Milk fat miRNome changes in response to LPS challenge in Holstein cows.

Mastitis is an inflammatory disease in dairy cows, causing economic losses and reducing animal welfare. In order to contribute for the discovery of early and noninvasive indicators, our objective was to determine the effects of a lipopolysaccharide (LPS) challenge on the microRNA profile (miRNome) of milk fat, using microarray analyses in cows. Cows were fed a lactation diet at ad libitum intake (n = 6). At 27 ± 3 days in milk, cows were injected with 50 µg of LPS Escherichia coli in one healthy rear mammary quarter. Milk samples were collected just before LPS challenge (LPS-) and 6.5 h after LPS challenge (LPS +) from the same cows. Microarray analysis was performed using customized 8 × 60 K ruminant miRNA microarrays to compare LPS- to LPS + miRNome. In silico functional analyses were performed using OmicsNet and Mienturnet software. MiRNome comparison between LPS- and LPS + identified 37 differentially abundant miRNAs (q-value ≤ 0.05). The predicted target genes of the 37 differentially abundant miRNAs are mostly involved in cell life including apoptosis, cell cycle, proliferation and differentiation and in gene expression processes. MiRNome analyses suggest that miRNAs profile is related to the inflammation response of the mammary gland. In conclusion, we demonstrated that milk fat might be an easy and rapid source of miRNAs that are potential indicators of early mastitis in cows.

Evaluation of the biofilm-forming ability and molecular characterization of dairy Bacillus spp. isolates.

Food processing lines represents a suitable environment for bacterial biofilm formation. One of the most common biofilm-forming genera in dairy processing plants is Bacillus, which includes species that may have a negative impact on safety and/or quality of dairy products. In the current study, we evaluated the biofilm forming ability and molecular characteristics of dairy Bacillus spp. isolates (B. cereus and B. subtilis). Reference strains (B. cereus ATCC 14579 and B. subtilis NCTC 3610) were also included in the experiment. All isolates were screened by micro-titer plate (96 wells) to assess their ability to form biofilm. Then, they were tested on two common food contact surfaces (polystyrene and stainless steel) by using 6-well plates and AISI 316 stainless steel coupons. Biofilm formation, expressed as biofilm production index (BPI), was higher on polystyrene than stainless steel (except for B. cereus ATCC 14579). These observations were further confirmed by scanning electron microscopy, which allowed the microscopy observation of biofilm structure. Moreover, a possible correlation among total viable cell counts (CFU) and BPI was examined, as well as a connection among biofilm formation and bacterial cell hydrophobicity. Finally, whole genome sequencing was performed highlighting a genetic similarity among the strains belonging to the same species. The presence of selected genes involved in biofilm formation was also examined showing that strains with a greater presence of these genes were able to produce more biofilm in the tested materials. Additionally, for B. cereus strains enterotoxin genes were detected.

MALDI-TOF mass spectrometry profiling of bovine skim milk for subclinical mastitis detection.

Introduction: Mastitis is one of most impacting health issues in bovine dairy farming that reduces milk yield and quality, leading to important economic losses. Subclinical forms of the disease are routinely monitored through the measurement of somatic cell count (SCC) and microbiological tests. However, their identification can be tricky, reducing the possibilities of early treatments. In this study, a MALDI-TOF mass spectrometry approach was applied to milk samples collected from cows classified according to the SCC, to identify differences in polypeptide/protein profiles. Materials and methods: Twenty-nine raw milk samples with SCC >200,000 cell/ml (group H) and 91 samples with SCC lower than 200,000 (group L) were randomly collected from 12 dairy farms. Spectral profiles from skim milk were acquired in the positive linear mode within the 4,000-20,000 m/z mass acquisition range. Results and discussion: Based on signal intensity, a total of 24 peaks emerged as significant different between the two groups. The most discriminant signals (4,218.2 and 4,342.98 m/z) presented a ROC curve with AUC values higher than 0.8. Classification algorithms (i.e., quick classifier, genetic algorithm, and supervised neural network) were applied for generating models able to classify new spectra (i.e., samples) into the two classes. Our results support the MALDI-TOF mass spectrometry profiling as a tool to detect mastitic milk samples and to potentially discover biomarkers of the disease. Thanks to its rapidity and low-cost, such method could be associated with the SCC measurement for the early diagnosis of subclinical mastitis.

Amoxicillin and thiamphenicol treatments may influence the co-selection of resistance genes in the chicken gut microbiota.

