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Reisinger N.,Biomin Research Center | Schaumberger S.,Biomin Research Center | Nagl V.,University of Natural Resources and Life Sciences, Vienna | Hessenberger S.,Biomin Research Center | Schatzmayr G.,Biomin Research Center
Toxins | Year: 2014

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 |ig/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 |ig/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application. © 2014 by the authors; licensee MDPI, Basel, Switzerland.


Vekiru E.,Christian Doppler Laboratory | Hametner C.,Vienna University of Technology | Mitterbauer R.,Christian Doppler Laboratory | Mitterbauer R.,Sandoz GmbH | And 5 more authors.
Applied and Environmental Microbiology | Year: 2010

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography- tandem mass spectrometry (LCMS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C 18H24O7, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]- and at m/z 375.1416 [M-HNa]+. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6- [(1E)-5-hydroxypent-1-en1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Jiang Z.,Purdue University | Schatzmayr G.,Biomin Research Center | Mohnl M.,Biomin Holding GmbH | Applegate T.J.,Purdue University
Poultry Science | Year: 2010

The acute phase response (APR) is characterized by inflammation, fever, and altered organ metabolism resulting in muscle catabolism and anorexia. Lipopolysaccharide (LPS)-induced APR may reflect depressed growth and appetite loss. Therefore, a 1-wk growth experiment was conducted to examine whether dietary supplementation of a multispecies probiotic (PoultryStar) would alleviate growth suppression and anorexia caused by LPS-induced APR. The experiment was designed with 4 treatments (n = 8 cages/treatment; 6 birds/cage) starting at 14 d of age. Before (0 to 14 d of age) and for the experiment (14 to 21 d of age), male broiler chicks were fed diets devoid of probiotic or were supplemented with 1.7 × 10 8 cfu/kg of probiotic. At 14 d of age, birds fed the diet devoid of probiotic were further divided into 3 treatments: an unchallenged positive control, LPS-challenged negative control (LPS- NC), and a treatment that was pair-fed to LPS-NC. The probiotic-fed birds were also then challenged with LPS. The LPS (Escherichia coli 055:B5) was injected intraperitoneally 4 times at 48-h intervals at 1 mg/kg of BW. The LPS challenge dramatically depressed BW gain from 14 to 21 d of age by 22% (P < 0.001). However, 41% of growth depression was attributable to factors other than feed intake reduction when compared with the pair-fed treatment. Probiotic supplementation recovered 17% of depressed growth (vs. LPS-NC; P = 0.068), but this improved growth was not due to improvements in feed intake (P = 0.47). However, recovery of feed intake of the probiotic + LPS birds occurred 48-h earlier than the LPS-NC birds. Growth depression induced by LPS administration resulted in an overall relative feed intake (vs. positive control) of 0.83 and also decreased net energy and protein accretion. Probiotic supplementation did not alleviate the reduction in net energy or protein accretion induced by LPS. In conclusion, APR (induced by LPS administration) diverted a large portion of consumed nutrients from tissue accretion. Probiotic supplementation lessened the anorexic effects of LPS resulting in a trend toward BW gain improvement versus the LPS-NC. © 2010 Poultry Science Association Inc.


Nagl V.,University of Natural Resources and Life Sciences, Vienna | Woechtl B.,University of Veterinary Medicine Vienna | Schwartz-Zimmermann H.E.,University of Natural Resources and Life Sciences, Vienna | Hennig-Pauka I.,University of Veterinary Medicine Vienna | And 3 more authors.
Toxicology Letters | Year: 2014

Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-β-d-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of in vivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116. μg/kg b.w.) and the equimolar amount of DON (75. μg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5. μg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24. h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), deoxynivalenol-15-GlcA (DON-15-GlcA) and deepoxy-deoxynivalenol (DOM-1) by UHPLC-MS/MS. After oral application of DON and D3G, in total 84.8. ±. 9.7% and 40.3. ±. 8.5% of the given dose were detected in urine, respectively. The majority of orally administered D3G was excreted in form of DON, DON-15-GlcA, DOM-1 and DON-3-GlcA, while urinary D3G accounted for only 2.6. ±. 1.4%. In feces, just trace amounts of metabolites were found. Intravenously administered D3G was almost exclusively excreted in unmetabolized form via urine. Data indicate that D3G is nearly completely hydrolyzed in the intestinal tract of pigs, while the toxin seems to be rather stable after systemic absorption. Compared to DON, the oral bioavailability of D3G and its metabolites seems to be reduced by a factor of up to 2, approximately. © 2014 The Authors.


Rampler E.,University of Natural Resources and Life Sciences, Vienna | Rampler E.,Biomin Research Center | Dalik T.,University of Natural Resources and Life Sciences, Vienna | Stingeder G.,University of Natural Resources and Life Sciences, Vienna | And 2 more authors.
Journal of Analytical Atomic Spectrometry | Year: 2012

For the first time, quantitative analysis of the proteinogenic sulfur containing amino acids methionine and cysteine was performed by LC-ICP-MS. A dedicated sample preparation procedure was implemented consisting of two steps, i.e. (1) protection of the redox sensitive amino acids by controlled oxidation of methionine, cystine and cysteine to methionine sulfone and cysteic acid, respectively, and (2) subsequent protein hydrolysis. Anion exchange chromatography enabled the separation of all relevant sulfur species within 10 min. Sulfur was detected on m/z 32S 16O using O 2 as the reaction gas. Absolute limits of detection in the pmol range were achieved for methionine sulfone and cysteic acid. The method offered the possibility of protein quantification. Absolute amounts of 2 μg of hydrolyzed protein (on column) were investigated by LC-ICP-MS. Both oxidized forms of amino acids showed excellent recoveries from lysozyme and myoglobin standards, enabling accurate quantification. A repeatability of <10% (n = 6 independently prepared samples) was found without the application of isotope dilution strategies. The limits of detection of <1 μM protein were comparable to the limits of detection achieved by spectroscopy based protein quantification assays. Moreover, the validity of the approach was shown by implementing HPLC in combination with fluorescence detection as a reference method for the quantification of proteinogenic amino acids in yeast. Both methods were in good agreement and met the theoretical value in a yeast reference material certified for the methionine content. © 2012 The Royal Society of Chemistry.


Grenier B.,Purdue University | Grenier B.,Biomin Research Center | Applegate T.J.,Purdue University
Toxins | Year: 2013

Mycotoxins are secondary metabolites of fungi that can cause serious health problems in animals, and may result in severe economic losses. Deleterious effects of these feed contaminants in animals are well documented, ranging from growth impairment, decreased resistance to pathogens, hepato- and nephrotoxicity to death. By contrast, data with regard to their impact on intestinal functions are more limited. However, intestinal cells are the first cells to be exposed to mycotoxins, and often at higher concentrations than other tissues. In addition, mycotoxins specifically target high protein turnover- and activated-cells, which are predominant in gut epithelium. Therefore, intestinal investigations have gained significant interest over the last decade, and some publications have demonstrated that mycotoxins are able to compromise several key functions of the gastrointestinal tract, including decreased surface area available for nutrient absorption, modulation of nutrient transporters, or loss of barrier function. In addition some mycotoxins facilitate persistence of intestinal pathogens and potentiate intestinal inflammation. By contrast, the effect of these fungal metabolites on the intestinal microbiota is largely unknown. This review focuses on mycotoxins which are of concern in terms of occurrence and toxicity, namely: aflatoxins, ochratoxin A and Fusarium toxins. Results from nearly 100 published experiments (in vitro, ex vivo and in vivo) were analyzed with a special attention to the doses used. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Reisinger N.,Biomin Research Center | Steiner T.,Biomin Holding GmbH | Nitsch S.,Biomin Holding GmbH | Schatzmayr G.,Biomin Research Center | Applegate T.J.,Purdue University
Journal of Applied Poultry Research | Year: 2011

