Vučković, Ivan

ABSTRACT The search for long‐standing, drought‐tolerant species with consistent annual yields presents both the demands of sustainable agriculture and a significant challenge. Hungarian clover, an underutilized fodder species with a long lifespan, may be a potential solution to these demands. This study represents the first report on the metabolomic profile of this species. We analyzed 52 samples of Hungarian clover and Swedish clover, used as a control species, employing 1 H NMR spectroscopy combined with multivariate data analysis. A total of 24 metabolites were identified, with dominant signals derived from sugars, amino, eucomic acids, and hydroxyeucomic acids. These acids contributed to the higher antioxidant capacity observed in Hungarian clover samples compared to Swedish clover. The application of nuclear magnetic resonance in the pre‐breeding evaluation of fodder species is a well‐justified approach, providing clear insight into the metabolic variations of the examined material and valuable guidance for future breeding activities.

Combined histopathological and metabolomic analysis of the same tissue specimen can reduce biological variability compared to independent analyses of similar samples. However, conventional workflows are incompatible: tissue fixation leads to metabolite loss through dilution and chemical reactions with fixative, while metabolomic extraction typically destroys the specimen. We propose a method that reconciles these conflicting requirements by enabling metabolite recovery from fixative while preserving tissue for histopathology. In this approach, tissue is initially fixed in a small volume of fixative; an aliquot of the fixative is then collected for nuclear magnetic resonance-based metabolomic analysis before additional fixative is added for standard processing. As a proof-of-concept, mouse kidneys from the same subject were analyzed using two protocols: standard perchloric acid extraction (ensuring complete metabolite recovery but destroying tissue) and metabolite extraction from fixative. Several metabolites were fully recoverable from the fixative, others partially, while some were lost due to chemical reactions. Despite some limitations, this strategy may be useful for analyzing precious specimens, such as human biopsies, that must remain intact for further studies. Moreover, using the same specimen for dual analyses reduces the number of animals required and enhances statistical robustness.