Fig. 4

Evaluation of sperm functional tests. Legend: Aniline blue, Acridin orange and BODIPY staining at a) 2 month and b) 4 months post-surgery. Aniline blue, acridine orange and bodipy tests all indicated a significant increase in sperm alterations at 2 M varicocele compared to the 2 M control and sham groups. At 4 months of VCL, the percentage of aniline blue, acridine orange and bodipy positive spermatozoa were significantly higher in the V.I-ALA⁻ group compared to the treated group (V.I-ALA⁺) and the 4 M control and sham groups. Statistical analysis (one way ANOVA) for each time point (2 or 4 M) and for each parameter was carried out between groups and p-value is provided for the groups which showed significant difference. c) a) aniline blue staining, sperm heads with dark blue staining indicate anilin blue positive and light blue staining indicates anilin blue negative. b) acridin orange positive sperm heads show orange-red fluorescence light indicate damaged chromatin, c) acridin orange negative sperm heads that expose green fluorescence light indicate intact chromatin. D) Evaluation of lipid peroxidation by bodipy C11 staining, 2-D flow cytometry dot plot of fluorescence intensity, for each sample, approximately 10,000 sperm cells were counted. d) shows unstained sperms while e) shows stained ones. The upper right quadrant, represents C11 Bodipy positive spermatozoa at 530 nm in green fluorescence with a FL1 band pass (BP).AB+: anilin blue positive, AB-: anilin blue negative, AO+: acridin orange positive, ALA: Alpha lipoic acid, Bodipy +: Bodipy positive, LL: Lower left, LR: lower right, UL: Upper left, UR: upper right, M: month post-surgery or varicocele induction (V.I), V.I-ALA⁺; varicocele rats treated with ALA for 2 to 4 month post induction of varicocele, V.I-ALA⁻: varicocele rats treated with water as solvent of ALA for 2 to 4 month post induction of varicocele. Magnification bar (50 μm)