Use of hepatic and testicular molecular biomarkers to detect growth promoters misuse in cattle: a preliminary application under field conditions

Introduction. Growth promoters (GPs) are forbidden at the European Community level. Nevertheless, GPs misuse in cattle still represent a major concern. In the past decade an increasing interest toward the set up and validation of molecular biomarkers to be used side by side with official analytical methods has been recorded (Nebbia, 2010). In preceding pilot studies, a number of tissue-specific responsive genes have been identified (Giantin, 2010; Lopparelli, 2011). In this study, these biomarkers were preliminarily tested under field conditions. Materials and methods. Ninety-five cattle testis and liver aliquots were collected by chance at slaughterhouses, placed in microtubes with RNAlater® and stored at -80°C until use. A robust set of negative controls (44 animals), from earlier pilot studies, were included in the study, too. Total RNA was extracted with TRIzol® Reagent and gene expression profiles measured by using a quantitative Real Time RT-PCR approach (qPCR). Seven and eight target genes were chosen for liver and testis, respectively. Results were elaborated (Hierarchical Clustering, HCL, and Principal Component Analysis, PCA) by using the GenEx software (Berkvist, 2010). Results. In liver, HCL clustered samples into three main groups, supported by PCA: negative controls and most of random samples were clustered together (“negatives”), while three animals were distinctly grouped in another cluster (“suspects”). Further nine samples, assigned to negatives by GenEx, generated a different cluster; therefore, they were classified as “doubtful”. In testis, three “suspects” and three “doubtful” were identified besides “negatives”. Considering both tissues as a whole, the software identified three “suspects” and two “doubtful”. Conclusions. This study aimed to test a set of candidate genes and a popular software for qPCR data processing and analysis upon a large number of random samples. The approach allocated samples into three different clusters, representing different expression profiles. Presented data suggest that transcriptome analysis and bioinformatic tools, coupled with a robust database of negative controls, might be helpful for tracking GPs abuse in cattle. Further studies are needed to confirm these promising results. References. Nebbia C., Urbani A., Carletti M., Gardini G., Balbo A., Bertarelli D., Girolami F., 2010. Novel strategies for tracing the exposure of meat cattle to illegal growth-promoters. The Veterinary Journal, 189: 34-42. Giantin M., Lopparelli R. M., Zancanella V., Martin P. G., Polizzi A., Gallina G., Gottardo F., Montesissa C., Ravarotto L., Pineau T., Dacasto M., 2010. Effects of illicit dexamethasone upon hepatic drug metabolizing enzymes and related transcription factors mRNAs and their potential use as biomarkers in cattle. Journal of Agricultural and Food Chemistry, 58: 1342-1349. Lopparelli R. M., Zancanella V., Giantin M., Ravarotto L., Pozza G., Montesissa C., Dacasto M., 2011 Steroidogenic enzyme gene expression profiles in the testis of cattle treated with illicit growth promoters. Steroids, 76: 508-516. Berkvist A., Rusnakova V., Sindelka R., Garda J. M. A., Sjogreen B., Lindh D., Forootan A., Kubista M., 2010. Gene expression profiling – Clusters of possibilities. Methods, 50: 323-335. Acknowledgements. Project supported by a grant from Regione del Veneto (Dgr 2888 07/10/2008) to M.D.