Assessment of Antimicrobial Effects on Broiler Gut Barrier Through Histopathology and Immunohistochemistry of Tight-Junction Proteins.

In recent years, antimicrobial (AM) use in poultry farming has been attracting attention worldwide mainly due to AM resistance spreading. The role of AM prophylaxis in the modulation of gut microbiota, as well as of gut health, is still not clearly understood. Therefore, this study aimed to investigate the role of different prophylaxis protocols in the modulation of the gut barrier in broilers by applying a histopathological approach. Intestinal tissue samples were collected from a total of 240 male broilers (Ross 306), reared and treated with different AM protocols. Haematoxylin and Eosin (HE) staining and a multiple scoring system were used to evaluate the presence of lesions in ileum, cecum and colon of treated broilers. Moreover, immunohistochemistry (IHC) was performed to assess the expression of claudin-3 and ZO-1 proteins in intestinal tissues. The application of a semi-quantitative scoring system was used in IHC stained samples. HE results revealed that intestinal tissues were mainly characterized by epithelial detachment and fusion of the intestinal villi, but also by the presence of lymphocytic infiltrate in the mucosa and submucosa of AM-treated broilers. However, the IHC approach for the evaluation of claudin-3 and ZO-1 proteins showed that their expression was not affected by the different AM treatments. Nevertheless, the presence of intestinal lesions highlighted by histopathology suggests that AM treatments could harm the gut health of broilers, inducing an inflammatory response and consequent epithelial lesions. In order to clarify the role of AM treatments in the modulation of gut barrier in broilers, further studies are needed.

Tissue and species identification in minced meat and meat products from Italian commercial markets by DNA microarray and histological approach.

Adequate testing and adulterant detection of food products are required to assure its safety and avoid fraudulent activities. Adulteration/substitution of costlier meat with a cheaper or inferior meat is one of the most common fraudulence in meat industry. Aim of this study was to check the correct labelling of meat and ready to cook bovine meat products, combining the DNA microarray approach to identify the animal species with the histological examination, to check the composition and safety of meat. One hundred and one samples of bovine minced meat (Group 1) and ready to cook meat products (Group 2) were collected from supermarkets in Turin, Italy. DNA microarray revealed that 25.7% of samples were positive for species not declared on the label, swine being the most common. Histology showed the presence of cartilage, bone and glandular tissue. A higher presence of bacteria and inflammatory cells was detected in Group 1. Bacterial cells associated to inflammatory cells were detected with a higher score in Group 2. Sarcocystis spp. were present in 83.3% samples of Group 1 and 49.1% of Group 2. This study confirmed that the mislabelling of meat products is not uncommon. The combination of DNA microarrays and histology can increase the monitoring capacity in bovine meat industry.

Molecular Evidence of Bartonella spp. in Rodents: A Study in Pianosa Island, Italy.

Wild rodents are reservoirs of several Bartonella species that cause human bartonellosis. The aim of this study was to assess the presence of Bartonella spp. DNA in wild rodents in Pianosa island, Italy. Rats (Rattus spp.; n = 15) and field mice (Apodemus spp.; n = 16) were captured and spleen DNA tested for the presence of Bartonella spp. by means of an initial screening using a qPCR amplifying a short segment of the 16S-23S rRNA gene intergenic transcribed spacer region (ITS, ~200 bp) followed by conventional PCR amplification of a longer ITS fragment (~600 bp) and of a citrate synthase (gltA, ~340 bp) gene segment. A total of 25 spleen DNA samples obtained from 31 rodent carcasses (81%) yielded positive qPCR results. Bartonella genus was confirmed by amplicon sequencing. By conventional PCR, eight out of 25 samples (32%) yielded bands on gels consistent with ITS segment, and 6/25 (24%) yielded bands consistent with the gltA locus. Amplicon sequencing identified B. henselae and B. coopersplainsensis in 1/25 (4%), and 4/25 (16%) samples, respectively. Moreover, 5/25 (20%) of Bartonella spp. positive samples showed gltA sequences with about 97% identity to B. grahamii. These results provide support to recently published observations suggesting that B. henselae circulates in wild rodent populations.

