Desenvolvimento de uma metodologia ex vivo para avaliação de aditivos antimicotoxinas para aves

Abstract

Inclusion of antimycotoxins additives (AMAs) in poultry feed is one of the main strategies to reduce the harmful effects of mycotoxins. AMAs are substances capable of adsorbing, inactivating, neutralizing or biotransforming mycotoxins. This work aimed to develop an ex vivo methodology to evaluate AMAs used in poultry production. Intestinal explants from broiler chickens destined to human consumption were used in two studies to test the efficacy of AMAs against aflatoxin B1 (AFB1) and deoxynivalenol (DON); the direct effect of DON upon intestinal tissue was also investigated. Four pairs of Ussing chambers with an intestinal contact area of 1.0 cm² were used at 37°C and bubbled with carbogen gas for 120 min. In study 1, six commercially available AMAs (antimycotoxins additive - AMA 1 to 6) had their ability to reduce intestinal absorption of AFB1 evaluated in order to assess efficacy. Jejunal explants (n=4/bird) were collected from 60 broilers at slaughter, totaling 240 samples (40 samples/AMA). The explants were subjected to two treatments per AMA: T1 (control) - 2.8 mg/L of AFB1, and T2 - 2.8 mg/L of AFB1 + 0.5% AMA. The AMAs were also tested in vitro to investigate AFB1 adsorption in artificial intestinal fluid. Study 2 analyzed the detrimental impact of DON on intestinal tissue as well as the efficacy of an AMA. Two experiments were conducted to examine histopathological and immunohistochemical parameters: Experiment 1) T1 (positive control) - buffer solution only, and T2 - 10 mg/L DON (two replicates/treatment); and Experiment 2) T1 (positive control) - buffer solution only, T2 (negative control) - 10 mg/L DON, T3 - AMA (0.5%) only, and T4 - 10 mg/L DON + 0.5% AMA. Jejunal explants (n=4/bird) were taken from 22 broilers at slaughter, totaling 88 samples (40 and 48 samples for experiments 1 and 2, respectively). Results of study 1 showed that AMA1 to AMA6 decreased intestinal absorption of AFB1 in the ex vivo assays by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively; as for the in vitro tests, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. No correlation was seen between ex vivo and in vitro findings. Regarding the second study, DON reduced the size of enterocytes as well as of their nuclei and increased cytoplasmic vacuolization, apical denudation of villi and the number of apoptotic cells in Experiment 1; the parameters investigated in Experiment 2 evidenced that the AMA mitigated the harmful effects of DON upon intestinal villi. The present assessment demonstrates that the ex vivo model using intestinal explants from broiler chickens mounted on Ussing chambers is a viable tool to complement in vitro and in vivo assays employed to evaluate the efficacy of AMAs and also to screen new compounds. Moreover, this methodology may be applied to determine the burden imposed by DON on the integrity of the intestinal epithelium of broilers.