g. swimming in small circles, the flagella force hardly displacing the head, or when only a flagella beat can be observed), and progressively motile (PR, spermatozoa moves actively, either linearly or in a large circle, regardless of the speed).25 The Wnt beta-catenin pathway percentage of the motile sperms was calculated according to a previous work conducted by Moreira et al.26 Lectin Histochemistry The smears were prepared Inhibitors,research,lifescience,medical from the aliquoted sperms. The smears were fixed with paraformaldehyde for 20 minutes and washed in phosphate buffered saline (PBS) for 30 minutes. Then, the sperms were incubated in FITC-conjugated lectins (10 µL/mL) for 2 hours in the

dark. The smears were, subsequently, washed in PBS and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 minutes. Inhibitors,research,lifescience,medical The specimens were observed under the fluorescent microscope (Nickon, Eclipse,

E600). Flow Cytometry All the samples were incubated in the media, LC or PF, and were washed with 800 μL of PBS and centrifuged at 1200 rpm for 10 minutes before they were fixed with 2% paraformaldehyde for 30 minutes at 4°C. The fixed samples were centrifuged and resuspended in PBS. Afterward, the samples were incubated in FITC-conjugated lectins (10 µL/mL) for 2 Inhibitors,research,lifescience,medical hours at 37°C in a humidified atmosphere in the dark. They were then washed twice in PBS, and the percentage of the lectin-reactive sperms was measured via FL1 channel flow cytometry. The percentage of the spermatozoa that reacted with FITC-conjugated lectins was analyzed by histogram using WINmdi 2.8 software. The mean of fluorescence intensity was also analyzed Inhibitors,research,lifescience,medical using FlowJo software. Statistical Analyses All the results are presented as mean±SE (standard error of mean). The statistical analyses were performed using the One Way Analysis of Variance (ANOVA) and the Least Significant Difference test (LSD) using SPSS version 15 for Windows. A p value less than 0.05 was considered a statistically significant difference. Results Sperm Motility Assay The data Inhibitors,research,lifescience,medical showed a significant increase in the percentage of the progressive sperms exposed to PF compared to the control sperms at 30 minutes (P=0.001) and 90 minutes (P=0.007) after incubation. There

was a significant decrease in the percentage of the immotile sperms and a significant increase in the percentage of the non-progressive sperms PDK4 in the presence of LC and PF compared with the control samples at 30 minutes (P=0.000 and P=0.000) and 90 minutes after incubation (P<0.001 and P<0.001), respectively. Table 1 summarizes the data for the sperm motility assay. Table 1 The comparison of the testicular sperm motility percentages (mean±S.E; n=8) at 30 and 90 minutes after incubation in the media (control), L-carnitine- and Pentoxifylline Lectin Histochemistry Distribution Pattern The data demonstrated no changes in the distribution pattern of the glycoconjugates in the testicular sperms in the presence of the additives compared with the media.