Thanatometabolomics in wildlife: Identifying potential metabolic markers of post-mortem intervals in wild boars by direct analysis in real time high resolution mass spectrometry (DART-HRMS)

Veterinarians, particularly in the fields of veterinary forensics and veterinary epidemiology, need to be able to estimate the amount of time that has elapsed since an animal died. A reliable estimate of post-mortem interval (PMI) of the earliest cadavers found would inform veterinary services about the possible time since the index case of a disease and the possible range of the epidemic front. We pictured, in a non-targeted manner, the metabolic changes that occurred in four wild boar cadavers placed in a forested area of the North-Eastern Italy. We sampled tissue from the hind limb adductor muscles at nine different time points. The metabolic signatures of decomposing tissues were then analyzed by direct analysis in real time high resolution mass spectrometry (DART-HRMS). A non-parametric ANOVA identified a suitable number of metabolic markers that were able to describe the post-mortem changes based on PMI. In order to go beyond a simple tentative annotation, these metabolites were identified by liquid chromatography tandem high resolution mass spectrometry (LC-HRMS/MS). We were able to identify specific biomolecules that have potential for use in PMI estimation, such as dipeptides, homocarnosine, hypoxanthine, and amino acids. Next, pathway analysis confirmed the extinction of energetic metabolism and a switch towards another source of fuel for the on-going decomposition processes. Our findings suggest that, by targeting a combination of compounds with different post-mortem stabilities, the decomposition of wild boars could be tracked by using an appropriate set of metabolites as revealed by our DART-HRMS study.