Thirty two adult male NMRI mice (8-10 weeks old) weighing 26±2 g were purchased from animal house of faculty of science Urmia University. All animals were kept under controlled environmental conditions at room temperature (22±2°C) with humidity of 50±10% and a 12/12 hr photoperiod. The animals were provided with standard diet pellets and water ad libitum.

Animals were randomly divided into four groups, containing 8 mice in each group. Group 1 (control) received distilled water 0.1 ml/mice/day (ip) for 30 day. Group 2 (MTX) received methotrexate at a dose of 20 mg/kg once a week (ip) for 30 days. Group 3 (EP) received ethyl pyruvate at a dose of 40 mg/kg/daily (ip) for 30 days. Group 4 (MTX+EP) received methotrexate at a dose of 20 mg/kg once a week (ip) concomitant with ethyl pyruvate administration at a dose of 40 mg/kg over a week.

24 hr after the end of the treatment period, animals were sacrificed by dislocation of cervical vertebrae. The caudal epididymis was removed after sacrificing the animals and placed into 1 ml of HTF medium with 4 mg/ml of Bovine Serum Albumin, then minced into small pieces to allow the sperm to swim out and incubated at 37°C/5% Co₂ for 30 min.

The obtained sperm suspension was centrifuged at 1000 rpm for 5 min. After centrifugation, 10 µl of the supernatant was taken and the epididymal sperm count was determined using Neubauerhemocytometer (²¹).

Sperm viability was evaluated as follows. 20 µl of 0.05% Eosin Y -Nigrosin were added into an equal volume of sperm suspension. After 2 min of incubation at room temperature, slides were viewed by light microscope with magnification of ×400. Dead sperm appeared to be pink and live sperm were not stained. In each sample, 400 sperm were counted and viability percentages were calculated (²²).

The motility was determined by placing 10 µl of the sperm suspersion on a clean pre-warmed microscopic slide, covered with a cover-slip and examined using a light microscope at 400× magnification (Nikon Labophot 2) equipped with a heated stage (37°C). The motility of each spermatozoon was graded as “RPFM” (rapid progressive forward movement), “SPFM” (slowly progressive forward movement) “RM” (residual motion) and “ML” (motionless) and percentages of motile and immotile sperm were obtained (²³).

Sperm DNA fragmentation has now become a new biomarker for male infertility diagnosis (²⁴). Acridine Orange (AO) staining is used, after challenge at a low pH, to distinguish between denatured, single stranded, native, and double-stranded DNA regions in sperm chromatin (²⁵). The results showed the cytogram patterns of the fluorescence intensity of denatured DNA (red) and native DNA (green). Thick smears were placed in Carnoy's fixative (methanol/ acetic acid 1:3) for 2 hr for fixation (²⁶). The slides were removed from the fixative and allowed to dry for a few minutes. After 5 min at laboratory temperature, the sperm were stained with stock solution consisting of 1 mg of AO in 1000 ml of distilled water and stored in the dark at 4°C. The staining solution was prepared as follows. 10 ml of the stock solution was added to 40 ml of 0.1 M citric acid and 2.5 ml of 0.3 M Na₂HPO₄ · 7H₂O (²⁸). After staining for 5 min, the slides were rinsed with deionized water. The sperm were analyzed by fluorescent light microscopy. Red and green sperm could be observed, green sperm were classified as normal DNA and yellow to red sperm were classified as damaged DNA (²⁶).

During histone replacement with protamines and chromatin condensation in spermatogenesis, normal sperm do not stain with aniline blue, but sperm with defective density or immature sperm absorb the stain (²⁸). Sperm samples were air-dried and fixed for 30 min in 3% glutaraldehyde in phosphate buffered saline (pH=7.2). Each smear was stained with 5% acidic aniline blue stain (5 g aniline blue (Sigma-Aldrich, USA), 4% acetic acid in double distilled water, pH=3.5 for 5 min). At least 200 spermatozoa under light microscopy (Olympus Co, Tokyo, Japan) were counted in each slide (²⁹).

In order to do this, 300 µl of 10% trichloroacetic acid was added to 150 µl of the sample and centrifuged at 1,000 rpm for 10 min at 4°C. 300 µl of supernatant was transferred to a test tube with 300 µl of 67% thiobarbituric acid and incubated at 100°C for 25 min. 5 min after cooling the solution, a pink color appeared because of MDA-TBA reaction and was evaluated using a spectrophotometer at a wavelength of 535 nm (³⁰).

After collecting blood, samples were centrifuged to isolate serum and kept at −80°C until biochemical analysis with immuneradiometric technique (WHO/Sigma Asso-RTGC-768/98) for testosterone measurement.

Results were shown as mean± standard error of mean (S.E.M.) for each group. For comparison between the groups, the results were analyzed by SPSS 16 software, using oneway ANOVA followed by a Bonferroni post hoc test. A p<0.05 was considered significant.

The results revealed that sperm count decreased significantly (p<0.05) in methotrexate group in comparison with the control group. Ethyl pyruvate+methotrexate group revealed significant enhancement (p<0.05) in sperm count which are presented in Table 1. There was no significant (p>0.05) difference in sperm maturity among groups. Treatment with anti-neoplastic drug (MTX) caused a significant decrease in viability and an increase in sperm with damaged DNA compared with the control group while Ethyl pyruvate caused improvement in viability and DNA fragmentation in MTX+EP group (Figures 1, 2, 3; Table 1).

In Table 2, we can see that regarding sperm motility, compared with the control group, RPFM decreased significantly (p<0.05) in methotrexate group. However, Ethyl pyruvate caused an increase in RPFM in MTX+EP group which was significantly (p<0.05) higher compared with the MTX group. SPFM results indicated that the difference between groups was not significant. RM and ML in MTX treatment group increased significantly (p<0.05). MTX treatment in EP supplemented animals showed a significant reduction (p<0.05) in RM and ML compared with MTX group alone (Table 2).

The results of biochemical analyses were shown in Table 3. Briefly, the level of MDA, a major degradation product of lipid peroxidation, was significantly increased in MTX treated mice compared with the control group (p<0.05). There was a significant restoration in MDA level in the groups that received EP treatment with MTX (p<0.05).

The level of testosterone was found to be significantly lower in MTX-treated group when compared with other groups (p<0.005). Ethyl pyruvate administration, after MTX, caused significant increase in testosterone concentrations when compared with MTX and control group alone (p< 0.05). On the other hand, EP+MTX group testosterone level was similar to the control group.