FKBP5 gene expression in skeletal muscle as a potential biomarker for illegal glucocorticoid treatment in veal calves.

For the current European legislation, the chemical analysis of drug residues is the exclusive accepted method to identify animals illicitly treated with growth promoters. Glucocorticoids and their metabolites are no detectable by LC/MS-MS methods in biological fluids when the growth promoter administration is discontinued several days prior to the slaughtering. The aim of this study was to elucidate the effect on the expression of genes belonging to the glucocorticoid pathway in three types of skeletal muscle of calves treated with prednisolone or dexamethasone in combination with estradiol. A gene expression change of glucocorticoid receptors (NR3C1 and NR3C2), their chaperones molecules (FKBP prolyl isomerase 4 and 5, FKBP4 and 5) and pre-receptor system (hydroxysteroid 11-beta dehydrogenases 1 and 2, HSD11B1 and 2) may indicate potential biomarkers of glucocorticoid treatment. In the biceps brachii muscle, the administration of dexamethasone with estradiol increased HSD11B2 (P < 0.01) and NR3C2 (P < 0.01) gene expression, whereas prednisolone administration increased HSD11B1 transcript levels (P < 0.05). In the longissimus lumborum muscle, NR3C2 gene expression decreased following prednisolone administration (P < 0.05). FKBP5 gene expression decreased in all considered muscles of calves administered with dexamethasone and estradiol (P < 0.01), whereas increased in the longissimus lumborum (P < 0.01) and vastus lateralis (P < 0.05) muscle of prednisolone-treated group (P < 0.05). The opposite effect of dexamethasone and prednisolone appears very promising to develop a low-cost screening test, because the expression analysis of a unique gene in a given tissue may distinguish the dispensed molecules.

Conscientious Objection to Animal Testing: A Preliminary Survey Among Italian Medical and Veterinary Students.

The use of animals for educational and research purposes is common in both veterinary and human medicine degree courses, and one that involves important ethical considerations. The aim of this study was to assess the extent of differences between the knowledge and attitudes of veterinary students and medical students on animal bioethics, on alternative strategies and on their right to conscientiously object to animal experimentation. To this end, a questionnaire was completed by 733 students (384 human medicine students (HMS) and 349 veterinary medicine students (VMS)). VMS were more aware than HMS (72.2% and 59.6%, respectively) of the existence of an Italian law on the right to conscientiously object to animal experimentation. However, very few of them had exercised this right. Many VMS (43.3%) felt that animal bioethics courses should be mandatory (only 17.4% of HMS felt the same way). More VMS than HMS (81.7% and 59.1%, respectively) expressed an interest in attending a course on alternatives to animal experimentation. The data suggest the need for appropriate educational interventions, in order to allow students to make choices based on ethical principles. Fostering close collaborations between departments of human medicine and veterinary medicine, for example, through shared study modules, could promote the development of ethical competence as a basic skill of students of both veterinary and human medicine courses.

A survey on zoo mortality over a 12-year period in Italy.

