Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves

Abstract

Corticosteroids such as Dexamethasone (DEX) are commonly licensed for therapy in meat animals due to their known pharmacological properties. However, their misuse aimed to achieve anabolic effects is often found by National Residues Control Plans. The setup of a complementary “biomarker based” methods to unveil such illicit practices is encouraged by current European legislation. In this study, the combined use of molecular and histological quantitative techniques was applied on formalin fixed paraffin embedded (FFPE) muscle samples to assess the effects of illicit DEX treatment on veal calves. A PCR array, including 28 transcriptional biomarkers related to DEX exposure, was combined with a histochemical analysis of muscle fiber. An analysis based on unsupervised (PCA) and supervised (PLS-DA and Kohonen’s SOM) methods, was applied in order to define multivariate models able to classify animals suspected of illicit treatment by DEX. According to the conventional univariate approach, a not-significant reduction in type I fibres was recorded in the DEX-treated group, and only 12 out of 28 targeted genes maintained their expected differential expression, confirming the technical limitations of a quantitative analysis on FFPE samples. However, the multivariate models developed highlighted the possibility to establish complementary screening strategies, particularly when based on transcriptional biomarkers characterised by low expression profiles.

RNAseq Analysis of Livers from Pigs Treated with Testosterone and Nandrolone Esters: Selection and Field Validation of Transcriptional Biomarkers

“…Moreover, the transcriptional signature related to Myodine treatment, identified using RNAseq, was characterized by a larger number of downregulated genes in comparison to the Sustanon signature (see B3GNT8, ATP1A2, CACNA1H, DUOX1, and TINAG profiles in Figure 5). As already seen in previous studies based on the profiling of transcriptional biomarkers to unveil growth promoters' administration in farm animals [38], the validation analysis of RNAseq/Microarray data using Real Time and/or Digital PCR has often revealed how dowregulated genes, especially those that are characterized by low basal expression levels, are more difficult to validate for reliable application in field/unknown samples, where optimal preanalytical conditions in sampling procedures are not always guaranteed [39].…”

“…The tested panel of transcriptional biomarkers has therefore confirmed, at least for the illicit administration of testosterone esters, an interesting potential for supplementary field investigations, which will need to be extended in order to be validated on an even larger number of pig specimens than that already shown in the present study. In this regard, more refined supervised classification tools [38][39][40] will need to be considered in light of ISO-oriented validation approaches based on multivariate analysis [41]. carried out in accordance with institutional guidelines and national legislation, Act.…”