Effet chez le porcelet d'une exposition à un régime co-contaminé en ...

These are not the final page numbersMol. Nutr. Food Res. 2011, 55, 1–11 3Table 1. Composition of the experimental dietIngredient (%)Wheat 47.50Soybean meal 24.30Barley 22.90Calcium phosphate 1.12Calcium carbonate 1.00Vitamin and mineral premix a) 0.50Vegetable oil 1.40Sodium chloride 0.40Phytase 0.01Lysine 0.465Methionine 0.165Threonine 0.195Tryptophan 0.045Composition b)Starch (g) 476.8Crude protein (g) 218.3Crude fiber (g) 37.5Ca (g) 10.5P (g) 6.5K (g) 8.7Net energy (MJ) 15.6a) Vitamin A, 2 000 000 IU/kg; vitamin D3, 400 000 IU/kg; vitaminE, 4000 mg/kg; vitamin C, 8000 mg/kg; vitamin B1, 400 mg/kg;vitamin K3, 400 mg/kg; iron, 20 000 mg/kg; copper, 4000 mg/kg; zinc, 20 000 mg/kg; manganese, 8000 mg/kg.b) Corresponding to 1000 g dry matter/kg.21 g/kg DON and FB, respectively. The extracts containingthe mycotoxins were mixed into the vitamin mineralsupplements and then incorporated into the cereal mixturebefore granulation.The feed was analysed for mycotoxin content by QuantasAnalytik (Tulln, Austria) and by using a multi-mycotoxinmethod [22]. DON, zearalenone and enniatin were found tobe naturally present in the cereals used, resulting inconcentrations of 500, 50 and 100 mg/kg of feed, respectively.All other mycotoxins, including aflatoxins, T-2 toxin, HT-2toxin and ochratoxin A were below the limit of detection.The mono-contaminated diets contained 2.8 mg of DON/kgof feed and 5.9 mg of FB/kg of feed (4.1 mg FB1/kg11.8 mgFB2/kg of feed) while the co-contaminated diet contained3.1 mg of DON and 6.5 mg of FB/kg of feed (4.5 mg FB1/kg12.0 mg FB2/kg of feed).2.3 Experimental design and sample collectionOn the 4 th and 16 th day of the experiment, all piglets wereimmunized by subcutaneous inoculation with 1 and 2 mg ofovalbumin (OVA), respectively (Sigma, St-Quentin Fallavier,France), dissolved in sterile PBS and mixed with incompleteFreund’s adjuvant (Sigma). At weekly time intervals, bloodsamples were aseptically collected from the left jugular vein.Blood was collected in tubes containing sodium heparin or& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimEDTA (Vacutainer s , Becton-Dickinson, USA) for blood cultureor blood formula, respectively. Plasma samples were obtainedafter centrifugation of heparinized blood and stored at 201Cfor later analysis. After 35 days of dietary exposure to mycotoxins,immediately after electrical stunning, pigs were killedby exsanguination. Samples of lungs, liver and kidneys werecollected from all groups and fixed in 10% buffered formalinfor histopathological analysis. In addition, a portion of thespleen was collected from euthanatized animals, flash-frozenin liquid nitrogen and stored at 801C until processed formeasurements of cytokine mRNA.2.4 Hematology and biochemistryHematological analysis was carried out using the impedancecoulter LH500 (Beckman Coulter, Villepinte, France). Subpopulationsof white blood cells (lymphocytes, monocytes,neutrophils, eosinophils and basophils) were also studiedand made manually on 100 leukocytes on May-Gr.unwaldGiemsa stained smears.Plasma concentrations of total proteins, albumin, urea,creatinin, cholesterol, triglycerides and activity of g-glutamyltransferase were determined by a Vitros 250 analyzer (OrthoClinical Diagnostics, Issy les Moulineaux, France) at theVeterinary School of Toulouse (France).2.5 HistologyThe tissue pieces were dehydrated through graded alcoholsand embedded in paraffin wax. Sections of 3 mm werestained with hematoxylin–eosin (HE) for histopathologicalevaluation. For each organ, three slides per animal wereprepared for analysis, and an area of 2000–2500 mm 2 perslide was observed. As displayed in Table 2, microscopicobservations led to the identification of different lesions inthe different organs, and allowed for establishing a lesionscore per animal. Based on a recent method published [23],we calculated the lesion score by taking into account thedegree of severity (severity factor) and the extent of eachlesion (according to intensity or observed frequency, scoredfrom 0 to 3). For each lesion, the score of the extent wasmultiplied by the severity factor. For each tissue, the minimalscores were 0 and the maximal scores were 21, 33 and15 for liver, lung and kidney, respectively (Table 2).2.6 Measurement of hepatocyte proliferationThe cellular proliferation activity was assessed by countingKi-67-positive nuclei on formalin-fixed embedded liversections as already described [24]. Briefly, the sections wereincubated with the primary antibody (Zymed (South SanFrancisco, CA, USA) Ki-67 Clone 7B11 – diluted 1:50) at 41Covernight in a humidity chamber; then the secondarywww.mnf-journal.com