2.10. Sperm Analysis

The seminal fluid was spread on a Neubauer hemocytometer and the sperm heads were counted manually by light microscope. About 200–300 head of sperms were counted in every rat and results were expressed as total sperm count per milliliter [29, 30].

The seminal fluid was loaded in preheated microscope slides. Ten microscopic fields at a final magnification of 400x randomly selected and 200–300 sperms per animal were evaluated. Sperm motility was classified as progressive motile, nonprogressive, and nonmotile according to the fifth edition of the World Health Organization guidelines [31]. The percentage of sperm motility was assessed by number of motile sperm × 100/total number of spermatozoa [29, 30].

For sperm viability, the smear of seminal fluid was stained with eosin and nigrosin dyes (Sigma–Aldrich) according to the World Health Organization guidelines. The heads of living sperms are not dyed, but the heads of dead sperms are dyed. The 200–300 stained and nonstained sperms per rat at a final magnification of ×400 in 10 random microscopic fields were evaluated. The percentage of live sperm was assessed by the number of live sperm × 100/sum of live and dead sperm [29, 30].

To assess sperm morphology, 40 μL sperm suspension was gently mixed with 10 μL solution containing 1% eosin Y and nigrosin in a test tube. The sperm was incubated at room temperature for 60 min to facilitate staining. Subsequently, the sperm was resuspended using a pipette. A light microscope examined 200 spermatozoa on each slide (magnification: 400x). The abnormalities were classified as head and tail defects (double head, flattened head, bent tail, curved neck). The percentages of normal sperms were determined [32].