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

Simple Summary Testosterone and nandrolone can be illegally administered to meat-producing animals as synthetic esters. To tackle the abuse of growth promoters, alternative approaches able to investigate specific changes induced in proteins, transcripts, and metabolites are becoming recommendable. This work aimed to characterize transcriptome perturbations related to the illicit administration of steroid esters in fattening pigs. Animals were treated with testosterone esters (Sustanon (R), Organon, Jersey City, NJ, USA) or nandrolone esters (Myodine (R), Le Vet Beheer B.V., Oudewater, Utrecht, The Netherlands). At the end of the trial, liver samples were collected for gene expression studies. Comparisons between treated and control groups using RNAseq allowed the identification of 491 differentially expressed genes (DEGs). Further analysis of DEGs characterized a smaller cluster of 16 genes. A field survey performed on liver samples collected from pigs belonging to different breeds and weight categories allowed the validation of the selected biomarkers using qPCR, confirming their specificity by comparison with testosterone residue profiles on respective serum samples.Abstract The use of anabolic-androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon (R)) and other nine with nandrolone esters (Myodine (R)). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.