Describing plasma targeted and untargeted metabolomic profiles in various pig breeds to infer metabolic adaptations and animal welfare conditions

Metabolomics is the study of the metabolome, which refers to the complete set of small molecules within a biological system. These metabolites represent intermediate or final products of metabolic pathways and reflect molecular phenotypes that link an organism’s genetic background to complex traits, by providing a general physiological picture of an entire animal. Therefore, metabolomics is a valuable tool for identifying biomarkers associated with biological perturbations associated to intrinsic conditions like the breed and sex of the animals, as well as external factors like stressing conditions and animal welfare issues. Particularly, differences in metabolite profiles between breeds primarily reflect genetic variation helping linking genotypes to metabolic patterns and their adaptation to production systems. Similarly, the investigation of sex-related metabolic differences is valuable, especially for understanding the physiological effects of practices such as castration and their implications for animal welfare. In this study, we focused on the identification of breed-related and sex-related metabolites by implementing a comparative metabolomic approach. We applied targeted and untargeted metabolomic platforms to analyse over 1,000 metabolites in the plasma of approximately 950 performance tested pigs from three breeds: Italian Large White (ILW), Italian Landrace (ILA) and Italian Duroc (IDU). The pigs were slaughtered when they reached at least nine months of age and a live weight of approximately 170 kg. Samples were collected just after jugulation in an authorized slaughterhouse and plasma was prepared within two hours of collection. The animals were not raised or treated specifically for this study, so no ethical evaluation was necessary. Plasma metabolomic profiles were compared between breeds and sexes using a machine learning approach (Random Forest). This analysis identified metabolites that could distinguish between pairs of breeds and also the sex of the animals within each breed, that when tested in an unsupervised manner clearly showed their discrimination and classification ability, reflecting the different genetic backgrounds and biological characteristics. In particular, we identified 100, 59, and 40 metabolites discriminating IDU from ILW, ILA from ILW, and castrated males from intact females, respectively. For example, we observed a higher content of lipids in IDU pigs, while ILW pigs had a higher plasma content of molecules belonging to the amino acid super-pathway. We also observed that more than half of the sexinfluenced metabolites were from the amino-acid super-pathway. Overall, this study provided a detailed overview of circulating metabolites that can be used to establish a baseline physiological profile and identify differences between pig breeds and sexes, potentially aiding in the discrimination of different welfare states in animals. Acknowledgments: This study has received funding from the European Union's Horizon Europe research and innovation programme under grant agreement No. 101059609 (Re-Livestock project).