Influence of accessions, metabolite ion features, storage and processing conditions on efficacy of Moringa plant extracts to inhibit enteric methane emission

Developing healthier and more practically applicable intervention strategies for methane (CH4) inhibition emitted from ruminants has been a priority issue globally. Among the strategies, the use of natural medicinal plants containing bioactive secondary plant metabolites (SPMs) is increasing owing to the cost-effectiveness, environmental suitability and safety value of producing healthy organic animal products of the interventions. Since multiple complex factors affect their bioactive SPMs and bioactivities, the study of wider influencing factors of the extract activities may probably increase the efficacy and sustainability of the final products during application. Thus, this study was initiated i) to investigate the relationships of agronomic traits and SPMs of Moringa accessions with in vitro gas production characteristics from ruminants, ii) to identify potential bioactive SPMs ion features responsible for high and low CH4 inhibition from ruminants, iii) to investigate the effectiveness of varying proportions of binary Moringa accession extract cocktails on in vitro CH4 inhibition, and iv) to evaluate the effects of postharvest processing and storage conditions on the efficacy of Moringa extracts to inhibit enteric CH4 production from ruminants. For the study, twelve Moringa accessions were raised at the University of Pretoria and transplanted in the field at the Roodeplaat experimental site of the ARC in Pretoria, South Africa. The leaf samples from individual plants from these accessions were harvested in the fifth month of transplanting to the field. Then, a series of studies were conducted to develop Moringa leaf extract products that were effective and consistent when used as additives for mitigating enteric CH4 emission in ruminants. In the study that investigated the relationships of agronomic traits of Moringa accessions with in vitro gas production characteristics, most of the agronomic traits, total flavonoids and total phenolics varied among these accessions of M. oleifera. All accessions reduced the total gas (TGP) and CH4 volume compared with the control, incubated without Moringa plant extracts. Hence, among the twelve accessions, those designated A3 (7633), A8 (7717) and A11 (Pretoria) exhibited superior in vitro antimethanogenesis and organic matter digestibility (OMD) with equivalent or superior performances in adaptability in the field. The subsequent study was aimed at identifying potential bioactive secondary plant metabolite ion features responsible for high and low CH4 inhibition from ruminants. In this study, the m/z ion features (MIFs) 4.44_609.1462 and 4.53_433.1112 were linked with higher CH4 inhibition, whereas the MIF 9.06_443.2317 and MIF 15.00_487.2319 were linked with lower CH4 inhibition. The secondary MIFs associated with higher CH4 inhibition can be considered potential secondary MIFs markers for the standardization of plant extracts and commercialization of Moringa varieties that provide extracts effective in antimethanogenic activity from ruminants. In another study, the effectiveness of varying proportions of binary Moringa accession extract cocktails of the two highest CH4-inhibiting accessions was investigated for their effect on in vitro fermentation parameters and CH4 inhibition. The cocktails were prepared by mixing the selected accessions extracts after freeze-drying, milling, and extracting with methanol as 100:0, 80:20, 60:40, 50:50, 40:60, 20:80, and 0:100 proportions (A3:A11) and applied to anaerobically incubated E. curvula hay with 50 mg extract per kg substrate. Thus, the application of Moringa accession extracts in the form of cocktails provided better inhibition of CH4 with comparable or greater in vitro organic matter digestibility (IVOMD) of the substrate feed compared with those values recorded for single Moringa accessions. Thus, a cocktail of A350A1150 showed favourable associative effects on propionate, CH4 inhibition, total volatile fatty acids (TVFA), CH4/TVFA and acetate to propionate ratio (C2/C3), whereas A360A1140 exhibited associative effects on TVFA, acetate, and CH4/TVFA. Thus, the binary combination of the two selected M. oleifera accession extracts at 50% mix exhibited higher benefits of antimethanogenesis and propionate production with a decrease in C2/C3 ratio than when were used the two Moringa accessions in a single form or other cocktails were mixed in various proportions. In the last study, the effects of various postharvest processing and storage conditions were evaluated for their effect on the efficacy of M. oleifera extract to inhibit CH4 production from ruminants. The studied postharvest factors include drying methods (sun, freeze, oven, and shade), drying temperatures (25, 45, 60, and 80 °C), extraction solvents (absolute methanol, 70% methanol, 70% ethanol, and 70% acetone), storage temperatures (4, 15, 25, and 35 °C) and storage light conditions (light and dark). The TGP, CH4 inhibition, total phenolics and total flavonoids differed significantly among the drying methods, drying temperatures and extraction solvents. The freeze- and sun-drying methods compared with shade- and oven-drying methods, drying temperatures of 25 °C and 45 °C compared with 60 °C and 80 °C, and 70% acetone among the extraction solvents recorded higher total phenolics and total flavonoids with better antimethanogenic activities. Storage temperatures of 4 °C and 15 °C also maintained higher total phenolics, total flavonoids and IVOMD with lower CH4/TGP and CH4/IVOMD than those recorded at 25 °C and 35 °C of storage. Dark storage showed lower TGP and CH4 production with better total phenolics, total flavonoids and IVOMD than light storage. Hence, among the postharvest factors, freeze-drying methods, drying temperature of 45 °C, 70% acetone, the storage temperature of 4 °C and dark storage conditions were preferable processing and storage conditions to maintain higher antimethanogenesis, total phenolics and total flavonoids with equal or improved IVOMD from the Moringa plant extract products. The variation recorded among these Moringa accessions when grown in the same environment in terms of SPMs, agronomic performances, and different in vitro fermentation parameters generally indicate the presence of wider genetic sources among Moringa varieties. These could in turn show that superior accessions can be further improved to develop commercial varieties suitable as a source of plant extract products that can be used for mitigating enteric CH4 production. The total phenolics, total flavonoids and the subsequent antimethanogenic activities are influenced crucially by the various postharvest treatments of Moringa leaf/powder. This infers the need for the establishment of appropriate postharvest processing methods, storage conditions and time for a specific purpose and application to utilize optimally Moringa as a dietary additive effectively throughout the year to inhibit CH4 emission from ruminants. However, further investigations are needed on the relationship of agronomic traits with gas production characteristics using long-term adaptability performance at different agro-ecologies, stages of plant growth, plant density, season of harvesting and parts of the plant. The identified MIFs as potential candidates for higher and lower CH4 inhibition of Moringa accessions needed to be studied for their pathways and mode of action. The different organic solvents with various extraction aqueous levels and each storage temperature for longer storage periods at various time points to fix a cut-off storage time for each storage temperature need also be conducted in the future. Keywords: agronomic traits; acetate to propionate ratio; drying methods; drying temperatures; extraction solvents; in vitro organic matter digestibility; storage light; total flavonoids; total gas; phenolics