BACKGROUND: The zoo is a unique environment in which to study animals. Zoos have a long history of research into aspects of animal biology, even if this was not the primary purpose for which they were established. The data collected from zoo animals can have a great biological relevance and it can tell us more about what these animals are like outside the captive environment. In order to ensure the health of all captive animals, it is important to perform a post-mortem examination on all the animals that die in captivity. METHODS: The causes of mortality of two hundred and eighty two mammals which died between 2004 and 2015 in three different Italian zoos (a Biopark, a Safari Park and a private conservation center) have been investigated. RESULTS: Post mortem findings have been evaluated reporting the cause of death, zoo type, year and animal category. The animals frequently died from infectious diseases, in particular the causes of death in ruminants were mostly related to gastro-intestinal pathologies. pulmonary diseases were also very common in each of the zoos in the study. Moreover, death was sometimes attributable to traumas, as a result of fighting between conspecifics or during mating. Cases of genetic diseases and malformations have also been registered. DISCUSSION: This research was a confirmation of how conservation, histology and pathology are all connected through individual animals. These areas of expertise are extremely important to ensure the survival of rare and endangered species and to learn more about their morphological and physiological conditions. They are also useful to control pathologies, parasites and illnesses that can have a great impact on the species in captivity. Finally, this study underlines the importance of a close collaboration between veterinarians, zoo biologists and pathologists. Necropsy findings can help conservationists to determine how to support wild animal populations.

A two-step immunomagnetic separation of somatic cell subpopulations for a gene expression profile study in bovine milk.

The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

Gene expression profile associated with thymus regeneration in dexamethasone-treated beef cattle.

Glucocorticoids (GCs) are illegally used as growth promoters in cattle, and the analytical methods officially applied most likely underestimate the precise frequency of the abuse. As a side effect, the administration of GCs causes fat infiltration, apoptosis, and atrophy of the thymus. However, gross and histological observations carried out previously showed that the thymus preserves an intrinsic ability to regenerate. The aim of this work was to study the transcriptional effects of GCs on genes likely involved in regeneration of the epithelial cell network in the cervical and thoracic thymus of beef cattle treated with dexamethasone (DEX) or prednisolone (PRD) in comparison with a control group. Moreover, the ratio of bax/bcl2 genes was examined to verify a possible antiapoptotic activity occurring at the same time. In the cervical thymus, DEX administration increased the gene expression of c-myc (P < 0.01), tcf3 (P < 0.05), tp63 (P < 0.01), and keratin 5 (krt5; P < 0.01). In the thoracic thymus of DEX-treated cattle, the gene expression of tcf3 (P < 0.01), tp63 (P < 0.01), and krt5 (P < 0.05) was increased. These results suggested that thymic regeneration is underway in the DEX-treated animals. However, the bax/bcl2 ratio was decreased in both cervical and thoracic thymus of DEX-treated cattle (P < 0.01 and P < 0.05, respectively), showing an antiapoptotic effect through the mitochondrial pathway. Conversely, PRD administration caused no change in the expression of all considered genes. These results sustain the hypothesis that regeneration occurs in the thymus parenchyma 6 d after the DEX treatment was discontinued. This hypothesis is also supported by the absence of alterations in the thymus of PRD-treated beef cattle. Indeed, previous studies showed the inability of PRD to induce macroscopic and microscopic lesions in the thymus. Therefore, in this context, it is not surprising that PRD induced no alteration of genes involved in the regeneration pathway.

Reference Gene Selection and Prednisolone Target Gene Expression in Adipose Tissues of Friesian Cattle.

Corticosteroids are frequently used in livestock production, and their use is permitted by the European Union for therapeutic purposes only. However, small doses of corticosteroids are often administered in meat-producing animals to improve zootechnical performance. Prednisolone is one of the most commonly used corticosteroids with a growth-promoting purpose in animal husbandry. This study proposes to identify a gene whose expression is significantly regulated by prednisolone in visceral and subcutaneous adipose tissues. The analysis was conducted on Friesian cattle treated with prednisolone (30 mg day-1). The reference gene expression stability and optimal number for gene expression normalization were calculated. Family with sequence similarity 107 member A (FAM107A) and pyruvate dehydrogenase kinase 4 are the prednisolone target genes identified in adipose tissue. FAM107A was downregulated by ∼2.9-fold by prednisolone in subcutaneous adipose tissue. This result suggests that FAM107A could be a possible indirect biomarker of prednisolone treatment in cattle and encourages a deeper investigation in this direction.

