First insights into serum metabolomics of trenbolone/estradiol implanted bovines; screening model to predict hormone-treated and control animals’ status

International audienceThe use of anabolic agents in livestock production is a subject of much concern. Although prohibited for more than 20&nbsp;years within the EU, growth promoting practices are still widely suspected. To meet the current challenges for detecting illicit practices, ‘omics’ strategies have recently been demonstrated as important new investigative tools. These investigations, based on the observation of physiological disturbances, mainly in urine, demonstrated the possibility to monitor biomarkers enabling high throughput determination of animal status in terms of hormonal treatment. In this context, serum was investigated for the first time as an alternative and potential complementary sample type. A metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry, was exploited in order to, highlight metabolic perturbations in serum of Revalor-XS® (trenbolone acetate/estradiol) implanted bovines. Univariate and multivariate statistical analyses were carried out to establish descriptive and predictive models. These models enabled the discrimination of anabolised from control animals, and highlighted a number of metabolites which contributed the most in the observed discrimination. Further, a screening model combining a set of selected markers intensities was generated and it successfuly classified animals according to their status, up to 4&nbsp;weeks post Revalor-XS® implant. This research indicates, for the first time, that serum metabolomics has an important role in screening to detect for anabolic misuse in bovines.</p

Aminoaciduria caused by fanconi syndrome in a heifer

A case study of renal tubular dysfunction consistent with idiopathic Fanconi syndrome is reported in an 18-month-old Holstein heifer. The clinical, biochemical, and histopathological features are described. The heifer had clinical signs of growth retardation, wasting, and persistent diarrhea. Biochemical blood analysis identified hypokalemia, hyponatremia, and hypo-chloremia. Urinalysis identified glycosuria, proteinuria, and acidic pH. Histological examination of the kidney disclosed mild tubular necrosis with proteinaceous casts in the lumina of renal tubules. We performed LC-HRMS on urine to confirm Fanconi syndrome. Using this technique, we identified severe generalized aminoaciduria suggestive of idiopathic renal Fanconi syndrome in this heifer

Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin

The administration of recombinant methionyl bovine somatotropin (rMbST) to dairy cows to increase milk yield remains a common practice in many countries including the USA, Brazil, Mexico, South Africa and Korea, whereas it has been forbidden within the European Union (EU) since 1999. A rapid screening immunoanalytical method capable of the unequivocal determination of rMbST in milk would be highly desirable in order to effectively monitor compliance with the EU-wide ban for home-made or imported dairy products. For decades, the production of specific antibodies for this recombinant isoform of bovine somatotropin (bST) has remained elusive, due to the high degree of sequence homology between both counterparts (e.g. methionine for rMbST in substitution of alanine in bST at the N-terminus). In this study, we compared several immunizing strategies for the production of specific polyclonal antibodies (pAbs), based on the use of the full-length recombinant protein, an rMbST N-terminus peptide fragment and a multiple antigen peptide (MAP) which consists of an oligomeric branching lysine core attached to the first two N-terminus amino acids of rMbST, methionine and phenylalanine (MF-MAP). The immunization with KLH-conjugated MF-MAP led to the production of the pAb with the highest rMbST/bST recognition ratio amongst the generated battery of antibodies. The pAb exhibited a specific binding ability to rMbST in a competitive antigen-coated ELISA format, which avidity was further improved after purification by rMbST N-terminus peptide-based affinity chromatography. These results suggest that immunodiscrimination between structurally related proteins can be achieved using immuno-enhanced immunogens such as MAPs

Hydrophilic interaction (HILIC) and reverse phase liquid chromatography (RPLC)–high resolution MS for characterizing lipids profile disruption in serum of anabolic implanted bovines

Glycerophospholipids have been highlighted as the major lipids class affected by trenbolone acetate/estradiol implant administration in bovines. Non-targeted hydrophilic interaction chromatography (HILIC) and reverse phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry have been successfully applied for characterizing lipid profile disruption in serum of implanted bovines. HILIC data pointed towards significant decrease of C22 fatty acids in implanted animals, i.e., C22:3 (p &lt; 0.05), C22:4 (p &lt; 0.05) and C22:6 (p &lt; 0.01), whilst RPLC data confirmed depletion of glycerophospholipids (phosphatidylglycerols, phosphatidylcholine, phosphatidic acid and phosphatidylethanolamine) with C22 fatty acid chains. Using these two complementary methods, complex lipids with the same alkyl chain have been putatively characterized and could further serve to understand the biological mechanism of illicit administration of trenbolone acetate/estradiol implant. These metabolites underpin glycerophospholipid metabolism as potential pathway, which was identified using metabolomic pathway analysis and MetExplore platforms. We hypothesized that implantation of exogenous androgenic and estrogenic steroids induces a depletion of glycerophospholipids with C22 FA chains, by decreasing high-density lipoproteins, the main transporters of glycerophospholipids in plasma

