION: The use of the Epiquatre software allowed us to establish regression curves for different morphometric parameters that can permit the detection of changes in their values under different pathological or experimental conditions.

Use of the Sperm-Class Analyser for objective assessment of human sperm morphology.

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.

Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques.

The recent development of automated systems for morphometric sperm head analysis has provided a series of objective parameters which have facilitated the standardization of morphological semen evaluation. This current work attempts to establish the optimum fixing conditions for the morphometric characterization of ram spermatozoa. Ejaculates were obtained from 5 Merino rams used for periodic collection of semen and were diluted at 1:50 with TEST medium. Air-dried smears were fixed either in ethanol-ether (1:1), 50% methanol, 2% glutaraldehyde or SUZA fixative, in which case the smear was pretreated with chloramine. The samples were then stained with commercial kit Hemacolor. Once the preparations had been mounted, they were analyzed with the Sperm Class Analyzer automatic sperm morphometry analysis system (ASMA). The minimum number of sperm cells analyzed per sample was 100. The parameters evaluated were the area, perimeter, length, width, shape factor and mass. The results showed significant differences in sperm head dimensions between the 4 fixation techniques, with the lowest values for all parameters corresponding to the SUZA fixative, followed by glutaraldehyde, methanol, and finally ethanol-ether. In addition, there were significant variations between animals. It can, therefore, be concluded that the working protocol must be defined when performing morphometric analysis of ram semen and that the results obtained under different conditions of fixation cannot be entirely extrapolated. Equally, the high variability among individuals suggests that, in a species like the ram with a low index of teratozoospermia, there is a need for a revision of the classic definition of normality, which should include morphometric data.

Morphometric analysis of human sperm morphology.

Fourteen morphological forms of human spermatozoa were analysed morphometrically using semi-automated image analysis techniques. Five basic (area, perimeter, length, width and mass) and five derived (ratio, length minus width, ellipticity, form and total mass) parameters were considered. Statistical analysis showed differences among all 14 types of human sperm heads. Basic parameters describing the size and shape were enough to distinguish most of the categories, whereas derived parameters as well as parameters dependent on stain intensity, were demonstrated to be useful for the discrimination of some morphological categories. The fact that statistical analysis showed differences among all 14 sperm types provides evidence for the reliability of our morphological classification. These results show that morphometry can be used for the fine study of sperm morphology and may serve as a database for future work dealing with sperm classification. As this is a pilot study to assess methodology, further studies will be required to validate the method in terms of its application and usefulness in assessing the fertilizing potential of human spermatozoa.