EFFECT OF SUSTAINED-RELEASE TRACE ELEMENT RUMINAL BOLUS ON PLASMA TRACE MINERAL PROFILES IN CAPTIVE BLESBOK ANTELOPES (DAMALISCUS PYGARGUS PHILLIPSI).

Nutritional deficiencies in mineral metabolism have been described or suspected in managed and wild ungulate populations. In blesboks (Damaliscus pygargus phillipsi), clinical signs of copper deficiencies have been described in the wild as well as in captivity. Plasma concentrations of cobalt (Co), copper (Cu), iodine (I), manganese (Mn), selenium (Se), and zinc (Zn) were measured over a 6-mon period by inductively coupled plasma mass spectrometry in two groups of five apparently healthy blesboks from a single zoological collection. The control group did not receive any treatment, whereas animals from the treatment group were given an oral drench in October with two sustained-release trace element ruminal boluses (Oligovet ovin-caprin 6 g bolus, Vetalis, 16100 Château Bernard, France). Plasma samples were obtained prior to the start of treatment (October) and in November, February, and April following treatment. No significant differences were found between treatment and control groups for any of the measured minerals over the course of the study. The plasma concentrations of Co, Cu, Se, and Zn were significantly different (P < 0.05) over time for all individuals, but this effect could not be linked to a change in the diet or husbandry. Copper plasma values fluctuated between deficient and normal ranges for cattle. Zinc plasma values were within a range consistent with deficiency in cattle. The great variability of these results should prompt caution in the interpretation of the efficacy of oral trace mineral intake or the expected effect of a dietary modification on trace mineral status based on plasma values.

Atypical actinobacillosis affecting hind limbs and lungs in a single beef cattle herd.

Actinobacillosis usually is a sporadic infection that affects the tongue in cattle ("wooden tongue") with possible spread to the digestive tract. Two 4-year-old Rouge-des-Prés cows from a single French beef herd were referred for chronic (2-6 months) swelling and cutaneous nodules in the distal hind limbs. In addition to cutaneous signs, physical examination disclosed cachexia, lameness, lymphadenitis of the hind limbs, and pneumonia in both cows. Cytologic examination of direct skin smears was inconclusive, and no parasites were observed in examination of multiple skin scrapings. Histopathological examination of skin and lung biopsy specimens identified chronic, diffuse, severe pyogranulomatous dermatitis, associated with Splendore-Hoeppli phenomenon and intralesional Gram-negative bacteria. Cultures from skin, lymph nodes, and lungs (both cows were euthanized for welfare reasons) identified a Pasteurellaceae organism, confirmed as Actinobacillus lignieresii by partial sequencing of the rpoB gene. This report emphasizes that actinobacillosis can appear as a small outbreak in cattle with cutaneous and respiratory signs.

Comprehensive steroid profiling by liquid chromatography coupled to high resolution mass spectrometry.

A steroidomics workflow has been developed in the objective of monitoring a wide range (n >150) of steroids in urine. The proposed workflow relies on the optimization of an adequate SPE extraction step followed by an UHPLC-HRMS/MS simultaneous analysis of both free and conjugated forms of C18, C19 and C21 steroid hormones. On the basis of 44 selected steroids, representative of main classes of steroids constituting the steroidome, the performances of the developed workflow were evaluated in terms of selectivity, repeatability (< 13%) and linearity (R2> 0.985 in the concentration range [0.01-10 ng/mL]). As metabolites identification and characterization constitute the bottleneck of such profiling approaches, a homemade database was created encompassing a large number of characterized free and conjugated steroids (n> 150) for putative steroid-like biomarkers identification purposes. The efficiency of the workflow in highlighting fine modifications within the urinary steroidome was assessed in the frame of an anabolic treatment involving an intra-muscular administration of boldenone undecylenate (2 mg/kg) to veals (n=6) and the investigation of potential steroid biomarkers. Besides monitoring known phase II metabolites of boldenone in the bovine specie, namely, boldenone glucuronide and sulfate, the applied strategy also permitted to observe, upon boldenone administration, a modified profile of epiboldenone glucuronide. Furthermore, 31 signals corresponding to non-identified steroid species could also be highlighted as impacted upon the exogenous steroid treatment. This study is the first to simultaneously investigate both free and conjugated C18, C19 and C21 steroid hormones in their native form using UHPLC-HRMS/MS and allowing their comprehensive profiling. This strategy was probed in-vivo.

Specific characterization of non-steroidal selective androgen peceptor modulators using supercritical fluid chromatography coupled to ion-mobility mass spectrometry: application to the detection of enobosarm in bovine urine.

