Effect of red clover isoflavones on ruminal microbial composition and fermentation in dairy cows

Abstract

Red clover isoflavones, particularly biochanin A and formononetin, are known for their benefits in enhancing feed efficiency and nitrogen utilization in ruminants. However, their specific effects on rumen fermentation and microbial diversity remain insufficiently explored. This study investigated the impacts of red clover isoflavones on rumen function and bacterial diversity in dairy cows, utilizing both in vivo and in vitro methodologies. In the in vivo study, 40 Holstein dairy cows were allocated to four groups, each receiving red clover isoflavones at doses of 0, 0.4, 0.8, and 1.6 g/kg. Rumen fluid was collected for analysis of fermentation parameters, enzyme activity, and microbial composition through shotgun metagenomic sequencing. Concurrently, an in vitro rumen fermentation trial was conducted to evaluate the effects of biochanin A and formononetin on urea hydrolysis. Results from the in vivo experiments showed that red clover isoflavones significantly decreased ammonia nitrogen (NH₃-N) concentrations and urease activity in the rumen (P < 0.05). Species level metagenomic analysis indicated a reduced abundance of proteolytic and ureolytic bacteria, such as Prevotella sp002317355 and Treponema_D bryantii_C, with a corresponding increase in cellulolytic bacteria, including Ruminococcus_D sp900319075 and Ruminococcus_C sp000433635 (P < 0.05). The in vitro trial further demonstrated that biochanin A and formononetin significantly reduced urea decomposition rates (P < 0.05), with biochanin A exerting a more pronounced effect. These findings align with the observed reduction in ureolytic and proteolytic bacteria, along with an increase in cellulolytic bacteria across both trials. In conclusion, biochanin A emerged as the primary active component of red clover isoflavones, modulating urea nitrogen hydrolysis and rumen fermentation. This study substantiates previous findings and highlights the potential of red clover isoflavones for enhancing rumen microbial fermentation, offering a promising strategy for future dairy industry applications. KEY POINTS: • Red clover isoflavones inhibit urease activity to decrease the abundance of urealytic bacteria. • Biochanin A reduces ammonia nitrogen and urease activity, promoting protein efficiency. • Red clover isoflavones may improve dairy cow rumen health and nitrogen utilization.

Keywords: Dairy cows; Fermentation; Microbial composition; Red clover isoflavones; Rumen microbiome.

Fig. 1

Effect of isoflavones on the ruminal bacterial diversity in vivo. Numbers (0, 0.4, 0.8, 1.6) indicate the dose of red clover isoflavones (g/kg DM). Alpha diversity of rumen microflora across the four treatment groups (A). Beta diversity as assessed by principal coordinates analysis (PCA) (B). The relative abundance of the top 20 bacterial species in the rumen at the species level (C). DM = dry matter

Fig. 2

Effect of isoflavones on the ruminal bacterial abundance in vivo. LEfSe analysis of the four treatment groups (A). Relative abundance of the representive bacterial species (B-G). DM = dry matter

Fig. 3

Effects of biochanin A and formononetin on in vitro rumen fermentation. Urea-N concentration (A), NH₃-N concentration (B). Different letters indicate significant differences (P < 0.05), and error bars represent the standard error of the mean (SEM)

Fig. 4

Effects of biochanin A and formononetin on in vitro rumen bacterial diversity and composition. Beta diversity based on principal coordinates analysis (PCoA) (A). The cluster analysis of abundance at genus-level (B). The changed bacteria at genus level (C-F). Different letters indicate significant differences (P < 0.05), with error bars representing SEM, (B) to (F) Treatment groups: color code as presented in subfigure A

Fig. 5

Effects of biochanin A and formononetin on in vitro rumen bacterial function. Functional predictions across the four treatments, displayed through KEGG functional abundance cluster analysis and heatmaps. Color intensity indicates abundance