The aim of this study was to assess the dynamics of microbial communities and antimicrobial resistance genes (ARGs) in the chicken gut following amoxicillin and thiamphenicol treatments and potential co-selection of ARGs. To this purpose, the microbial community composition, using 16S rRNA NGS, and the abundance of ARGs conferring resistance to ß-lactams and phenicols, using qPCRs, were determined. Results revealed that the administered antimicrobials did not significantly reduce the gut microbiota diversity, but changed its composition, with taxa (e.g. Gallibacterium and Megamonas) being enriched after treatment and replacing other bacteria (e.g. Streptococcus and Bifidobacterium). Positive correlations were found between ARGs (e.g. cmlA, blaCMY-2, and blaSHV) and the relative abundance of specific taxa (e.g. Lactobacillus and Subdoligranulum). The selective pressure exerted by both amoxicillin and thiamphenicol resulted in an increased abundance of ARGs conferring resistance to ß-lactams (e.g. blaTEM-1, blaSHV, and blaCTX-M1-like) and phenicols (e.g. floR and cmlA). These findings, together with the co-occurrence of genes conferring resistance to the two antimicrobial classes (e.g. blaTEM-1 and cmlA), suggest a possible interaction among antimicrobials on resistance emergence, possibly due to the presence of mobile genetic elements (MGEs) carrying multiple resistance determinants.

A Practical Application of Genomic Predictions for Mastitis Resistance in Italian Holstein Heifers.

Heifers are a fundamental resource on farms, and their importance is reflected in both farm management and economy. Therefore, the selection of heifers to be reared on a farm should be carefully performed to select only the best animals. Genomic selection is available nowadays to evaluate animals in a fast and economic way. However, it is mainly used on the sire line and on performance traits. Ten farms were selected based on their 5-year records of average somatic cell count and evenly classified into high (>300,000 cells/mL) and low somatic cell count (<150,000 cells/mL). Genomic indexes (regarding both wellness and productive traits) were evaluated in 157 Italian Holstein heifers reared in the selected ten farms (90 from high-cells farms and 67 from low-cells ones). Linear mixed models were fitted to analyze the effects of the abovementioned genomic indexes on related phenotypes. Results have shown that farms classified into low somatic cell count had an overall better animal genomic pool compared to high somatic cell count ones. Additionally, the results shown in this study highlighted a difference in wellness genomic indexes in animals from farms with either a high or a low average somatic cell count. Applying genomic tools directly to heifer selection could improve economic aspects related to herd turnover.

Removal of antibiotics from milk via ozonation in a vortex reactor.

Antibiotics (ABX) residues occur frequently in milk, causing considerable wastage of medicated milk and serious economic losses, and making the issue a burden for the dairy industry. Improper disposal of medicated milk harms dairy production, animal welfare, and the environment. This work studies the use of ozonation in a vortex reactor for removing ceftiofur hydrochloride (CEF), sulfamonomethoxine sodium (SMM), marbofloxacin (MAR) and oxytetracycline (OTC) from milk. In terms of residual concentration, O3 efficiency and the degradation kinetics of the various O3-involving processes in the vortex reactor, ABX removal via ozonation is better using stronger vortexing, which induces hydrodynamic cavitation. CEF undergoes the fastest degradation, followed by SMM, MAR, and OTC. High ABX hydrophobicity favors ABX degradation via ozonation, O3/H2O2, and O3/Na2S2O8. ABX oxidation by •OH at the O3 gas-bubble/milk interface is the principle degradation pathway, except for MAR. ABX degradation follows pseudo-first-order kinetics and is affected by initial ABX concentration, O3 concentration/flow rate, reaction temperature, and milk components to varying degrees. Under optimal ozonation conditions, ABX residues meet the maximum limits as set by the European Commission and no antimicrobial activity was observed. The decontaminated milk was therefore suggested to be reused as calf food, animal feed, organic fertilizer, etc.

Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine.

Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen® was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden's index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/µl of cDNA with 1 copies/µL and 0.5 copies/µL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids.

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.

Coping with Tissue Sampling in Suboptimal Conditions: Comparison of Different Tissue Preservation Methods for Histological and Molecular Analysis.

