Louis, MO, USA) All other chemicals used were obtained from stan

Louis, MO, USA). All other chemicals used were obtained from standard commercial suppliers. The Z-VAD-FMK order stain used for the blood smear was the quick panoptic (Laborclin Produtos para Laboratório Ltda, Pinhais, PR, Brazil). ZEA was prepared in olive oil, immediately before administration. Mice were weighed and randomly divided in two groups which received one administration of ZEA (40 mg/kg – 8% of LD50) or olive

oil by gavage (10 ml/kg). Forty eight hours after ZEA or vehicle administration the animals received a dose of pentobarbital (180 mg/kg, i.p.), and blood was collected by cardiac puncture into tubes containing heparin (1 UI/μl). The liver, kidneys and testes were removed, weighed and homogenized in Tris–HCl 50 mM, pH 7.4 for the determination of enzymatic and non-enzymatic indicators of oxidative stress. The epididymis were weighed and used for determining the number and motility of spermatozoa. The open field task is a simple assessment used to determine HER2 inhibitor general activity levels, gross locomotor activity and exploration habits in rodents. Two days (48 h) after the treatment with ZEA or vehicle, mice were submitted to the open field test. Mice were placed in a wooden box (20 × 30 cm) with the floor divided in twenty-one identical squares, and the number

of squares crossed with all paws, the number of rearings and the time of cleaning were counted during 10 min. In order to evaluate any possible toxic action of acute ZEA administration, the body and vital organs relative weight were determined. Mice were weighted before, and two days (48 h) after the treatment with ZEA and some vital and reproductive organs (lungs, liver, spleen, kidneys, testes and epydidymis) were weighted relatively to the body weight. Total leucocyte count was performed using 25 ul of blood and 500 ul of solution Turkey in a Neubauer chamber with

the aid of optical microscope with a 40× objective (Nikon Eclipse 50i). Subsequently, SB-3CT we applied the technique of blood smears for differential counts of neutrophils (segmented and sticks), eosinophils, lymphocytes and monocytes with 5 ul blood. After performing the same, the slides were stained (panotico fast) and viewed under a microscope according to the method described by (Failace et al., 2009). Assessment of spermatozoa count and motility was performed according to Freund and Carol (1964). The two cauda epididymides from each mouse were homogenized in 2 mL of warmed (37 °C) saline solution (0.9% NaCl). Briefly, 10 μL of the diluted spermatozoa suspension was transferred to each counting chamber of the hemocytometer and was allowed to stand for 5 min. The cells settled during this time were counted with the help of light microscope at 200× magnification (Nikon Eclipse 50i).

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