Bright field optical microscope

Sperm viability was determined through eosin-nigrosin staining [32 (link)]. Briefly, 10 µL of each sperm sample was placed on a slide, previously warmed to 37 °C, and 10 µL of the eosin-nigrosin stain was added. Subsequently, a smear of the mixture was prepared on the slide using a glass rod. Samples were air dried at room temperature. A minimum of 200 sperm/sample were evaluated under a bright field optical microscope (Carl Zeiss, Göttingen, Germany) at 1000× magnification using immersion oil. Three technical replicates were evaluated, and the percentage of viable spermatozoa (eosin negative) was recorded.

The stain preparations were made according to the following procedure: a drop of semen (5 µL) was placed on a slide preheated to 40 °C and mixed with the same volume of the dye mixture (one part 5% bluish eosin solution (Carl Roth Gmbh+Co. KG, Karlsruhe, Germany) to four parts of 10% nigrosin aqueous solution (Sigma-Aldrich, Saint Louis, MO, USA) using a glass rod to produce a smear on the slide. The samples were air-dried at room temperature. Samples were evaluated under a bright field optical microscope (Carl Zeiss, Göttingen, Germany) at ×1000 magnification using immersion oil. Three replicates were made for each ejaculate and the mean was calculated. All slides were scored blind with a minimum of 200 sperm/sample counted and classified for morpho-abnormalities.

To assess sperm morphology, ejaculate smears were prepared, fixed by methanol (during 1 min) and stained using Diff-Quick kits (Abris plus, Russia) according to the “WHO manual…” [2 ]. Spermatozoa were examined for morphology using the bright-field optical microscope (Carl Zeiss, Germany) at ×1000 magnification with oil-immersion. The microscope was equipped by the digital camera AxioScope with a special software (AxioVision 9.0) enabling to perform sperm morphometry. Two hundred sperm for each semen sample were assessed. Percentage of normal sperm was estimated twice in random and blinded order by two experienced investigators. Spearman correlation coefficient between between percentages sperm with normal morphology obtained by first and second count was 0.78, statistically significant difference was not observed. The differences between first and second counts of normal sperm morphology were acceptable according criteria provided by «WHO laboratory manual…» [2 ] for each sample. The mean value for percentage of normal sperm was calculated.
Percentages of sperm morphological defects were estimated in random and blinded order by the only experienced researcher (M.K.).