A 2 × 2 factorial experiment (8 pens/treatment, 26 birds/pen) was conducted with 2 doses of a coccidial vaccine [1× or 5× (Paracox-5, schering-Plough animal Health, Uxbridge, UK)], administered at 1 d of age) with or without supplementation (125 g/1,000 kg) with a phytogenic feed additive containing a blend of essential oils from oregano, anise, and citrus peel [Biomin P.e.P. 125 poultry (Biomin gmbH, Herzogenburg, austria); eO]. Within each pen, 13 birds received the coccidial vaccine, whereas the remainder were naturally exposed through the recycling of oocysts in the litter. in pens of birds receiving the higher (5×) dosage of coccidial vaccine, the BW of broilers decreased by up to 2.7% (P = 0.08), but feed-to-gain ratio (FCR) and mortality at 27 d of age (P > 0.05) were not affected. Between d 14 and 27 within pens of birds receiving the 1× dosage of coccidial vaccine, those that were fed eO had a 5.6% increase in BW gain (P < 0.05). supplementation with eO did not affect feed intake or FCR (P > 0.05). Birds naturally exposed in pens receiving the higher (5×) dosage of coccidial vaccine had an 11% reduction in ileal crypt depth (P < 0.05) and a 19% quicker enterocyte turnover rate (P = 0.086), but there was no effect on ileal villus length or number of goblet cells (P > 0.05). in naturally exposed birds with pen-mates given the 1× dosage of coccidial vaccine, birds fed EO had 12% longer villi than did un-supplemented birds (P < 0.05), with 30% more goblet cells and 23% more goblet cells/10 μm of villus length (P < 0.05). in conclusion, the dosage of coccidial vaccine had an impact on BW, intestinal turnover rate, and crypt depth, whereas dietary supplementation with the EO additive increased villus length and goblet cell density, which may have created an improved barrier against pathogens during a mild coccidial exposure. © 2011 Poultry science association, inc.


Applegate T.J.,Purdue University | Klose V.,University of Natural Resources and Life Sciences, Vienna | Steiner T.,Biomin Holding GmbH | Ganner A.,Biomin Research Center | Schatzmayr G.,Biomin Research Center
Journal of Applied Poultry Research | Year: 2010

Removal and restriction of subtherapeutic antibiotics from poultry diets in many parts of the world has amplified interest in improving intestinal health and nutrient utilization. Some probiotic (direct-fed microbials) and plant-derived (phytogenic) feed additives are gaining market presence. Defined probiotic cultures have the potential to succeed, in large part because of in vitro screening and selection. However, regulatory approval delays, particularly in Europe, have stymied the commercial application of some microorganisms in poultry diets. Phytogenic feed additives have demonstrated ranges of antimicrobial activities in vitro and are building a track record of improvements in bird performance. Hesitation by nutritionists to incorporate these feed additives are due in part to 1) unfamiliarity, 2) the overselling of plausible effects by industry, 3) product inconsistency, 4) a lack of documented physiological and microbiological effects in vivo, and, in the case of probiotics, 5) a lack of documentation of persistence. © 2010 Poultry Science Association, Inc.


Mountzouris K.C.,Agricultural University of Athens | Tsitrsikos P.,Agricultural University of Athens | Palamidi I.,Agricultural University of Athens | Arvaniti A.,Agricultural University of Athens | And 3 more authors.
Poultry Science | Year: 2010

