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A total of 333 spermatozoa were evaluated, in random fields. To be classified as normal, a sperm cell must have a hook-shaped head and no defects of head, neck or tail. Otherwise, spermatozoa were considered abnormal. A combined extraction of polar and apolar metabolites was performed as previously described (Alves et?al., 2011b). The aqueous phase containing the water-soluble metabolites was http://www.selleckchem.com/products/INCB18424.html lyophilized. NMR spectra was acquired as described previously (Alves et?al., 2011b). Sodium fumarate was used as an internal reference (6.50?ppm) to quantify the following metabolites (multiplet, ��, ppm): lactate (doublet, 1.33?ppm); alanine (doublet, 1.45?ppm); acetate (singlet, 1.90?ppm); creatine (singlet, 3.0?ppm); glycine (singlet, 3.54?ppm). http://www.selleck.cn/products/U0126.html The relative areas of 1H-NMR resonances were quantified using the curve-fitting routine supplied with the NUTSpro NMR spectral analysis program (Acorn, CA, USA). Semi-quantitative PCR was performed to analyse lactate dehydrogenase A (LDH A), monocarboxylate transporter 4 (MCT4), GLUT1, GLUT3, phosphofrutokinase 1 (PFK1) and lactate dehydrogenase C (LDHC) mRNA expression as described by Oliveira et?al. (2011). Both optimal annealing temperature and the amplification size of fragments are shown in Table?1. mRNA expression was normalized with 18S gene expression and expressed as fold variation (induction/reduction) vs the control group. Western Blot procedure was performed as previously described by Alves et?al. (2011a). The resulting membranes were incubated with rabbit anti-GLUT1 (1?:?300, CBL242), rabbit anti-GLUT3 (1?:?1000, ab41525), rabbit anti-PFK1 (1?:?500, Sc67028), rabbit anti-MCT4 (1?:?1000, Sc50329), rabbit anti-LDH (1?:?10000, ab52488) and rabbit anti-ALT (1?:?500, Sc99088). Mouse anti-tubulin (1?:?5000, A5441) was used as protein loading control for testicular tissue. The http://www.selleckchem.com/products/PLX-4032.html immunoreactive proteins were detected separately with goat anti-rabbit IgG-AP (1?:?5000, Sc2007) or goat anti-mouse IgG-AP (1?:?5000, Sc2008). Membranes were reacted with ECF detection system. The densities from each band were obtained using the Quantity One Software (Bio-Rad, Hertfordshire, UK), divided by the respective tubulin band density and then normalized against the respective control. Lactate dehydrogenase (LDH) activity was determined using a commercial assay kit (Promega, Madison, WI, USA) and following the manufacturer's instructions. Phosphofructokinase 1 (PFK1) activity was determined as previously described (Vaz et?al., 2012). The enzyme activity was expressed as units per milligram of protein. The attained activities were expressed as fold variation vs. the control group. The statistical significances of differences of all experimental data were assessed by Student-t test (GraphPad Software, San Diego, CA). All experimental data are shown as mean?��?SEM; p?