Currently under development for therapeutic purposes in human medicine, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth associated pathways. As such, they present a potential for abuse in sports and food-producing animals as interesting alternative anabolic substances. Forbidden since 2008 by the World Anti-Doping Agency (WADA) these compounds are however easily available and could be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detection of these compounds in biological matrices. Using an innovative analytical platform constituted of supercritical fluid chromatography coupled to ion mobility-mass spectrometry, the present study enabled efficient separation and identification in urine of 4 of these drugs (andarine, bicalutamide, hydroxyflutamide, and enobosarm) in accordance with European Union criteria (Commission Decision 2002/657/EC). Besides providing information about compounds structure and behaviour in gas phase, such a coupling enabled reaching low limits of detection (LOD < 0.05 ng.mL-1 for andarine and limits of detection < 0.005 ng.mL-1 for the three others) in urine with good repeatability (CV < 21 %). The workflow has been applied to quantitative determination of enobosarm elimination in urine of treated bovine (200 mg, oral). Copyright © 2016 John Wiley & Sons, Ltd.

Selective androgen receptor modulators: comparative excretion study of bicalutamide in bovine urine and faeces.

Besides their development for therapeutic purposes, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth-associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food-producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non-steroidal SARM used in human treatment of non-metastatic prostate cancer because of its anti-androgenic activity exhibiting no anti-anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd.

Analytical strategies to detect enobosarm administration in bovines.

Selective androgen receptor modulators (SARMs) are a novel class of androgen receptor ligands. They are intended to exhibit the same kind of effects as androgenic drugs, like anabolic steroids, but be much more selective in their action, targeting particular tissues without any undesirable effects on others. While the main applications of these synthetic substances are for therapeutic purposes, they also have a high potential for misuse in veterinary practice and the sporting world. In order to guarantee for consumers with food from animal origin that it is free from any residues of such compounds, analytical strategies are required to ensure safe food and also to enable fair trade between producers. In this context an animal experiment involving bovines administered with enobosarm was conducted to provide the study with biological matrices. Different animal matrices (urine and faeces) were investigated to select the most appropriate matrix for use for control purposes, in terms of metabolite relevance and detection time window. Based on ultra-high-pressure liquid chromatography (UHPLC) with tandem mass spectrometry (LC-MS/MS) this work highlighted the presence of sulfonated and glucuronated-conjugated forms of the molecule in the urine of treated animals. Enobosarm could be detected in urine up to 9 days after the administration when samples underwent phase II hydrolysis. Faeces was demonstrated to be the main matrix of excretion of enobosarm since values up to 500 times higher compared with urine could be detected for 21 days. There was no difference between the kinetic profiles when a deconjugation step was or was not was applied.

Laparoscopic Evaluation of Umbilical Disorders in Calves.

OBJECTIVE: To describe a laparoscopic technique for evaluating umbilical disorders in calves, including feasibility, visualization of umbilical structures, and related complications. STUDY DESIGN: Prospective clinical study. ANIMALS: Male calves (15 Holstein, 2 Montbeliard) with umbilical disorders (n=17). METHODS: Calves <2 months old with obvious umbilical disease were assessed by clinical examination and ultrasonography of the umbilical structures. Laparoscopic evaluation was performed in dorsal recumbency under subarachnoid lumbosacral anesthesia and sedation. An open insertion technique with short 60 mm cannulas was used after creating 2 portals 10 cm cranial to the umbilicus (one 5 cm left of midline for the laparoscope and one 5 cm right of midline as an instrument portal). After laparoscopy, abnormal tissues were resected by laparotomy during the same anesthetic period. RESULTS: Laparoscopic evaluation of umbilical structures was performed quickly (mean surgery time 7.1 ± 2.5 minutes). Umbilical structures could be completely visualized in all calves without intraoperative complications. In addition to abnormalities previously detected on ultrasound, laparoscopy enabled detection of adhesions 7 calves that were not suspected on ultrasound, as well as focal enlargements of the umbilical arteries and urachus close to the bladder in 5 calves. Laparoscopy failed to detect abnormalities observed with ultrasound or laparotomy in 4 calves, including small hernias and omphalitis. CONCLUSION: Laparoscopic evaluation of umbilical structures was performed safely and quickly in young calves and allowed complete evaluation of intra-abdominal umbilical structures and may, therefore, be a useful adjunct to physical examination and ultrasound to fully assess the abdomen in calves.

Tuberculoid nodular thelitis in a dairy goat flock.

