As the global population continues to rise, more than a third of the protein demand for humans, such as meat and milk, depends on animal agriculture, thus resulting in livestock production intensification and consequently causing the agricultural sector to be a major source of greenhouse gas (GHG) generation (Sakadevan and Nguyen, 2017). The main greenhouse gas emitted from the animal sector is carbon dioxide (CO₂), methane (CH₄)_, and nitrous oxide (N₂O). Controlling GHG emissions is key to stabilizing the global climate system because its excess in the atmosphere leads to frequent natural disasters, increases the spread of pathogenic diseases, and poses a risk to the health and survival of humans and animals alike. The contribution of methane to global warming is more prominent than N₂O because it constitutes about half of the entire greenhouse gas in the atmosphere (Stocker et al., 2013; Myhre et al., 2014; Haque, 2018), with most of the methane emanating from ruminants. Therefore, reducing CH₄ emissions from enteric fermentation in ruminants is an effective way to mitigate greenhouse gas emissions in the livestock industry (Vaghar Seyedin et al., 2022).

CH₄ produced by ruminants is mainly from the enteric fermentation process initiated by rumen microorganisms through the CO₂ reduction pathway under the action of methanogenic bacteria while reducing the partial buildup of rumen H₂, maintaining the stability of rumen pH and ensuring normal rumen fermentation and the methane produced is released into the atmosphere through feces or burp. Therefore, it is crucial to mitigate CH₄ by regulating the rumen microbiota. Research has found that adding ionic carriers such as monensin to the diet could alter the rumen microbiome, inhibit the production of available substrates by methanogenic bacteria, and reduce CH₄ production (Marques and Cooke, 2021). However, with longer feeding times, ion carriers could develop resistance in some bacteria (Vendramini et al., 2016) and have therefore been banned in many countries. The addition of methanogenic inhibitors such as nitro derivatives, on the other hand, though could reduce CH₄ production via rumen microbiome alteration, but there are very few reports on their safety assessment (Teng and Kim, 2021).

Plant extracts are gaining popularity due to their safe and non-toxic benefits. Literature has reported that plant extracts have colony-modulating effects (Jubair et al., 2021; Vaou et al., 2021) and that flavonoids extracted from plants not only reduce the number of methanogenic bacteria in the rumen but also reduce the number of protozoa attached to the methanogens to reduce CH₄ production (Ku-Vera et al., 2020). Seradj et al. (2014) observed that adding flavonoid extract inhibited hydrogenotrophic methanogenic archaea and Methanosarcina in the rumen. Likewise, the flavonoid-rich extract obtained from Brazilian spinach (Alternanthera sissoo; BS) was found to decrease CH₄ production and protozoa counts while elevating total volatile fatty acid and propionic acid levels during in vitro fermentation (Sommai et al., 2021).

Silibinin (C₂₅H₂₂O₁₀), an active ingredient isolated from the fruit of Silybum marianum (chrysanthemum plant), is single in composition and chemistry as a dihydro flavonol compound compared to some other flavonoids extracted from plants. According to recent scientific investigations, gram-positive bacteria are more susceptible to the inhibitory effects of silibinin as compared to their gram-negative counterparts (Janssen and Kirs, 2008; Miller, 2015; Mahmoudi-Rad et al., 2022). Therefore, the potential of silibinin to effectively regulate the activity and population of gram-positive bacteria associated with methane synthesis is critical for methane regulation. Additionally, the mechanism by which silibinin regulates gram-positive bacteria is mainly by inhibiting protein and RNA synthesis (Lee et al., 2003). Some reports have shown that silibinin has a modulating effect on the activity of Staphylococcus aureus (Cai et al., 2018), Bacillus cereus (Mahmoudi-Rad et al., 2022), and Prevotella intermedia (Lee et al., 2012). Also, its anti-inflammatory effects are exerted by regulating the microbial flora (Rakelly de Oliveira et al., 2015).

