The eosin-nigrosin staining method (differential staining). The preparations for analyses were made according to the following methodology: a drop of semen was placed on a slide preheated to 40°C and mixed with double volume of the dye mixture (one part 5% bluish eosin solution (Carl Roth Gmbh+Co. KG, Germany) to four parts of 10% nigrosin aqueous solution (Sigma-Aldrich, USA)) using a glass rod to produce a smear on the slide. The samples were air-dried at room temperature.
The eosin-gentian staining method. Thin, fat-free semen smears, heated up to 36°C, were prepared. After drying, the smears were fixed in 96% ethanol. Then, they were rinsed with water and counterstained with 10% blue eosin solution (Carl Roth Gmbh+Co. KG, Germany) for 20–60 s. Next the slides were rinsed with water again, and coloured for 3 min in gentian pigment (Sigma-Aldrich, USA). After colouring, the slides were washed and dried, leading to a clean background and thus to good contrast against the stained spermatozoa. The slides were prepared and assessed microscopically at the same time and by the same person.
The eosin-gentian staining method. Thin, fat-free semen smears, heated up to 36°C, were prepared. After drying, the smears were fixed in 96% ethanol. Then, they were rinsed with water and counterstained with 10% blue eosin solution (Carl Roth Gmbh+Co. KG, Germany) for 20–60 s. Next the slides were rinsed with water again, and coloured for 3 min in gentian pigment (Sigma-Aldrich, USA). After colouring, the slides were washed and dried, leading to a clean background and thus to good contrast against the stained spermatozoa. The slides were prepared and assessed microscopically at the same time and by the same person.