The aim of this work was to investigate the effect of inclusion levels of a 5-bacterial species probiotic in broiler nutrition. Five hundred twenty-five 1-d-old male Cobb broilers were allocated in 5 experimental treatments for 6 wk. The experimental treatments received a corn-soybean coccidiostat-free basal diet and depending on the addition were labeled as follows: no addition (C), 108 cfu probiotic/kg of diet (P1), 109 cfu probiotic/kg of diet (P2), 1010 cfu probiotic/kg of diet (P3), and 2.5 mg of avilamycin/kg of diet (A). Each treatment had 3 replicates of 35 broilers each. Treatment effects on broiler growth performance and biomarkers such as ileal and total tract nutrient digestibility, plasma Ig concentration, and cecal micro-flora composition were determined. Differences among treatments were considered significant when P ≤ 0.05. Overall BW gain was significantly higher in treatment P1 (2,293 g) compared with P2 (2,163 g), C (2,165 g), and P3 (2,167 g), with A (2,230 g) being intermediate and not different from P1. Overall feed conversion ratio values were similar and significantly better for P1 (1.80) and A (1.80) compared with P2 (1.87), C (1.89), and P3 (1.92). Ileal apparent digestibility coefficients (ADC) of CP and ether extract were higher in A. Generally, treatments A and P1 showed an improved total tract ADC for DM, organic matter, ash, ether extract, and AMEn values. The total tract ADC of CP was higher in P1, C, and P2. There were no differences between treatments regarding plasma Ig in 14-and 42-d-old broilers. Treatments P2 and P3 were effective at beneficially modulating cecal microflora composition. In particular, the lower cecal coliform concentration (log cfu/g of wet digesta) was seen in P2 (6.12) and P3 (4.90) in 14-and 42-d-old broilers, respectively, whereas at 42 d, P3 and P2 had the highest Bifido-bacterium (8.31; 8.08) and Lactobacillus concentrations (8.20; 7.86), respectively. It is concluded that probiotic inclusion level had a significant effect on broiler growth responses, nutrient ADC, AMEn, and cecal microflora composition. © 2010 Poultry Science Association Inc.


PubMed | Biomin Research Center and Purdue University
Type: Journal Article | Journal: Poultry science | Year: 2016

A 20-day trial was conducted to determine the impact of aflatoxin B1 (AFB1) and dietary protein concentration on performance, nutrient digestibility, and gut health in broiler chicks. The 6 dietary treatments were arranged in a 2 3 factorial with 3 crude protein (CP) concentrations (16, 22, and 26%) with or without 1.5 mg/kg AFB1 Each diet was fed to 6 replicate cages (6 chicks per cage) from zero to 20 d of age. Endogenous N and amino acid loss were estimated from birds fed a N-free diet with or without 1.5 mg/kg AFB1 A significant interaction between AFB1 and CP concentration was observed for growth performance, where reduction of BW gain, feed intake, gain:feed ratio, and breast muscle weight by AFB1 were most profound in birds fed the 16%-CP diet, and were completely eliminated when birds were fed the 26%-CP diet (AFB1 by CP interaction; P 0.023). Similarly, AFB1 reduced serum albumin, total protein, and globulin concentrations in birds fed 16 and 22% CP diets, but not in those fed the 26%-CP (AFB1 by CP interaction; P 0.071). Gut permeability was increased in birds fed AFB1-contamiated diets as measured by serum lactulose/rhamnose ratio (main effect; P=0.04). Additionally, AFB1 tended to increase endogenous N loss (P=0.09), and significantly reduced apparent ileal digestible energy and standardized ileal N and amino acid digestibility in birds fed the 16%-CP diet, while birds fed higher dietary CP were not affected (AFB1 by CP interaction; P 0.01). Further, AFB1 increased the translation initiation factor 4E-binding protein(4EBP1), claudin1, and multiple jejunal amino acid transporters expression (main effect; P 0.04). Results from this study indicate that a 1.5 mg AFB1/kg diet significantly impairs growth, major serum biochemistry measures, gut barrier, endogenous loss, and energy and amino acid digestibility. Aflatoxicosis can be augmented by low dietary CP, while higher dietary CP completely eliminated the impairment of performance, serum proteins, and nutrient digestibility from aflatoxicosis in zero to 20 d broiler chicks.

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