F. Spada

Corticosteroid Hormone Receptors and Prereceptors as New Biomarkers of the Illegal Use of Glucocorticoids in Meat Production

Despite the European ban on the use of growth promoters in cattle, veterinary surveillance reports indicate that the illicit use of corticosteroids persists both alone and in combination with anabolic hormones and β-agonists. Current control strategies should be informed by research into the effects of corticosteroids on bovine metabolism and improved through the development of specific, sensitive diagnostic methods that utilize potential molecular biomarkers of corticosteroid treatment. The actions of corticosteroids on target tissues are principally regulated by two receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The effects of these steroids are modulated by prereceptor enzyme-mediated metabolism: the two isoforms of the 11β-hydroxysteroid dehydrogenase (11β-HSDs) enzyme catalyze the interconversion between active glucocorticoids, such as cortisol, into inactive compounds, such as cortisone. This study aimed to determine whether the expression of the prereceptor system and of the corticosteroid receptors could be regulated in different target tissues by the administration of dexamethasone and prednisolone in cattle. It was observed that greater up-regulation of the GR and MR genes followed dexamethasone treatment in the muscle tissues than in the kidney, liver, and salivary glands; up-regulation of GR and MR expression following prednisolone treatment was higher in adipose tissue than in the other tissues. The thymus seemed to respond to dexamethasone treatment but not to prednisolone treatment. Both treatments significantly down-regulated 11β-HSD2 gene expression in the adrenal tissues, but only dexamethasone treatment down-regulated 11β-HSD2 expression in the bulbourethral and prostate glands. Together, these data indicate that the combination of GR, MR, and 11β-HSD2 could provide a useful biomarker system to detect the use of illicit glucocorticoid treatment in cattle.

Progesterone receptor gene expression in the accessory sex glands of veal calves

This study investigated progesterone receptor (PR) cDNA expression in the testes, prostate and bulbourethral glands of prepubertal calves treated experimentally with high and low doses of 17β-oestradiol and with testosterone. Tissue samples were examined histologically and immunohistochemically for PR. Western blot analysis and quantitative PCR against PR was performed on cDNA and protein extracted from the same tissues. Bulbourethral glands from animals treated with low and high dosages of 17β-oestradiol had 39- and 429-fold increases of PR transcript, respectively, compared with controls. In the prostate there were 7.5- and 16-fold increases, respectively. Animals treated with testosterone showed no increases in PR transcript. The results demonstrate that 17β-oestradiol specifically induces marked overexpression of the PR gene and protein, particularly in the bulbourethral gland.

Thymus atrophy and regeneration following dexamethasone administration to beef cattle

Thymus atrophy and regeneration were studied in 13- to 22-month-old beef calves treated with dexamethasone (DMT), using anabolic dosages and implementing different withdrawal times. Two trials were conducted. In trial 1, group A (n=6) received 0.7 mg/day DMT orally for 40 days, group B (n=6) received 1.4 mg/day orally for 40 days and group C (n=6) was the control. In trial 2, group D (n=6) received 0.7 mg/day DMT orally for 40 days, group E (n=6) received 1.4 mg/day orally for 40 days and group K (n=6) was the control. DMT withdrawal times before slaughter were six days (groups A and B) and 26 days (groups D and E). At slaughter, thymus atrophy was severe and progressive in animals from groups A and B. In contrast, thymus weight and volume of the animals from groups D and E were almost normal. Slight atrophy was also detected in the calves in these groups. Histological changes and Ki67 immunostaining revealed a large number of positive lymphoid cells, mostly in the cortical area, associated with higher expression of apoptosis in the medulla compared with controls. This demonstrated that the thymus of beef cattle is still able to regenerate following DMT administration.

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.

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.