# Ruminal Degradation of Puerarin and Its Effects on In Vitro Ruminal Fermentation, Methane Emission, and Microbial Community Structure

## TL;DR

This study shows that puerarin, a natural compound, can reduce methane emissions from ruminants by altering rumen fermentation and microbial communities.

## Contribution

The study provides new insights into puerarin's degradation and its effects on methane reduction and rumen fermentation in vitro.

## Key findings

- Puerarin degrades rapidly in ruminal fluid, with 70% degraded within 6 hours.
- Puerarin reduces methane production and the acetate-to-propionate ratio in vitro.
- Puerarin increases microbial protein synthesis and alters the abundance of certain rumen bacteria.

## Abstract

Currently, animal husbandry is a major contributor to global agricultural greenhouse gas emissions, with a substantial portion of its carbon footprint originating from methane produced by microbial fermentation in the rumen of ruminants. To mitigate this issue, one of the most effective strategies involves the application of natural feed additives. Puerarin (PE), a flavonoid compound with inherent bioactivities such as antibacterial properties, exhibits potential for reducing methane emissions. Therefore, this study aims to assess the effects of PE on nutrient digestibility and methane emissions, as well as its degradation dynamics in ruminal fluid. The results indicate that dietary inclusion of PE alters rumen fermentation and reduces methane emissions without affecting fiber degradability.

Few studies have examined the effects of puerarin (PE) on ruminant parameters and methane production. Therefore, we determined the degradation of PE in the rumen and evaluated the effect of PE on in vitro fermentation, methanogenesis, and microbial community structure. A completely randomized design was used for the in vitro fermentation, and 4 gradient dosages of PE (0 mg/kg, 50 mg/kg, 100 mg/kg, and 150 mg/kg of DM) were applied in this trial. The in vitro fermentation was carried out in three runs at 6 h and 48 h, with four replicates per treatment per time point. Each run included 40 samples: eight treatments × four replicates and eight blank samples. Based on the fermentation results, both the PE treatments and the control group (CON) at 48 h were chosen for further analysis to explore the effects of PE on the bacterial community structure. Meanwhile, we determined the degradation rate and degradation products of PE in vitro ruminal fluid using high-performance liquid chromatography (HPLC). In this trial, PE may be isomerized into daidzin by rumen microorganisms; the in vitro degradation results of PE indicated that 70% of PE was degraded within 6 h, with the degradation rate reaching nearly 85% by 12 h. The concentrations of NH3-N and microbial crude protein (MCP) significantly increased linearly with the PE doses at 6 h (p = 0.01). The concentrations of MCP (p = 0.02) and propionate (p = 0.04) demonstrated a linear increase with increasing PE doses at 48 h. In contrast to microbial protein (MCP) and propionate, the acetate-to-propionate ratio decreased linearly with increasing PE doses at 48 h (p = 0.05). Additionally, the addition of PE linearly decreased methane production at 48 h (p = 0.01). Meanwhile, the relative abundances of g_UBA1217 (p = 0.03), g_UBA2810 (p = 0.04), and g_Succiniclasticum (p = 0.03) were significantly lower compared with the CON group. The results showed that PE can be degraded by rumen microflora. Furthermore, it can improve rumen fermentation parameters, increase the amount of protein synthesized by rumen microorganisms, and reduce methane production and the acetate-to-propionate ratio. PE could potentially be an effective strategy for methane mitigation; however, further research is needed to assess its in vivo effects in dairy cows over a longer period.