Screening of Candidate Bioactive Secondary Plant Metabolite Ion-Features from Moringa oleifera Accessions Associated with High and Low Enteric Methane Inhibition from Ruminants

This study evaluated the relationship of secondary bioactive plant metabolite ion-features (MIFs) of Moringa oleifera accessions with antimethanogenesis to identify potential MIFs that were responsible for high and low methane inhibition from ruminants. Plant extracts from 12 Moringa accessions were evaluated at a 50 mg/kg DM feed for gas production and methane inhibition. Subsequently, the accessions were classified into low and high enteric methane inhibition groups. Four of twelve accessions (two the lowest and two the highest methane inhibitors), were used to characterize them in terms of MIFs. A total of 24 samples (12 from lower and 12 from higher methane inhibitors) were selected according to their methane inhibition potential, which ranged from 18% to 29%. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and untargeted metabolomics with univariate and multivariate statistical analysis with MetaboAnalyst were used in the study. Although 86 MIFs showed (p < 0.05) variation between higher and lower methane inhibition groups and lay within the detection ranges of the UPLC-MS column, only 14 were significant with the volcano plot. However, Bonferroni correction reduced the candidate MIFs to 10, and their R-2-value with methane production ranged from 0.39 to 0.64. Eventually, MIFs 4.44_609.1462 and MIF 4.53_433.1112 were identified as bioactive MIFs associated with higher methane inhibition, whereas MIF 9.06_443.2317 and 15.00_487.2319 were associated with lower methane inhibition with no significant effect on in vitro organic matter digestibility of the feed. These MIFs could be used by plant breeders as potential markers to develop new M. oleifera varieties with high methane inhibition characteristics. However, further investigation on identifying the name, structure, and detailed biological activities of these bioactive metabolites needs to be carried out for future standardization, commercialization, and application as dietary methane mitigation additives.