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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 β2-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 β2-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.

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 11β-hydroxysteroid dehydrogenases (11βHSDs), 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 5β-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 11βHSD 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.

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 β-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 β-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 β-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 β-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 β-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.

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17β-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17β-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CCα, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17β-oestradiol (5 mg week(-1) per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17β-oestradiol (group C; 20 mg week(-1) per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CCα on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CCα and they were classified as being suspected of 17β-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium.

Prednisolone is a synthetic corticosteroid acting on both hydrosaline balance and metabolism that is liable to fraudulent administration to meat-producing animals for growth-promoting purposes. Its use outside strict therapeutic control and prescription is banned by the European legislation, but official controls are hampered by its negligible direct excretion into the urinary matrix. Recent studies reported on a potential endogenous origin of prednisolone in animals subjected to stressful conditions, accounting for its occasional detection in control urines. The objective of the present study was the identification and quantification of prednisolone urinary metabolites to be used as illicit treatment biomarkers in place of the parent drug. An LC-MS/MS screening was conducted on urine samples collected from a bullock intramuscularly administered with prednisolone acetate by using a therapeutic protocol (2 × 0.52 mg kg(-1) at 48-hour interval). Four prednisolone metabolites were identified: 20β-dihydroprednisolone, 20α-dihydroprednisolone, 6β-hydroxyprednisolone and 20β-dihydroprednisone; the first was detected at relatively high concentrations. An existing quantitative LC-MS/MS method was expanded and revalidated to include these metabolites. The new analytical method proved sensitive (LODs: 0.35-0.42 ng mL(-1)) and specific and was applied to urine samples collected from eight beef cattle subjected to low-dosage oral administration of prednisolone acetate for a 35-day period, as in standard growth-promoting treatments. 20β-Dihydroprednisolone was detected in all urine samples collected during the treatment, at relatively high concentration (1.2-27 ng mL(-1)), whereas the prednisolone concentration was virtually negligible (<0.7 ng mL(-1)). 20β-Dihydroprednisolone was no longer present in almost all samples collected 6 days after the end of the treatment, but trace amounts of this metabolite were found in two urine samples from control animals. 20β-Dihydroprednisolone is proposed as an effective biomarker to test illegal growth-promoting treatments with prednisolone in meat cattle, alternatively to the parent drug.

The monitoring of gene regulation via mRNA levels to detect anabolic sex steroid administration in cattle is a novel approach to detecting the illicit treatment of livestock in meat production. A previous study revealed that progesterone receptor (PR) gene expression levels were increased in the bulbourethral glands and prostates of 17β-oestradiol-treated prepubertal calves, suggesting that the PR can be used as a specific molecular biomarker for oestrogen treatment. The aim of this study was to verify the specificity and applicability of the PR to detect the illegal use of 17β-oestradiol in sexually mature beef cattle. Accessory sex glands were sampled from 42 male beef cattle that were divided into six experimental groups, including two control groups, K1 and K2. Group A cattle were treated with 17β-oestradiol (five weekly intramuscular doses of 20 mg), and group B cattle were treated with dexamethasone (40 daily doses of 0.7 mg per os). Group C cattle received an implant of Revalor-200 (200 mg of trenbolone acetate and 20 mg of 17β-oestradiol), and group D cattle received Revalor-200 plus dexamethasone (0.7 mg daily per os). 17β-Oestradiol, either alone or in combination with other steroids, up-regulated the PR gene and protein expression, even in the absence of detectable histological changes in the accessory sex glands, confirming the high sensitivity of PR gene expression as an indirect diagnostic screening tool to detect illicit oestrogen treatment in sexually mature male bovine., (© 2011 Taylor & Francis)

17beta-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17beta-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17beta-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17beta-estradiol, 17alpha-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.