Abstract
The rumen primary and secondary metabolite content is intimately related to its community of bacteria, protozoa, fungi, archaea and bacteriophages, ingested feed and the host. Despite the myriad of interactions and novel compounds to be discovered, few studies have explored the rumen metabolome. Here, we present the first study using ultra-high performance liquid chromatography tandem mass-spectrometry and Molecular Networking approach, and various extraction methods on the cell-free rumen fluid of a non-lactating Holstein cow. Putative molecules were annotated based on accurate fragmentation matching the Global Natural Products Social Molecular Networking library, public spectral libraries, or annotated manually. The combination of five extraction methods resulted on 1,882 molecular features observed. Liquid-liquid extraction resulted on the highest molecular features abundance, 1,166 (61.96% of total). Sixty-seven compounds were annotated using Global Natural Products Social Molecular Networking library and public libraries, such as hydrocinnamic and azelaic acid, and monensin. Only 3.56% of molecular features (67) observed had positive match with available libraries, which shows the potential of the rumen as reservoir of novel compounds. The use of untargeted metabolomics in this study provided a snapshot of the rumen fluid metabolome. The complexity of the rumen will remain long unknown, but the use of new tools should be encouraged to foster advances on the rumen metabolome.
Highlights
The rumen is a complex anaerobic environment enriched in bacteria, protozoa, fungi, archaea and bacteriophages[1]
Artegoitia et al.[9] analyzed rumen fluid collected after slaughter of beef cattle using liquid chromatography-mass spectrometry (LC-MS) to explore potential metabolite markers related to average daily gain
The combination of five extraction methods in the rumen fluid of a non-lactating Holstein cow resulted on 1,882 molecular features analyzed by UHPLC-MS/MS
Summary
The rumen is a complex anaerobic environment enriched in bacteria, protozoa, fungi, archaea and bacteriophages[1]. The first study to employ different analytical methods, including mass spectrometry (MS), to explore the rumen metabolome was performed by Saleem et al.[7] The rumen fluid of 8 dairy cows fed different amounts of barley were evaluated and a total of 93 molecular features were observed. Artegoitia et al.[9] analyzed rumen fluid collected after slaughter of beef cattle using liquid chromatography-mass spectrometry (LC-MS) to explore potential metabolite markers related to average daily gain. The association between extraction methods, analytical techniques and annotation tools are essential to cover metabolites from various chemical classes, especially for complex samples. Different extraction methods aimed to extract metabolites varying in chemical properties, such as polarity and pKa. we present the first study using MS-based MN approach to explore the rumen fluid metabolome
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