Prevention of deoxynivalenol toxicity in broiler chickens by means of detoxifying agents

Mycotoxins are considered a very important public health issue because of their adverse effects on animals and humans. Deoxynivalenol (DON) is the most frequent mycotoxin in cereals worldwide. DON contamination leads to great economic losses in poultry industry due to their cereal-based diets. The most commonly used method to counteract the negative impact of mycotoxins on animals is the addition of mycotoxin detoxifying agents (mycotoxin detoxifiers) to feed. These feed additives, so-called mycotoxin binders or mycotoxin modifiers, either adsorb or biotransform mycotoxins in the gastrointestinal tract, respectively. These detoxifying agents should be tested not only in vitro, but also in vivo on their ability to bind or modify mycotoxins. At the time being, no reliable in vivo models in chicken are available to evaluate the efficacy of mycotoxin detoxifiers based on specific biomarkers.
The aim of this thesis was first to develop an in vivo model then to investigate the efficacy of detoxifying agents, based on specific and nonspecific indicators, in broiler chickens.
The general introduction of this doctoral research starts with an overview of DON mycotoxin. Next, specific and nonspecific biomarkers of DON toxicity in chickens have been reported. Finally, an overview of binding and biotransforming agents against DON has been carried out.
The first chapter shows the results of a DON toxicokinetic study in broiler chickens’ plasma, performed via oral bolus or intravenous injection of 0.75 or 2.25 mg DON/kg of body weight (BW). This toxicokinetic study was done with the objective to identify which biological compartment the DON was likely to reach and to specify in what form (initial compound or metabolites) it does so. The analysis of plasma by LC-MS/MS revealed that DON could not be quantified after oral bolus application, indicating the very low bioavailability of DON in broiler chickens. The evaluation of toxicokinetics parameters after the intravenous injection revealed the metabolization of DON in DON-3 sulphate, its rapid clearance and excretion in broiler chickens.
Chapter 2 and 3 show an in vivo model set-up to study the effect of a DON contaminated diet on specific and nonspecific relevant biomarkers on broiler chickens. Forty-five 1-day-old male broiler chickens (Ross 308) were fed diets during 42 d, distributed into 3 experimental groups:distribuidos en 3 grupos experimentales: control group (T1), DON contaminated feed (5 mg/kg feed) (T2), or DON contaminated feed (15 mg/kg feed) (T3).

Plasma, liver or excreta concentrations of DON and DON-3 sulphate were used as specific indicators (chapter 3). The nonspecific parameters evaluated were performance parameters, relative organ weights, morphology and histology of small intestine, serum biochemistry profile, fear behavior and leg color (chapter 2), blood hematology, response to common vaccines, plasma IL-8, relative gene expression of IL-6, IL-1β, IFN-γ and IL-10, stress index (heterophil to lymphocyte ratio), and plasma corticosterone (chapter 3).
DON was only quantified in excreta, suggesting low bioavailability, limited accumulation and rapid excretion of DON. DON-3S was quantified in all biological matrices, indicating that DON-3S is the most suitable metabolite of exposure of DON in broiler chickens. At 5 mg DON/kg feed, creatine kinase decreased and IL-1β, IL-6, and IFN-γ were upregulated. At 15 mg DON/kg feed, feed conversion ratio was impaired and blood cholesterol and red blood cells decreased. At both levels assayed relative weights of gizzard and thymus, the length of small intestine, and plasma IL-8 increased. However, the relative weight of small intestine, colon, and bursa of Fabricius, the density (weight/length) of small intestine, hemoglobin and plasma corticosterone were reduced. It can be concluded that specific and nonspecific parameters affected by the contaminated feed could be suitable to evaluate the efficacy of the mycotoxin detoxifying-agents in broiler chickens.
Chapter 4 shows the in vivo efficacy model used to test 3 mycotoxin detoxifiers (MFA, IMP and MDE) based on biomarkers selected on chapters 2 and 3, as well as other biomarkers (DOM-3 sulphate and DOM-1 in excreta, serum triglycerides, relative expression of IL-8 and TNF-α in jejunum tissues, and corticosterone in feathers). Three hundred eighty-four 1-d-old male broiler chickens (Ross308) were fed for 42 d with diets formulated as non-contaminated feed (control), contaminated feed, control+0.2% MFA, contaminated feed+0.2% MFA, control+0.0125% IMP, contaminated feed+0.0125% IMP, control+0.15% MDE, or contaminated feed+0.15% MDE. DON was the main mycotoxin of the contaminated feed and concentrations varied around 7 mg/kg feed. Studied biomarkers were evaluated at 10 and 42 d. DON, DON-3S, and DOM-3S were detected in excreta from contaminated groups. The addition of MDE to contaminated feed increased the excretion of DON but decreased the excretion of the metabolite (DOM-3S). The addition of MFA to contaminated diet increased the excretion of DON, suggesting that this product is effective to detoxify this mycotoxin. At d 10, DON impaired feed conversion ratio, increased serum cholesterol and triglycerides levels. The effect on feed conversion ratio was prevented by IMP addition to the contaminated diet. The effect on serum cholesterol level was reversed by MFA or IMP supplementation to the contaminated feed. At d 10, moreover, DON reduced hematocrit, hemoglobin, red blood cells, and monocytes levels. The addition of IMP to the contaminated diet counteracted the effect observed on blood hematocrit and monocytes levels. At 42 d, DON improved the feed conversion ratio, reduced the relative weight of liver, and blood lymphocytes level. At 42 d, furthermore, DON increased white blood cells counts, stress index (heterophils to lymphocytes ratio) and feather corticosterone. The effect on stress index was counteracted by the addition of MFA to the contaminated diet.
It can be concluded that the specific parameters selected are suitable to evaluate the efficacy of DON detoxifying agents in broiler chickens, and the product MFA partially counteracted the negative effects of DON.