Supplementary Material: Influence of saponin extracts on enteric methane emission and rumen fermentation: A meta-analysis of in vitro experiments

Background: Saponin extracts have been extensively studied as natural modifiers for the rumen. This meta-analysis aims to quantify the dose-response effects of saponin extract supplementation on rumen fermentation profiles, digestibility, microbial populations, and methane production under in vitro conditions.Results: A review of peer-reviewed in vitro studies from Scopus, PubMed, and Semantic Scholar identified 36 articles with 379 datapoints and 24 saponin sources. Meta-regression analysis found quadratic responses of the levels of saponin extract with dry matter degradability (DMD), gas production, reduced methane production, protozoa abundance, and. NH3 concentration. Subgroup meta-analysis revealed that the effects of saponin crude extract on in vitro rumen fermentation were source-dependent, with significant differences across most parameters. Subgroup meta-analysis demonstrated that DMD reduced by 8.0–19.1% (P<0.001), except Kometa lucerne which increased DMD by +14.3% (P<0.001), Total VFA increased with Calendula officinalis (+20.5%), Knautia arvensis (+9.2%), and tea saponin (+28.8%) with increased on propionate by selected saponin sources (+7.7% to +46.9%). Methane production was consistently reduced by 11–40.6%, with the greatest reduction observed for Sapindus mukorossi (P<0.001). Protozoa were largely unaffected except for a modest reduction (−10.5%; P=0.028) with ivy. Conclusions: modulating effects of saponin extracts on rumen fermentation and methane production are dependent on the origins and dose-dependent manner, suggesting diverse phytochemical characteristics of saponin from different sources. Certain sources of saponin extracts with potential methane-reducing properties were identified such as Sapindus mukorossi, Albizia lebbeck, Calendula officinalis, Chenopodium quinoa, Sapindus saporia, Saponaria officinalis, Saponaria vaccaria, and Sesbania grandiflora. However, optimized levels remain to be determined for specific saponin sources along with their specific phytochemical characteristics and specific mechanism how they modulates rumen fermentation.