A high quality of samples is crucial for the success of the analysis and diagnostic purposes, and therefore the right method of conservation is vitally important for an optimal preservation of tissues. Indeed, the time to deliver the sample to the laboratory could be remarkably long, especially under suboptimal conditions, and the use of specific fixatives or cold storage may not be possible. Moreover, the portability and cost of storage equipment, their toxicity, and their ease of use play a central role when choosing the correct preservation method. The aim of this study was the identification of a reliable and economic method for tissue preservation, to be used in "in-field" sampling, suitable for both histological and molecular analysis. Punch biopsies were collected from six cattle livers. Comparisons among methods of preservation using RNAlater, silica beads, and under-vacuum was carried out. These methods were tested through considering different times and temperatures, assuming three days as a maximum time interval from sampling to laboratory and choosing 4 °C and 24 °C as references for refrigeration temperature and room temperature, respectively. Histologically, the integrity of nucleus, cytoplasm, preservation of liver structure, and easiness of recognition of inflammatory infiltrate were evaluated. The integrity of the extracted DNA and RNA was evaluated through PCR and by means of an automated electrophoresis station, respectively. RNAlater and silica beads poorly preserved the histological parameters evaluated, independently from the temperature. Conversely, the vacuum-sealed samples showed a good grade of preservation until 48 h. DNA quality was acceptable for each sample. RNA integrity showed promising results only for samples preserved with silica beads.

16S rRNA Sequencing Analysis of the Gut Microbiota in Broiler Chickens Prophylactically Administered with Antimicrobial Agents.

In poultry production, gut microbiota (GM) plays a pivotal role and influences different host functions related to the efficiency of production performances. Antimicrobial (AM) use is one of the main factors affecting GM composition and functions. Although several studies have focused their attention on the role of AMs as growth promoters in the modulation of GM in broilers, the consequences of higher AM concentrations administered during prophylactic treatments need to be better elucidated. For this purpose, 16S rRNA gene sequencing was performed to evaluate the impact of different prophylactic AM protocols on the composition and diversity of the broiler GM. Diversity analysis has shown that AM treatment significantly affects alpha diversity in ileum and beta diversity in both ileum and caecum. In ileal samples, the Enterobacteriaceae family has been shown to be particularly affected by AM treatments. AMs have been demonstrated to affect GM composition in broiler. These findings indicate that withdrawal periods were not enough for the restoral of the original GM. Further studies are needed for a better elucidation of the negative effects caused by an altered GM in broilers.

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.

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.

LC-MS/MS analyses of bile and histological analyses of thymus as diagnostic tools to detect low dose dexamethasone illicit treatment in beef cattle at slaughterhouse.

Dexamethasone (DXM) is a synthetic adrenal corticosteroid with anti-inflammatory properties used for therapeutic purposes in a wide range of pathologies and of the most common corticosteroids used for anabolic purposes in beef cattle. It is proven that DXM induces histological changes, traceable as increasing fatty infiltration of the thymus associated with a concurrent decrease of the cortex-medulla ratio, so the histological examination of the thymus gland has been established as an indirect morphological biomarker. The aim of the present study is to compare thymus histology and DXM concentrations in biological fluids collected at slaughterhouse after 1 month of DXM treatment. Our findings demonstrate that a low dosage of DXM administered to 12 months-old-Chianina beef cattle induces severe thymic atrophy with concurrent reduction of the cortex/medulla ratio, demonstrable even when DXM residues are not found in serum and urine samples. It is worth to note that, at the slaughterhouse, DXM residues are detectable in bile samples, indicating the ability of this biological fluid to bio-concentrate the administered drug if compared to serum and urine. Therefore, bile could be candidates as new liquid matrix for the screening programs planned to contrast the illegal use of anabolic substances.

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.

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.

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.

Scrotal granulomatous aspergillosis in a dromedary camel (Camelus dromedarius).

BACKGROUND: This report describes a case of primary subcutaneous aspergillosis in a 7-year-old neutered male dromedary camel (Camelus dromedarius). CASE PRESENTATION: The animal developed a large nodular lesion in the right scrotum two years after surgical intervention for neutering. The mass had a firm consistency and was painful at palpation. Histopathology revealed dermal granulomatous inflammation with a necrotic centre, surrounded by plasma cells, macrophages, neutrophils, and sparse fungal hyphae characterised by parallel cell walls, distinct septa, and dichotomous branching. Fungal culture was not performed, but a panel of mono- and polyclonal antibodies specific for different fungal genera identified the hyphae as Aspergillus sp. CONCLUSIONS: The occurrence of subcutaneous lesions is a rare manifestation of aspergillosis in animals, and this appears to be the first case reported in the dromedary camel.

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.

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.