Development and Application of a Screening Method of Absolute Quantitative PCR To Detect the Abuse of Sex Steroid Hormone Administration in Male Bovines.

A methodology for the absolute quantification of regucalcin gene through quantitative PCR was set up to confirm that the decrease of regucalcin gene expression in the testis is an effective biomarker for tracing sex steroid hormone treatment in bovine husbandry. On the basis of TaqMan technology, an external standard curve was generated. Using in vivo experiments, a ROC curve was developed to calculate the criterion value, specificity, and sensitivity for this potential biomarker. Then, regucalcin gene expression was assessed in veal calves and beef intended for human consumption. In 11 of 54 calves and in 5 of 70 beef cattle the regucalcin gene was expressed under their respective cutoff. Additionally, a mild decrease of regucalcin protein expression was revealed by immunohistochemistry in subjects tested positive via qPCR. These preliminary results suggest that this transcriptomics test may be employed as a novel diagnostic screening tool, improving significantly the overall efficacy of food control.

17ß-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes.

BACKGROUND: The endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17ß-estradiol. METHODS: The skeletal muscle cell line C2C12 was treated with 17ß-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes. RESULTS: It was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17ß-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17ß-estradiol and oxytocin. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism. DISCUSSION: Oxytocin and its receptor are physiologically regulated along differentiation. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17ß-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism.

Effects and detection of Nandrosol and ractopamine administration in veal calves.

The present study describes different effects of the selective androgen receptor modulator (SARM) nandrolone phenylpropionate (Nandrosol) and the ß-agonist ractopamine administration in veal calves, and it investigates different strategies applied to trace these molecules. Morphological changes of gonads and accessory glands attributed to androgen effects, such as testicular atrophy, seminiferous tubule diameter reduction and hyperplasia of prostate epithelium, were detected, although SARMs are not described to cause these lesions. The gene expression analysis showed an anabolic activity of Nandrosol in Longissimus dorsi muscle, where myosin heavy chain (MYH) was significantly up-regulated. An IGF1 increase was weakly significant only in Vastus lateralis muscle. In conclusion, the anatomo-histopathological observations and the MYH mRNA up-regulation in Longissimus dorsi muscle confirm the androgenic treatment in experimental animals. The biosensor assay was not enough sensitive to detect residues in urines and only the direct chemical analysis of urine samples confirmed both ß-agonist and SARM treatment.

Bovine teeth as a novel matrix for the control of the food chain: liquid chromatography-tandem mass spectrometry detection of treatments with prednisolone, dexamethasone, estradiol, nandrolone and seven ß2-agonists.

Veterinary drugs usually have rapid clearance rates in the liver and kidney, hampering their detection in conventional matrices such as the liver or urine. Pharmacological principles such as esterification may be applied to facilitate the administration of veterinary drugs and increase drug half-life. Prednisolone, whose therapeutic administration is regulated for food producing animals in the EU, is available in its acetate form as well as nandrolone, a banned anabolic steroid, which may be obtained as nandrolone phenylpropionate and estradiol as a benzoyl ester. While the distribution and accumulation of lipophilic and hydrophilic substances in human teeth have been well documented, studies on residues in bovine teeth are lacking. We hypothesised that analysis of bovine teeth could be used to detect both regulated and banned veterinary drugs. Steroids may be illegally used as growth promoters in food producing animals, alone or combined with ß2-agonists; therefore, we developed, and validated, in accordance with the Commission Decision 2002/657/EC, two analytical confirmatory LC-MS/MS methods to detect these classes of compounds following a unique liquid extraction procedure. Finally, we analysed teeth from three male Friesian veal calves treated with intramuscular estradiol benzoate, oral prednisolone acetate or intramuscular nandrolone phenylpropionate in combination with oral ractopamine, respectively, and from seven bovines from the food chain. Teeth from treated animals were positive for their respective drugs, with the exception of nandrolone phenylpropionate. One sample from a food chain bovine was positive for isoxsuprine, one of the seven ß2-agonists studied. Non-esterified forms of the steroids were not found. These results demonstrate that bovine teeth are a suitable matrix for the determination of pseudoendogenous substances or illicit administration of veterinary drugs.

