The specimens were fixed with 99% methanol Navitoclax mouse and kept at room temperature until fluorescence staining. For staining, slides were incubated with 20 μg/ml Alexa Fluor-488-PNA (peanut agglutinin) at 37 °C for 30 min, washed with PBS, and analyzed by using epifluorescent microscopy with an appropriate filter. The images of stained sperm samples were classified into two groups: Sperm displaying intensive and moderate bright fluorescence in the acrosomal region were considered to be intact, whereas sperm displaying weak, patchy, or no fluorescence in the acrosomal region were considered to be damaged

[52]. 100 sperm on each slide were evaluated to determine the proportion of sperm with intact acrosomes. The sperm MMP was evaluated using the JC-1 fluorescent dye (M34152, Molecular Probes Inc.) by the modified method that was previously described by Guthrie and Welch [18]. The JC-1 fluorescent dye was used to distinguish spermatozoa with poorly and highly functional mitochondria. In poorly functional mitochondria, JC-1 remains in the monomeric state and fluoresces green. However, in highly functional mitochondria, JC-1 forms aggregates that fluoresce orange. For evaluation of MMP in spermatozoa, 300 μl the washed (prepared before acrosomal integrity

analysis) sperm suspensions (1–2 × 106 spermatozoa/ml) were mixed with 10 μl Cobimetinib order JC-1 (0.75 μg final concentration). The mixture was incubated at 37 °C for 30 min and then 100 sperm per sample were analyzed by using epifluorescent microscope with a dual fluorescence filter (Nikon Eclipse 600). Statistical analyses were performed using SPSS software (version 11.5 for Windows; SPSS Inc., Chicago, IL). The data were analyzed to determine the effects of extenders and freezing rate on motility, membrane and acrosome plasma integrity and MMP. Parametric data were analyzed by analysis Avelestat (AZD9668) of variance (Two-Way ANOVA) and if there were significant differences, Tukey test for multiple comparisons was used for post hoc analysis. The non-parametric data were

analyzed Kruskal–Wallis and if there were significant differences between groups, Mann–Whitney test was used to determine the differences in groups. Statistical significance was set at P < 0.05. Values were presented as the mean ± standard error of the mean (SEM). Most of the extenders tested were effective in maintaining motility after equilibration. Motility of diluted, equilibrated and frozen-thawed SD rat sperm for different extenders and cooling rates are given in Table 1, Table 2 and Table 3. Fresh and diluted sperm motility before equilibration was between 60.0% and 76.7% for SD rats. Equilibration caused less than 10% motility loss in sperm samples diluted in extenders with the exception of m-KRB in 40 °C/min group. After freezing, sperm motility ranged between 3.7% and 32.5% (p < 0.05). Sperm samples that were frozen in TES-S extender retained the highest motility (32.