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

Abstract

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.

Regucalcin Expression in Bovine Tissues and Its Regulation by Sex Steroid Hormones in Accessory Sex Glands

“…In particular, the estrogen administration caused a decrease of RGN expression in bulbo-urethral glands of veal calves, but not in beef cattle. This marked difference is likely due to physiological levels of the sex steroid hormones in adult male animals, as previously described for the expression of other estrogen-controlled genes [58]. Moreover, the different treatment schedule could influence the RGN expression.…”

“…This approach has led to the identification of specific biomarkers for use in screening analyses to identify animals treated with sex steroid hormones. In recent years, for example, PR gene expression in the bulbo-urethral glands and prostate has been used as a biomarker for the illicit estrogen treatment of veal calves and beef cattle [53] , [57] – [58] . Similarly, the variation of oxytocin gene expression in beef cattle muscle is indicative of estrogen and glucocorticoids illegal treatment [87] .…”

“…Recent studies have shown that progesterone receptor (PR) gene expression levels were increased in the bulbo-urethral and prostate glands of 17β-estradiol-treated calves and beef cattle [53] , [57] . For potential use in food safety monitoring, a quantitative PCR (qPCR) method has been developed for the detection of up-regulated PR gene expression in the bulbo-urethral glands of beef cattle and veal calves illegally administered 17β-estradiol [58] .…”

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

“…The transcriptomic techniques are based on the use of certain transcriptional markers for the low-cost screening analysis of GP administration in livestock production . A number of novel gene expression profiles have been examined in different bovine tissues, and some of these findings could be applicable in the field as routine screening methods. ,− For instance, the expression of progesterone receptor (PR) gene in the prostate and bulbo-urethral glands has been identified as a successful biomarker for the detection of beef cattle and veal calves illegally treated with estrogens. ,, In the same way, gene expression change of oxytocin (OXT) in beef cattle muscle may suggest an illicit treatment with glucocorticoids and estrogens . The University of Turin (Italy) patented PR- and OXT-based screening tests, due to their ability to detect illegally treated animals long after GP withdrawal. , …”

“…2,14−17 For instance, the expression of progesterone receptor (PR) gene in the prostate and bulbo-urethral glands has been identified as a successful biomarker for the detection of beef cattle and veal calves illegally treated with estrogens. 3,12,18 In the same way, gene expression change of oxytocin (OXT) in beef cattle muscle may suggest an illicit treatment with glucocorticoids and estrogens. 4 The University of Turin (Italy) patented PR-and OXT-based screening tests, due to their ability to detect illegally treated animals long after GP withdrawal.…”