An unusual outbreak of teat/udder skin lesions occurred in a dairy goat flock in France. Lesions first appeared as circular, indurated, erythematous areas of skin and progressed to form dark raised haemorrhagic crusts and ulcerative plaques. Histopathological examination revealed marked granulomatous dermatitis with multifocal ulceration. The granulomatous inflammation, with frequent Langhans type multinucleated cells and central caseous necrosis, was indicative of mycobacterial infection. The presence of non-cultivable mycobacteria was confirmed by sequencing PCR products from DNA extracted directly from the lesions and sequences matched a novel mycobacterial pathogen closely related to M. leprae and M. lepromatosis and previously identified in cattle thelitis. The association of nodular gross lesions and tuberculoid granulomas on the teat and lower udder, and the presence of mycobacteria DNA support a diagnosis of tuberculoid nodular thelitis in goats due to mycobacterial infection.

C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle.

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes.

Clinical biochemical and hormonal profiling in plasma: a promising strategy to predict growth hormone abuse in cattle.

Recombinant bovine somatotrophin (rbST) is widely used in some countries to increase milk production. Since 1994, both marketing and use of this substance have been prohibited within the European Union. In this context, the targeted plasma biochemical and hormonal profiling was assessed as a potential screening strategy to highlight rbST (ab)use in cattle. Twenty-one routinely measured clinical blood parameters, representative of main biological profiles (energetic, proteic, etc.), were measured in the plasma of six lactating cows before and after rbST treatment throughout a 23-day study period. Appropriate multivariate statistical analyses [principal component analysis (PCA) and orthogonal partial least square (OPLS)] enabled discriminating animal samples before and after treatment (days 0 vs. 2 to 9, P = 2.10(-9)) and highlighted the five most relevant blood parameters in this discrimination. Based on each five-analyte contribution, a simple mathematically weighted equation was suggested to predict the status of samples. A suspicious threshold was proposed, and the model was further tested with the status prediction of the supplementary samples from untreated (n = 20) and treated cows (n = 22). The calculated false-positive (10%) and false-negative (4.5%) rates were in accordance with the EU requirements for screening methods. Although the model needs to be further validated with additional samples, such targeted plasma biochemical and hormonal profiling already appears as a potential promising screening strategy to highlight rbST (ab)use in cattle.

Gas chromatography coupled to mass spectrometry-based metabolomic to screen for anabolic practices in cattle: identification of 5α-androst-2-en-17-one as new biomarker of 4-androstenedione misuse.

The use of anabolic steroids as growth promoters for meat-producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC-MS aiming to reveal potential biomarkers signing a fraudulent administration of 4-androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono-conjugates urinary metabolites were acquired by GC-MS in the full-scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4-AED. After a first evaluation of the signal robustness using additional and independent non-compliant samples, 17 steroid-like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC-MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α-androstane-3ß,17α-diol and 5α-androst-2-en-17-one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle.

Screening of 4-androstenedione misuse in cattle by LC-MS/MS profiling of glucuronide and sulfate steroids in urine.

The use of anabolic agents in food producing animals is prohibited within the European Union since 1988. The illegal use of natural steroid hormones control is however still a current challenge, especially regarding the limitations of existing screening methods. In this context, the present study aimed to develop a new screening approach based on the emerging 'untargeted profiling' concept, but with a special emphasis on steroids phase II conjugated metabolites, in the scope of revealing potential biomarkers signing a fraudulent administration of 4-androstenedione. After extraction and separation of the urinary glucuronide and sulfate steroid fractions, each one was analyzed separately by UPLC-MS/MS using the precursor ion scan acquisition mode. This approach was carried out in order to monitor product ion characteristic of sulfate (m/z 97) and glucuronide (m/z 113) functional groups, and then to fish for any potential conjugated steroid leading to these ionic species after fragmentation. After statistical analysis, 86 metabolites (33 from steroid compounds and 53 from other unknown substances) were highlighted as potential biomarkers of 4-androstenedione abuse. After application of several robustness criteria, 26 metabolites (whom 5 were unambiguously structurally identified), were finally selected to build a statistical model which could be used as new diagnostic tool for screening purposes.

Targeted phase II metabolites profiling as new screening strategy to investigate natural steroid abuse in animal breeding.

The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new "fishing" strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC-MS/MS in MRM mode showing a rapid (between 4h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate.

A new reliable sample preparation for high throughput focused steroid profiling by gas chromatography-mass spectrometry.

The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography-mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5ß-androstanedione, 6α-hydroxyestradiol and 7ß-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 µg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5-15 µg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10-75 µg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3ß,17α-diol, 5ß-androstane-3α,17α-diol and 5-androstene-3ß,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC-MS based steroid profiling and metabolomic.

Elimination kinetic of recombinant somatotropin in bovine.

Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form. In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC-ESI(+)-MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL(-1) during the kinetic.