The use of medicinal plants and their extracts has recently attracted the attention of many researchers as a methane (CH4) mitigation strategy. This study evaluated the relationship of agronomic traits of Moringa accessions with in vitro gas production measurements and feed digestibility from ruminants. Twelve Moringa accessions were grown at the Roodeplaat experimental site of the Agricultural Research Council in Pretoria, South Africa. Agronomic traits, such as seedling survival rate, leaf yield, canopy and stem diameter, plant height, number of primary branches, plant vigor, greenness, chlorosis, disease and pest incidences were recorded. The leaves were harvested in the fifth month after transplanting to the field. Freeze-dried leaves were extracted with methanol, and their total phenolic and total flavonoid contents were determined. The extract was applied at a dose of 50 mg/kg of dry matter (DM) feed for in vitro gas production studies. Most of the growth and agronomic traits, i.e., seedling survival rate, leaf yield, canopy diameter, plant height, number of primary branches, the score of plant vigor, and greenness, total phenolics and flavonoids were significantly different among the accessions except for stem diameter and chlorosis score. All accession leaf extracts significantly reduced the total gas and CH4 production compared with the control with equal or higher in vitro organic matter digestibility. Higher CH4 inhibition was obtained in Moringa oleifera (M. oleifera) A3 (28.4%) and A11 (29.1%), whereas a lower inhibition was recorded in A1 (17.9%) and A2 (18.2%). The total phenolic (0.62) and total flavonoid (0.71) contents as well as most agronomic traits of the accessions were positively correlated with the CH4 inhibition potential of the accessions. Moringa oleifera accessions A3, A8 and A11 resulted in higher in vitro CH4 inhibition potential and improved organic matter digestibility of the feed with equal or higher adaptability performances in the field. Thus, there is a possibility of selecting Moringa accessions for higher antimethanogenic activity without compromising the feed digestibility by selecting for higher total phenolics, total flavonoids and agronomic performances traits. There is a need for further study to determine the long-term adaptability of promising accessions in the study area with concurrent antimethanogenesis efficacy when used in the diet of ruminant animals.

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 R2-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.