Synergistic effects of bioactive plant extract mixtures on methane reduction and rumen fermentation of eragrostis curvula hay in vitro

Building on prior research indicating the methane-mitigating potential of specific medicinal plants for ruminant animals. This study aimed to investigate the associative effects of combining six medicinal plant extracts known for their methane-reducing properties: Aloe vera (AV), Carica papaya (CP), Azadirachta indica (AZ), Tithonia diversifolia (TD), Jatropha curcas (JA), and Moringa oleifera (MO). Methanolic extracts of the plants were combined in equal proportions into two-way mixtures and applied at a concentration of 50 mg/L to 400 mg Eragrostis curvula hay, followed by a 48-hour in vitro incubation. Phytochemical profiling of individual plant extracts was performed using LC-MS and HPLC methods. Evaluated parameters included methane (CH₄) production, total gas production (TGP), in vitro organic matter digestibility (IVOMD), ammonia nitrogen, and volatile fatty acids (VFA). Phytochemical profiling revealed diverse bioactive compounds such as flavonoids, saponins, anthraquinones, phenols, alkaloids, and terpenoids in all extracts, with AZ showing the highest phenolic content. The mixtures significantly reduced CH4 production by over 50%, individual plant extracts generally showed greater improvements in IVOMD compared to mixtures. Moreover, the mixture displayed positive associative effects on various parameters, including TVFA, CH₄/IVOMD, CH₄/TGP, and CH₄/TVFA production. Two-way mixture containing AV or CP (AV + CP, AV + JA) notably increased propionic acid concentration, differentiating them from single plant extracts, monensin, and control treatments. The study highlights that specific combinations of these medicinal plant extracts can significantly reduce methane emissions while positively modulating rumen fermentation parameters, indicating their potential as natural additives for sustainable livestock production.