The body weights of each animal were recorded on days 0 and 9. Faecal samples

were collected directly from the rectum of each animal on days 0, 5 and 9 to perform FECs (Gordon and Whitlock, 1939). The generic identification of the nematode population was determined by coproculture (Ueno and Gonçalves, 1998) of individual faecal samples that were collected prior to the start and at end of the treatment. Blood samples were collected from each animal by puncturing the jugular vein on Selleck Ixazomib days 0 and 9 of the experiment. The blood samples, collected in vacuum tube containing EDTA, were used to perform haemograms and to determine total plasma protein by refractometry (Jain, 1993). The serum activities of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) and alkaline phosphatase, as well as the concentrations of creatinine and urea were measured using commercial kits (DOLES®) and spectrophotometry. One week after the end of the treatment, six Trametinib supplier animals from each group were separated randomly and euthanized. The euthanisation procedure followed the recommendations of the Federal Council of Veterinary Medicine (Brasil, 2002). Subsequently, the animals were necropsied. For histopathological examination, fragments of the liver, kidney,

abomasum and intestine were collected and fixed in formalin (10%), and then paraffin-embedded sections were prepared (Prophet et al., 1992). Five-millimetre histological sections were stained with haematoxylin–eosin (Luna, 1968). Aliquots (10%) of the contents of the abomasum and

else the small intestine from each animal were analysed. The number of nematodes, which were categorised according to the genus, was multiplied by ten. The contents of the large intestine were examined completely (Ueno and Gonçalves, 1998). The identification of GINs species were determined according to Soulsby (1982). The anthelmintic efficacy was estimated by calculating the percent egg or larvae reduction, using the following formula: PR = 100 (1−T/C), where PR is the percent reduction, T and C are the arithmetic means of the eggs or larvae in the treated and negative control animals, respectively ( Coles et al., 1992). The results for the body weight, haematological and biochemical analyses, which demonstrated a normal distribution, were compared by ANOVA followed by Tukey’s test (5%). For parameters that did not show a normal distribution (egg, L3, L4 and adult worms, basophils, leukocytes and segmented rods), non-parametric analysis was performed: the Kruskal–Wallis test followed by Dunn’s multiple comparison test (5%). All analyses were performed using SAS, version 9.1 (SAS, 2004). An aliquot of the aqueous extract was extracted with isobutanol to remove small water-soluble molecules such as sugar. The iso-butanolic extract (BE) was analysed by 1H NMR (400 MHz, DMSO-d6 as the deutered solvent).