Specificity of monoclonal antibodies generated against arabinoxylans of cereal grains

TY - JOURInternational audienceXylooligosaccharides substituted by arabinose have been produced by degradation of wheat flour arabinoxylans with an endoxylanase. These oligosaccharides were coupled to carrier proteins (KLH and BSA) and three monoclonal antibodies were isolated. The specificity of antibody recognition was studied using arabino-xylo-oligosaccharides exhibiting different pattern of substitution by arabinose.ELISA competition tests and molecular modelling suggest that the conformation adopted by beta-(1->4) linked xylose residues is an antigenic determinant recognized by the different antibodies. Arabinose was not specifically involved in the interaction of antibody and epitope

Occurrence du Déchlorane plus et de composés apparentés chez des silures (Silurus spp.) provenant de rivières françaises

International audienceDechlorane related compounds (DRCs), including Dechlorane Plus (syn-DP and anti-DP), Dechlorane-601,-602, -603 and Chlordene Plus (CP), constitute a group of polychlorinated flame retardants (FRs) that are still of industrial use. In particular, DRCs have been detected in various nvironmental matrices and in different aquatic and terrestrial biota, thus exhibiting bioaccumulation and biomagnification potentials. The present study aimed at producing first occurrence data of a range of DRCs in Silurus spp. samples from different rivers located in France. Determination was carried out by gas chromatography high-resolution mass spectrometry after a sample clean-up based on a multilayer silica column and gel permeation chromatography. The concentration of monitored SDRCs ranged from 1.58 to 408 pg.g-1 wet weight (54 - 11100 pg.g-1 lipid weight). The fractional abundance of syn- and anti-DP stereoisomers was similar to that reported by other studies with an average equal to 0.60. Dec-601 was not detected in any sample. Detection frequencies ranged between 34 and 100% for other DRCs. Investigated correlations between DRCs and polychlorobiphenyls (PCBs) suggest a link with lipid content but independent contamination sources

Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves

Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based on mass spectrometry detection have been developed to ensure the control of such veterinary drug residues. Nevertheless, the use of “cocktails” composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention. New analytical tools able to detect such abuse are today mandatory. In this context, metabolomics may represent new emerging strategies for investigating the global physiological effects associated to a family of substances and therefore, to suspect the administration of steroids. The purpose of the present study was to set up, assess and compare two complementary mass spectrometry-based metabolomic strategies as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such approaches. The protocols were developed in two European laboratories in charge of residues analysis in the field of food safety. Apart from sample preparation, the global process was different in both laboratories from LC-HRMS fingerprinting to multivariate data analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-MetAlign strategies. The reproducibility of both sample preparation and MS measurements were assessed in order to guarantee that any differences in the acquired fingerprints were not caused by analytical variability but reflect metabolome modifications upon steroids administration. The protocols were then applied to urine samples collected on a large group of animals consisting of 12 control calves and 12 calves administrated with a mixture of 17ß-estradiol 3-benzoate and 17ß-nandrolone laureate esters according to a protocol reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid administration have been evaluated in this context and proved the suitability of the approach for discriminating anabolic treated animals from control ones. Such an approach may therefore open a new way for the screening of anabolic steroid administration through targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study

Toward a new comprehension of cheese ripening

Metabolic fingerprinting is an un targeted approach used in many scientific areas, which has shown potential to investigate food quality and safety . So far, this approach has not been applied to cheese .This study aimed to investigate the ability of mass spectrometry (MS) metabolic fingerprinting to characterizethe modifications induced by bacterial metabolism in cheese over time . Metabolic fingerprints were acquired after 0, 8 and 48 hours of incubation two different fractions of the cheese metabolome :i) a water -soluble fraction, analyzed using liquid chromatography - high resolution - MS and ii) a volatile fraction analyzedusing gas chromatography - MS . MS metabolic fingerprints were found to differsignificantly depending on the incubation time , pointing out the capacity of this approach to study the evolution of bacterial metabolismwithin cheese . Forty five metabolites were identified on the basis of an internal data bank [3]. Variations over time of a large diversity of well - known cheese metabolites such as 12 amino acids and 25 volatile compounds,but also less studied ones such as 4 vitamins and L-carnitine were highlighted. These results showed the relevance of cheese MS fingerprinting s to detect even slight differences and to possibly generate new insights on little - known cheese metabolites. As perspectives, these complementary untargeted “omic” approachescould be applied to explore strain biodiversity and the influence of ripening factors on microbial metabolis

Cert of Leadership Development for Education &amp; Training Managers

Bridging the divide: older learners and new technologies ICT and older learners: strategies and case studie