Effects of Exposure to Low Zearalenone Concentrations Close to the EU Recommended Value on Weaned Piglets' Colon.

Pigs are the most sensitive animal to zearalenone (ZEN) contamination, especially after weaning, with acute deleterious effects on different health parameters. Although recommendations not to exceed 100 µg/kg in piglets feed exists (2006/576/EC), there are no clear regulations concerning the maximum limit in feed for piglets, which means that more investigations are necessary to establish a guidance value. Due to these reasons, the present study aims to investigate if ZEN, at a concentration lower than the EC recommendation for piglets, might affect the microbiota or induce changes in SCFA synthesis and can trigger modifications of nutritional, physiological, and immunological markers in the colon (intestinal integrity through junction protein analysis and local immunity through IgA production). Consequently, the effect of two concentrations of zearalenone were tested, one below the limit recommended by the EC (75 µg/kg) and a higher one (290 µg/kg) for comparison reasons. Although exposure to contaminated feed with 75 µg ZEN/kg feed did not significantly affect the observed parameters, the 290 µg/kg feed altered several microbiota population abundances and the secretory IgA levels. The obtained results contribute to a better understanding of the adverse effects that ZEN can have in the colon of young pigs in a dose-dependent manner.

Yeast-Fermented Rapeseed Meal Extract Is Able to Reduce Inflammation and Oxidative Stress Caused by Escherichia coli Lipopolysaccharides and to Replace ZnO in Caco-2/HTX29 Co-Culture Cells.

(1) The present study tested in vitro the capacity of a fermented rapeseed meal extract to reduce medicinal ZnO, which will be banned at the EU level from 2023 onwards because of its potential to cause environmental pollution and the development of Zn resistance in gut bacteria. Rapeseed meal could be an important ZnO substitute as it has antioxidant/radical scavenging properties due to its content of bioactive compounds (e.g., polyphenols). (2) Protein array and flow cytometry were used to detect apoptosis, oxidative stress production, and inflammatory and signaling-related molecules in Caco-2 and goblet HT29-MTX co-culture cells challenged with Escherichia coli lipopolysaccharides and treated with ZnO and FRSM. (3) LPS induced cell death (21.1% vs. 12.7% in control, p < 0.005); apoptosis (16.6%); ROS production; and overexpression of biomarkers related to inflammation (63.15% cytokines and 66.67% chemokines), oxidative stress, and signaling proteins when compared to untreated cells. ZnO was effective in counteracting the effect of LPS, and 73.68% cytokines and 91.67% of chemokines were recovered. FRSM was better at restoring normal protein expression for 78.94% of cytokines, 91.67% of chemokines, and 61.11% of signaling molecules. FRSM was able to mitigate negative effects of LPS and might be an alternative to ZnO in pig diets.

Dietary Grape Seed Meal Bioactive Compounds Alleviate Epithelial Dysfunctions and Attenuates Inflammation in Colon of DSS-Treated Piglets.

Inflammatory Bowel Diseases (IBD) are chronic inflammations associated with progressive degradation of intestinal epithelium and impairment of the local innate immune response. Restoring of epithelial integrity and of the mucosal barrier function, together with modulation of inflammatory and innate immune markers, represent targets for alternative strategies in IBD. The aim of our study was to evaluate the effects of a diet including 8% grape seed meal (GSM), rich in bioactive compounds (polyphenols, polyunsaturated fatty acids (PUFAs), fiber) on the markers of colonic epithelial integrity, mucosal barrier function, pro-inflammatory, and innate immunity in DSS-treated piglets used as animal models of intestinal inflammation. Our results have demonstrated the beneficial effects of bioactive compounds from dietary GSM, exerted at three complementary levels: (a) restoration of the epithelial integrity and mucosal barrier reinforcement by modulation of claudins, Occludin (OCCL) and Zonula-1 (ZO-1) tight junction genes and proteins, myosin IXB (MYO9B) and protein tyrosine phosphatase (PTPN) tight junction regulators and mucin-2 (MUC2) gene; (b) reduction of pro-inflammatory MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) genes and activities; and (c) suppression of the innate immune TLR-2 (Toll-like receptor-2) and TLR-4 (Toll-like receptor-4) genes and attenuation of the expression of MyD88 (Myeloid Differentiation Primary Response 88)/MD-2 (Myeloid differentiation factor-2) signaling molecules. These beneficial effects of GSM could further attenuate the transition of chronic colitis to carcinogenesis, by modulating the in-depth signaling mediators belonging to the Wnt pathway.

