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) Carmela Zacometti a,1 , Andrea Massaro a,1 , Roberto Stella b,* , Alba Leone a , Roberto Celva c , Giammarco Assirelli d , Graziana Da Rold c , Alessia Franzoso e , Alice Casara f , Ranieri Verin f , Giuseppe Rocca g , Alessandro Sadocco h , Federica Obber c , Carlo Citterio c , Nicola Pozzato d,2 , Alessandra Tata a,*,2 a Laboratorio di Chimica Sperimentale, Istituto Zooprofilattico Sperimentale delle Venezie, Viale Fiume, 78, 36100 Vicenza, VI, Italy b Laboratorio Farmaci Veterinari e Ricerca, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Universit` a, 10, 35020 Legnaro, PD, Italy c Centro Specialistico Fauna Selvatica, Istituto Zooprofilattico Sperimentale delle Venezie, Via Fiorenzo Tomea, 5, 32100 Belluno, BL, Italy d Centro Specialistico Medicina Forense Veterinaria, Istituto Zooprofilattico Sperimentale delle Venezie, Viale Fiume, 78, 36100 Vicenza, VI, Italy e SCS4 Epidemiologia veterinaria, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Universit` a, 10, 35020 Legnaro, PD, Italy f University of Padua, Department of Comparative Biomedicine and Food Science, Viale dell’Universit` a, 16, 35020 Legnaro, PD, Italy g Parco Regionale dei Colli Euganei, via Rana Ca’ Mori, 8, 35042 Este, PD, Italy h AULSS 6 Euganea, Via E. degli Scrovegni, 14, 35131 Padova, Italy A R T I C L E INFO Keywords: DART-HRMS Post-mortem interval Boar Metabolomics Ambient mass spectrometry Time of death ABSTRACT 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. 1. Introduction In the veterinary legal context, it is sometimes crucial to determine the post-mortem interval (PMI) as it provides valuable information about the time of an animal’s death. The PMI can aid the investigation process by distinguishing true pathological changes from post-mortem artifacts, and it can be used to eliminate suspects involved in an ani- mal death based on their activity timelines [1]. In veterinary epidemi- ology, robust estimate of the PMI can also be of paramount importance in the passive surveillance of disease in free-ranging animal populations * Corresponding authors. E-mail addresses: rstella@izsvenezie.it (R. Stella), atata@izsvenezie.it (A. Tata). 1 The two authors contributed equally to this work. 2 The two authors share the last authorship. Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc https://doi.org/10.1016/j.microc.2025.113296 Received 18 December 2024; Received in revised form 17 February 2025; Accepted 7 March 2025 Microchemical Journal 212 (2025) 113296 Available online 8 March 2025 0026-265X/© 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.