2.4. Sperm Mobility, Concentration, Morphology, and Lipid Peroxidation

To determine sperm mobility, 10 μl of the sperm suspensions extracted from the cauda epididymides was placed on a slide and then evaluated under a light microscope (CX31 OLYMPUS; ×40 magnification). Two hundred spermatozoa were observed from random microscopic fields, and mean motility was recorded. A Makler chamber was used to determine sperm concentration, which was presented as millions of cells per ml.

Sperm morphology was evaluated by staining 20 μl of washed sperm with 40 μl eosin at room temperature. After 5 min, 60 μl of nigrosine dye was added. Smears were prepared with 20 μl of the preparation [15]. Two hundred spermatozoa on each slide were randomly evaluated on an optical microscope (CX31 OLYMPUS, magnification ×100), and the total percentage of abnormal forms was determined.

The BODIPY® 581/591 C11 test was used to assess sperm membrane lipid peroxidation. The procedure was based on the protocol described by Aitken et al. [21]: two million washed spermatozoa were exposed to the BODIPY C11 probe at a final concentration of 5 mM/ml for 30 min at 37°C. A positive control was performed for each sample by adding H₂O₂ to the sample. The samples were then washed twice with PBS 1x to remove the unbound BODIPY C11 probe (500 g for 5 min). Finally, a flow cytometer FACSCalibur (Becton Dickinson, San Jose, CA, USA) was used to assess lipid peroxidation. The percentage of BODIPY-positive spermatozoa was recorded for each sample.