Application of absolute qPCR as a screening method to detect illicit 17β-oestradiol administration in male cattle

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.     

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle

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.     

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle

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.     

Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures

Levels of several natural urinary steroids have been determined in the urine of a large number of animals of different cattle categories in the context of steroid abuse in beef production. Bovine animals of different breeds, sex and age included in the Slovene national residue detection plan for steroid abuse were studied. Urine from 120 males and 174 females was analysed. Urinary boldenone, boldione, androstenedione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone, epitestosterone, 17β-estradiol, testosterone, and nandrolone were determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of animals containing testosterone and androstenedione increased with age. Testosterone was not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not age dependent. The ratio of testosterone to epitestosterone thus increased with age, from 0.13 ± 0.09 at 1–7 months to 0.42 ± 0.10 at 25–38 months. It was significantly (p < 0.01) higher in bulls above 13 months than in younger animals. In contrast to males, no urinary testosterone was found in females, whereas epitestosterone, androstenedione, progesterone and estradiol were present. The proportion of animals of various age groups in which epitestosterone was detected ranged from 68% to 100%, but the differences were not significant. The presence of both estradiol and progesterone in the same sample was not observed in any animal. The results of this study could be helpful in determining physiological urinary steroid levels in order to provide a baseline for the control of steroid abuse in beef production.     

A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17β-estradiol

17β-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 17β-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 17β-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17β-estradiol, 17α-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17β-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.     

Fate of endogenous steroid hormones in steer feedlots under simulated rainfall-induced runoff

Steroid hormones pose potential risks to fish and other aquatic organisms at extremely low concentrations. To assess the factors affecting the release of endogenous estrogenic and androgenic steroids from feedlots during rainfall, runoff, and soil samples were collected after simulated rainfall on a 14-steer feedlot under different rainfall rates and aging periods and analyzed for six steroid hormones. While only 17α-estradiol, testosterone, and progesterone were detected in fresh manure, 17β-estradiol, estrone, and androstenedione were present in the surficial soil after two weeks. In the feedlot surficial soil, concentrations of 17α-estradiol decreased by approximately 25% accompanied by an equivalent increase in estrone and 17β-estradiol. Aging of the feedlot soils for an additional 7 days had no effect on estrogen and testosterone concentrations, but androstenedione concentrations decreased substantially, and progesterone concentrations increased. Androstenedione and progesterone concentrations in the surficial soil were much higher than could be accounted for by excretion or conversion from testosterone, suggesting that other potential precursors, such as sterols, were converted after excretion. The concentration of androgens and progesterone in the soil were approximately 85% lower after simulated rainfall, but the estrogen concentrations remained approximately constant. The decreased masses could not be accounted for by runoff, suggesting the possibility of rapid microbial transformation upon wetting. All six steroids in the runoff, with the exception of 17β-estradiol, were detected in both the filtered and particle-associated phases at concentrations well above thresholds for biological responses. Runoff from the aged plots contained less 17α-estradiol and testosterone, but more estrone, androstenedione, and progesterone relative to the runoff from the unaged plots, and most of the steroids had a lower particle-associated fraction.     

Effects of transport age and calf and maternal characteristics on health and performance of veal calves

The objective of this study was to investigate effects of calf transport age (14 vs. 28 d) and calf (e.g., sex and breed) and dam characteristics (e.g., parity and ease of birth) on health and performance of veal calves until slaughter age. Calves (n = 683) originated from 13 dairy farms in the Netherlands and were transported at either 14 or 28 d of age from the dairy farm to 8 Dutch veal farms. A health assessment of calves was performed on a weekly basis at the dairy farm and in wk 2, 10, 18, and 24 at the veal farm. Body weight of calves was measured on a weekly basis at the dairy farm and upon arrival at the veal farm. At the veal farm, use of antibiotics and other medicines during the rearing period (both at herd and individual level) was recorded and carcass weights were obtained from the slaughterhouse. Body weight upon arrival (Δ = 11.8 kg) and carcass weight at slaughter (Δ = 14.8 kg) were greater, and mortality risk (Δ = ?3.1%) and prevalence of animals treated with medicines other than antibiotics (e.g., antiinflammatories, multivitamins, and anticoccidial drugs; Δ = ?5.4%) were lower in calves transported at 28 d compared with calves transported at 14 d. Crossbreds other than Belgian Blue × Holstein Friesian received a higher number of individual treatments with antibiotics and other medicines (Δ = 14.8% and Δ = 15.1%, respectively) at the veal farm compared with Belgian Blue × Holstein Friesian calves. These findings suggest that calves transported at 28 d were more robust compared with calves transported at 14 d.     

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 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 (r² > 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.     

Regulation of Cortisol Uptake in Mammary Tissue of Cows

Mammary tissue explants from four nonlactating, nonpregnant cows were placed into culture with media containing various combinations of insulin, prolactin, growth hormone, 17β-estradiol, dexamethasone, and progesterone. Combinations of insulin, prolactin, growth hormone, or 17β-estradiol had no effect on cytoplasmic or nuclear uptake of tritiated cortisol compared with values at zero time. Combinations containing dexamethasone or progesterone reduced cytoplasmic and nuclear uptake of tritiated cortisol. To examine inhibition by progesterone of binding of tritiated cortisol, mammary tissue from each of four lactating, nonpregnant and four nonlactating, nonpregnant cows were placed in flasks containing tissue culture medium 199, tritiated cortisol (2 ng/ml), and progesterone at concentrations of 0, 10^?12, 10^?11, 10^?10, 10^?9, 10^?8, 10^?7, 10^?6, or 10^?5 × 6.4 M. Cytoplasmic uptake of tritiated cortisol into nonlactating tissue decreased linearly as progesterone increased, whereas tritiated cortisol uptake in lactating tissue did not decrease until progesterone exceeded 10^?7 M. We postulated progesterone is sequestered in milk fat of cytoplasm of lactating tissue whereas in nonlactating tissue progesterone is available to compete with cortisol at sites of cortisol binding.