The acridine orange (AO) test, which uses a cationic fluorescent dye, was performed to detect damage to sperm DNA. In brief, a smear of washed sperm was prepared on a slide and fixed with Carnoy's solution at 4°C. The slides were then stained with AO in citrate-phosphate buffer (80 ml 0.1 M citric acid+5 ml 0.3 M NaH2PO4, pH 2.5). The slides were washed with PBS 1x. Two hundred sperm were counted on each slide using a fluorescent microscope (B-383LD2 OPTIKA). Spermatozoa with a red or orange nucleus (see supplementary figure 1) were considered to show DNA damage [25].

The aniline blue (AB) test was used to assess the level of compaction of the spermatic nucleus. Aniline blue is an acid dye that interacts with lysine-rich histones. In brief, washed sperm cells were spread on slides, and after drying at room temperature, the slides were fixed with 3% glutaraldehyde [25]. Slides were stained with aniline blue (5% aqueous) in 4% ethanoic acid (5 min) and then washed with PBS 1x. A light microscope (×100 magnification, CX31 OLYMPUS, Japan) was used to randomly count 200 sperm cells in different fields for each slide. The percentage of spermatozoa with dark blue nuclei was noted and considered to be sperm cells exhibiting abnormal chromatin packaging (see supplementary figure 1).

Chromomycin A3 (CMA3) was used to evaluate the sperm nuclear protamine content. Washed spermatozoa were fixed with Carnoy's solution (methyl alcohol : ethanoic acid (3 : 1; v : v)) and stored at 4°C for 5 min. Then, 20 μl of fixed spermatozoa solution was spread on slides and left to dry at room temperature. The slides were stained for 90 min with 150 μl CMA3 solution (0.25 mg/ml CMA3 (St. Louis, MO, USA) in McIlvaine buffer containing 7 ml 0.1 M citric acid+33 ml 0.2 M Na2HPO47H2O, pH 7.0, 10 mmol/l MgCl2). The slides were then rinsed in PBS 1x and covered with a cover slip. A fluorescent microscope (OPTIKA, CX31 OLYMPUS, Japan) was used to randomly count 200 spermatozoa in different fields. The percentage of spermatozoa with CMA3-labelled nuclei was noted (×100 magnification).

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