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Metabolomics for Detecting Banned Steroid Growth Promoters in Fattening Animals

Due to the increasing global meat production and associated fattening costs, the use of growth promoters in livestock farming is rising, primarily to achieve higher fattening efficiency and higher feed conversion. This is particularly true for beef and pork, where improved feed conversion rates ultimately decrease the number of animals required to produce the same amount of meat. As a result, fewer animals are kept, thereby reducing the negative impact on the environment. This rationale is largely advocated in the USA today, with growth promoters primarily consisting of substances based on synthetic anabolic steroids most commonly administered via injection or slow release subcutaneous implants.

However, the situation on the European market is different. The fundamental principle of European food policy is food safety, which guarantees health protection for both consumers and animals. The EU prioritizes the principle of food safety over economic interests. Based on this principle, EU legislation banned the use of selected hormonally active substances in the fattening of food animals as early as 1981. Furthermore, in 1996, the EU banned the use of all growth promoters in the production of animal-derived foods. However, the continued findings of banned growth promoters in fattening livestock across Europe, as published in the regular annual reports of the European Food Safety Authority (EFSA), show that the situation remains very topical and serious. The possibility for illegal use of growth promoters for fattening purposes is further exacerbated by the removal of trade barriers, the easy availability of these substances on the black market (e.g. via the internet or various food supplement sellers), and the significant drop in selling prices by several orders of magnitude compared to the 1990s.

The five-year project entitled "Metabolomics of steroid hormones with a strong anabolic effect as the basis for new analytical control methods intended for confirmation of the practices of banned substances abuse in fattening livestock", funded by the National Agency for Agricultural Research of the Ministry of Agriculture under the No. QK1910311 from 2019 to 2023, focused on this issue. The Faculty of Agronomy of Mendel University in Brno and the company Steinhauser, s.r.o. participated in its implementation under the leadership of the Veterinary Research Institute.

The team of researchers proposed a new solution for confirmation of use of risk growth promoters based on a precisely selected and optimized combination of modern non-targeted analytical "-omics" techniques, namely metabolomics and proteomics. These new methods monitor precursors and their metabolites at the level of the whole organism, tissues and body fluids, down to the cellular level. In particular, analytical techniques based on liquid chromatography-high-resolution-mass spectrometry (LC-(HR)MS) were used for the measurements. By combining these omics methods, it was possible to obtain comprehensive knowledge of the response of the model organism to the stimulus, i.e. the administration of the substance used for growth stimulation, from the level of proteins up to the metabolites of the administered substance of the selected anabolic steroids. The introduction of combined non-targeted omics methods will significantly enhance the capability to detect the abuse of anabolic steroids and growth promoters in fattening food animals, including products imported from third countries.

For the overall complexity of the project implementation, changes at the organ level and the overall clinical health of the experimental animals were monitored. It is clear that the use of growth promoters is reflected in a wide range of tissues and organs, even those unrelated to muscle growth. Unsurprisingly, these include the testes and accessory sex glands, but also the kidneys, liver and heart. Significant cellular or tissue damage could be detected in these tissues. The project used precision microscopy as an innovative approach to detect the effects of growth promoters on tissues and organs, evaluating the complex findings using various histological methods. This detailed microscopic analysis provided valuable feedback on the physiological state of the organism, complementing the overall assessment of the impact of growth promoters on the body.

The outcomes of the project implementation resulted in numerous applications and publications. Major outcomes were published in several international scientific journals and domestic journals for professionals. They were also presented at international and Czech conferences and seminars, and discussed with professional breeders and unions of breeders, such as livestock breeders, producers and processors of animal-derived foods, veterinarians, and inspectors of the State Veterinary Administration of the Czech Republic at workshops and other meetings. The main result of the completed project was the identification and detection of two new prospective metabolites demonstrating the illegal use of the androgenic anabolic steroid nandrolone (19-nortestosterone), which is banned in fattening food animals by Council Directive 96/22/EC. For confirmatory analytical determination of these two new metabolites, a new Certified Methodology "Identification and quantitative determination of nandrolone metabolites - norandrosterone and noretiocholanolone glucuronides - by liquid chromatography-high-resolution-mass spectrometry in pig urine" was developed. This methodology was subsequently transferred to the National Reference Laboratory of the Institute for State Control of Veterinary Biologicals and Medicines, where it was accredited. Subsequently, these new metabolites of nandrolone were included in the strategic document of the State Veterinary Administration of the Czech Republic "Plan of Official Controls of Residues of Pharmacologically Active Substances, Pesticides and Contaminating substances in the Food Chain for 2024".