94 Associative effects of two potent anti-methanogenic <i>Moringa oleifera</i> accessions mixed at various proportions on in vitro rumen fermentation and methane inhibition

Abstract Moringa is an important anti-methanogenic plant with a potential to develop as multipurpose feed additives. Previous studies identified two potent accessions (accession 07633 designated as A3 and accession collected from Pretoria designated as A11) with high methane (CH4) inhibition characteristics. Two metabolite ion features (MIFs) 4.44_609.1462 and MIF 4.53_433.1112 were identified as bioactive MIFs associated with greater methane inhibition in Moringa accession A3 and A11, respectively. Two separate studies were conducted with an aim to name the MIF associated with methane inhibition and evaluate the benefit of mixing plant extract from A3 and A11 in different proportions to exploit their additive or synergistic effect in inhibiting CH4 emission. In study 1, software and online tools such as MS-DIAl, MS-FINDER, and the mass bank database were used to identify the MIF 4.44_609.1462 and MIF 4.53_433.1112, and the two MIFs were tentatively named as Hesperidin and Isovitexin, respectively (Figure 1). In study 2, the Moringa plant extracts from A3 and A11 were mixed in the following proportion A3100:A110, A380:A1120, A360:A1140, A350:A1150, A340:A1160, A320:A1180, and A30:A11100 proportions (A3:A11), and applied at a rate 50 mg/kg DM of Eragrostis curvula hay incubated in an Incoshake incubator to determine their effect on in vitro gas production and CH4, and in vitro organic matter digestibility (IVOMD) of the hay. In each incubation run, the sole or a mixture of the Moringa plant extract treatments were added to the test feed and incubated in 125 mL serum bottles with four replications per treatment, and the incubation was repeated for three runs. A Chi–square (☐2) test were used to determine the presence of an associative effect for mixtures combined at different proportion. Generally, the mixture resulted in less (P &amp;lt; 0.05) CH4 volume, slightly improved or no effect on IVOMD, propionate concentration decreased acetate while increasing propionate concentration, with higher CH4 inhibition, and % CH4 inhibition was significantly greater when the two accessions were mixed in 50% proportion (Figure 2). Specifically, associative effects were recorded when the two accessions are mixed in 50% proportion in terms of greater CH4 inhibition, propionic acid concentration and total volatile fatty acid production (TVFA), as well as reduction in CH4 per unit TVFA and C2:C3 ratio (Table 1). Therefore a 50% binary cocktail of M. oleifera accession A3 and A11 (A350:A1150) plant extracts can be used as feed additive for CH4 inhibition without affecting the feed fermentation adversely when compared with the sole accession leaf extracts or other proportions of binary cocktails. Detailed mechanisms of action, however, need to be confirmed.