Zearalenone and the Immune Response.

Zearalenone (ZEA) is an estrogenic fusariotoxin, being classified as a phytoestrogen, or as a mycoestrogen. ZEA and its metabolites are able to bind to estrogen receptors, 17ß-estradiol specific receptors, leading to reproductive disorders which include low fertility, abnormal fetal development, reduced litter size and modification at the level of reproductive hormones especially in female pigs. ZEA has also significant effects on immune response with immunostimulatory or immunosuppressive results. This review presents the effects of ZEA and its derivatives on all levels of the immune response such as innate immunity with its principal component inflammatory response as well as the acquired immunity with two components, humoral and cellular immune response. The mechanisms involved by ZEA in triggering its effects are addressed. The review cited more than 150 publications and discuss the results obtained from in vitro and in vivo experiments exploring the immunotoxicity produced by ZEA on different type of immune cells (phagocytes related to innate immunity and lymphocytes related to acquired immunity) as well as on immune organs. The review indicates that despite the increasing number of studies analyzing the mechanisms used by ZEA to modulate the immune response the available data are unsubstantial and needs further works.

The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A.

The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1-basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2-the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3-basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, CYP1A2 was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of CYP1A2, CYP2A19, CYP2E1, and CYP3A29, but we did not detect important changes in the expression level of CY4A24, MRP2 and GSTA1 genes.

Grape Seed Waste Counteracts Aflatoxin B1 Toxicity in Piglet Mesenteric Lymph Nodes.

Aflatoxin B1 (AFB1) is a mycotoxin that frequently contaminates cereals and cereal byproducts. This study investigates the effect of AFB1 on the mesenteric lymph nodes (MLNs) of piglets and evaluates if a diet containing grape seed meal (GSM) can counteract the negative effect of AFB1 on inflammation and oxidative stress. Twenty-four weaned piglets were fed the following diets: Control, AFB1 group (320 µg AFB1/kg feed), GSM group (8% GSM), and AFB1 + GSM group (8% GSM + 320 µg AFB1/kg feed) for 30 days. AFB1 has an important antioxidative effect by decreasing the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and total antioxidant status. As a result of the exposure to AFB1, an increase of MAP kinases, metalloproteinases, and cytokines, as effectors of an inflammatory response, were observed in the MLNs of intoxicated piglets. GSM induced a reduction of AFB1-induced oxidative stress by increasing the activity of GPx and SOD and by decreasing lipid peroxidation. GSM decreased the inflammatory markers increased by AFB1. These results represent an important and promising way to valorize this waste, which is rich in bioactive compounds, for decreasing AFB1 toxic effects in mesenteric lymph nodes.

Diet containing grape seed meal by-product counteracts AFB1 toxicity in liver of pig after weaning.

Liver is the earliest target for AFB1 toxicity in both human and animals. In the last decade, plant derived by-products have been used in animal feed to reduce AFB1 induced toxicity. In the present study we investigated whether the presence of 8% grape seed meal by-product is able to counteract the hepatotoxic effects produced by AFB1 in liver of pig after weaning exposed to the toxin through the contaminated feed for 28 days. Twenty four weaned cross-bred TOPIGS-40 piglets with an average body weight of 9.13±0.03 were allocated to the following experimentally treatments: control diet without AFB1 (normal compound feed for weaned pigs); contaminated diet with 320 mg kg-1 AFB1; GSM diet (compound feed plus 8% grape seed meal) and AFB1+GSM diet (320 mg kg-1 AFB1 contaminated feed plus 8% grape seed meal). Pigs fed AFB1 diet had altered performance, body weight decreasing with 25.1% (b.w.: 17.17 kg for AFB1 vs 22.92 kg for control). Exposure of piglets to AFB1 contaminated diet caused liver oxidative stress as well as liver histological damage, manly characterized by inflammatory infiltrate, fibrosis and parenchyma cells vacuolation when compared to control and GSM meal group. 94.12% of the total analysed genes (34) related to inflammation and immune response was up-regulated. The addition of GSM into the AFB1 diet diminished the gene overexpression and ameliorate histological liver injuries and oxidative stress. The protective effect of GSM diet in diminishing the AFB1 harmful effect was mediated through the decreasing of gene and protein expression of MAPKs and NF-κB signalling overexpressed by AFB1 diet. The inclusion of grape seed by-products in the diet of pigs after weaning might be used as a novel nutritional intervention to reduce aflatoxin toxicity.