Morphological Examination and Transcriptomic Profiling To Identify Prednisolone Treatment in Beef Cattle.

In livestock production corticosteroids are licensed only for therapy; nevertheless, they are often illegally used as growth promoters. The aim of this study was to identify morphological or biomolecular alterations induced by prednisolone (PDN) in experimentally treated beef cattle, because PDN and its metabolites are no longer detectable by LC-MS/MS methods in biological fluids. Moreover, PDN does not induce any histological alterations in the thymus, different from dexamethasone treatments. Therefore, a marker of illicit treatment for this growth promoter could be useful. Eight male Italian Friesian beef cattle were administered prednisolone acetate 30 mg day-1 per os for 35 days, and seven beef cattle represented the control group. Six days after drug withdrawal, the animals were slaughtered. Morphological and morphometric modifications were evaluated in the epididymis and testis, whereas transcriptomic changes induced by PDN administration were investigated in peripheral blood mononuclear cells (PBMCs) at different sampling times and in skeletal muscle and testis sampled at slaughtering. In the epididymis, spermatozoa number decreased in PDN-treated animals, and in some cases they were totally absent. Correspondingly, in the testis of treated animals, down-regulation for serine/threonine kinase 11 (STK11) gene expression was detected (p < 0.01). DNA microarray analysis revealed a total of 133 differentially expressed genes in skeletal muscle and testis, and 907 and 1416 in PBMCs after 33 days of treatment and at slaughtering, respectively. Histological investigations on epididymal content could represent a promising marker for PDN treatment in beef cattle and could be used as a screening method to identify animals worthy of further investigation with official methods. Moreover, the clear transcriptomic signature of PDN treatment evidenced in PBMCs supported the possibility of using this matrix to monitor the illicit treatment in vivo during ranching.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment.

The effects of long-term administration of low doses of dexamethasone (DX) and prednisolone (PL) on the metabolism of endogenous corticosteroids were investigated in veal calves. In addition to cortisol (F) and cortisone (E), whose interconversion is regulated by 11ß-hydroxysteroid dehydrogenases (11ßHSDs), special attention was paid to tetrahydrocortisol (THF), allo-tetrahydrocortisol (aTHF), tetrahydrocortisone (THE) and allo-tetrahydrocortisone (aTHE), which are produced from F and E by catalytic activity of 5α and 5ß-reductases. A specifically developed HPLC-ESI-MS/MS method achieved the complete chromatographic separation of two pairs of diastereoisomers (THF/aTHF and THE/aTHE), which, with appropriate mass fragmentation patterns, provided an unambiguous conformation. The method was linear (r(2) > 0.9905; 0.5-25 ng ml(-1)), with LOQQ of 0.5 ng ml(-1). Recoveries were in range 75-114%, while matrix effects were minimal. The experimental study was carried out on three groups of male Friesian veal calves: group PL (n = 6, PL acetate 15 mg day(-1) p.o. for 31 days); group DX (n = 5, 5 mg of estradiol (E2) i.m., weekly, and 0.4 mg day(-1) of DX p.o. for 31 days) and a control group (n = 8). Urine was collected before, during (twice) and at the end of treatment. During PL administration, the tetrahydro-metabolite levels decreased gradually and remained low after the suspension of treatment. DX reduced urinary THF that persisted after the treatment, while THE levels decreased during the experiment, but rebounded substantially after the DX was withdrawn. Both DX and PL significantly interfered with the production of F and E, leading to their complete depletion. Taken together, the results demonstrate the influence of DX and PL administration on 11ßHSD activity and their impact on dysfunction of the 5-reductase pathway. In conclusion, profiling tetrahydro-metabolites of F and E might serve as an alternative, indirect but reliable, non-invasive procedure for assessing the impact of synthetic glucocorticosteroids administration.

A Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Antimicrobial Agents from Seven Classes in Calf Milk Replacers: Validation and Application.

Calf milk replacers are low-cost feeds that contain available, digestible protein. During their reconstitution, however, the addition of drugs, such as antibiotics, could make them a very simple route for illicit treatment for therapeutic, preventive, or growth-promoting purposes. We developed an HPLC-MS/MS method, preceded by a unique extraction step, able to identify 17 antibiotics from seven classes (penicillins, tetracyclines, fluoroquinolones, sulfonamides, cephalosporins, amphenicols, and lincosamides) in this matrix. Prior to solid phase extraction (SPE), the sample underwent deproteinization and defatting. The method was fully validated according to Commission Decision 2002/657/EC. Decision limits (CCα) and detection capability (CCß) were in the ranges of 0.13-1.26 and 0.15-1.47 ng/mL, respectively. Thirty-eight samples were finally analyzed, showing the occasional presence of marbofloxacin (six samples) and amoxicillin (one sample).

An ethics for the living world: operation methods of Animal Ethics Committees in Italy.

INTRODUCTION AND OBJECTIVES: Coinciding with the recent implementation in Italy of the "Directive 2010/63/EU, regarding the protection of animals used for scientific purposes", the Authors would like to analyse the topic of the introduction of ethical committees for animal experimentation in Italy. This paper furthermore aims to underline some critical aspects concerning the actions taken by Italian institutions to comply with the provisions of EU. RESULTS AND DISCUSSION: The implementation of the recent Italian law (Decreto Legislativo n. 26 on 4 March 2014 Implementation of the Directive 2010/63/EU on the protection of animals used for scientific purposes) leans towards a restrictive interpretation of the European provisions about composition and responsibilities of "Ethical Committee for Animal Experimentation". In the composition of the bodies mentioned, we note a tendency to restrict the composition to few professional figures contemplated by Italian law, without guaranteeing the independence of each committee; also, an absence of hierarchical relationship between a research institution and his committee is apparent. Moreover, a critical aspect is the lack of decision-making powers of these new organisms in terms of ethical evaluation of protocols and research projects. CONCLUSIONS: What EU legislation imposes on the member states is to set up an animal-welfare body (art. 26). This represents a strong incentive for Italy to follow the steps of many other European Countries, where ad hoc ethical committees have been working for a long time. The proper functioning of these bodies may contribute to guarantee the safety and welfare of the animals inside the laboratories, and to balance the protection of animal life and the interests of research.

Regucalcin Expression as a Diagnostic Tool for the Illicit Use of Steroids in Veal Calves.

It has been previously demonstrated that sex steroid hormone treatment down-regulates regucalcin gene expression in the accessory sex glands and testis of prepubertal and adult male bovines. The aim of this study was to investigate whether low doses of sex steroid hormones combined with other drugs significantly affect regucalcin gene expression in the accessory sex glands and testis of veal calves. The regucalcin expression was down-regulated in the bulbo-urethral glands of estrogen-treated calves, whereas it was up-regulated in the prostate of estrogen-treated calves. Only the testis of androgen-treated calves showed a down-regulation of the regucalcin expression. Thus, the administration of sex steroid hormones, even in low doses and combined with other molecules, could affect regucalcin expression in target organs. Particularly, the specific response in the testis suggests regucalcin expression in this organ as a first molecular biomarker of illicit androgen administration in bovine husbandry.

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle.

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17ß-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50% (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC-MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.

Pseudoendogenous presence of ß-boldenone sulphate and glucuronide in untreated young bulls from the food chain.

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is considered fully compliant up to 2 ng ml(-1). Greater attention has been placed on ß-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and ß-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of ß-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of ß-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that ß-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a frequency of 78% and 46%, respectively, showing the endogenous origin of boldenone.