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.

Keywords:

• absolute qPCR
• gene expression
• progesterone receptor
• 17β-oestradiol
• bovine

Acknowledgements

This work was partially funded by the Regione Piemonte Direzione Sviluppo dell’Agricoltura project entitled ‘Prevenzione dell’uso di anabolizzanti in zootecnia: le biotecnologie nello sviluppo di disciplinari per la qualità e salubrità della carne e derivati’ (Project n.600, 7/08/2008) and by the Ministero della Salute project entitled ‘Sviluppo di un protocollo diagnostico integrato per il controllo relativo a trattamenti illeciti con promotori di crescita nei ruminanti’ (Projects RF-IZP-2006-364645). The authors would like to thank COOP ITALIA, Domenico Palmerini and Alessandra Sereno for the technical support provided, as well as the ‘Bruno Maria Zaini’ Reference Center of Comparative Pathology of the Faculty of Veterinary Medicine.