Even though flavonoids have been shown to have regulatory effects on rumen microflora and amino acids metabolic pathways such as tyrosine metabolism and proline metabolism (Yu et al., 2023), there are no studies on the use of silibinin as an additive in dairy cows. Based on the above, this study hypothesized that silibinin could affect rumen fermentation by regulating rumen microbiota and metabolites, thereby reducing methane production. Therefore, using in vitro fermentation method, we assessed rumen fermentation characteristics and gas production, analyzed the relative abundance and diversity of the rumen microbiome, and revealed the metabolites and metabolic pathways involved. This experiment provides a sufficient theoretical basis and data support for the application of silibinin in regulating ruminal methanogenesis in ruminants.

In this experiment, based on the linear effects of silibinin on most indicators, we selected the maximum dosage group as the research subject for analyzing 16S amplicons and non-targeted metabolomics. By comparing this group with the control group, we aimed to investigate the impact of silibinin on the rumen microbial community, metabolic products, and metabolic pathways to unveil its mechanism in regulating rumen gas production and fermentation parameters. After thoroughly analyzing the results, we discovered that high-dose silibinin influences methane production in the rumen directly and indirectly.

First, an indirect effect of silibinin is its ability to reduce the concentration of methane precursors, thereby indirectly influencing methane production. Ruminal methane production results from the combined effect of various microorganisms within the rumen, and microorganisms, such as protozoa, bacteria, and fungi, play important roles in methane generation within the rumen (Hook et al., 2010). In the rumen, almost all ciliated protozoa are associated with methane-producing bacteria; there is a portion of methane-producing bacteria in the cytoplasm of rumen ciliate protozoa, forming a mutualistic or symbiotic relationship between them (Finlay et al., 1994) which facilitates methanogens in utilizing the H₂ produced by the protozoa to generate methane. Isotricha and Ophryoscolex belong to ciliate protozoa (Schrenk and Bardele, 1987; Ivan et al., 2000), and in this experiment, compared to the control group, the relative abundance of these two protozoa was significantly reduced, which may be one of the reasons for the significant decrease in Methanosphaera. Related research has also shown that plant extracts rich in flavonoids have an inhibitory effect on rumen ciliate protozoa (Kim et al., 2015), consistent with the results of this experiment. Furthermore, rumen ciliate protozoa can adhere to feed particles and degrade cellulose, hemicellulose, and starch (Williams et al., 1984; Mendoza et al., 1993), ultimately producing CO₂ and H₂ for methanogens to utilize. Additionally, the absence of protozoa has been found to reduce organic matter degradation (Newbold et al., 2015). Moreover, silibinin, chemically belonging to flavonoids, has been found to disrupt the cell membrane of rumen protozoa and reduce their population (Ku-Vera et al., 2020), thereby reducing hydrogen transfer between protozoa and methane-producing bacteria. Based on these findings, we speculate that high doses of silibinin may have acted in a similar way on protozoa, leading to the decreased relative abundance of Isotricha and Ophryoscolex observed. This decrease in abundance could have resulted in changes in metabolite production and a reduction in the concentrations of CO₂ and H₂, ultimately affecting methane levels (Ranilla et al., 2007).

In addition, other indirect effects of silibinin include increased synthesis of propionate and reduced methane production by competitively utilizing substrate H₂. Bacteria and fungi in the rumen play a crucial role in fiber degradation, leading to an increase in dry matter degradation rate and the production of VFAs, CO₂, and H₂. After adding high doses of silibinin, we observed a significant increase in the relative abundance of Succiniclasticum and NK4A214_group. Succiniclasticum has been noted to promote the metabolism of succinate derived from carbohydrate fermentation into propionate (Van Gylswyk, 1995). The production of propionate reduces H₂ partial pressure and ensures normal rumen fermentation. However, the relationship between NK4A214_group and propionic acid production remains unclear, and its specific role in the rumen is still not well understood (Pacifico et al., 2021). Further, N6-(1,2-Dicarboxyethyl)-AMP, an upregulated differential metabolite, can be converted into fumarate, which is a precursor of propionate metabolism, by adenosylsuccinate lyase (Timpani et al., 2020). Fumarate acts as an alternative hydrogen sink in the rumen and competes with methanogenic bacteria for H₂, thereby reducing CH₄ production (Newbold and Rode, 2006). Based on the above analysis, we speculate that the increase in the proportion of propionate in the rumen is likely attributable to the increased relative abundance of Succiniclasticum. It is equally important to note that after adding a high dose of silibinin, there was a significant decrease in the relative abundances of Prevotella, Orpinomyces, and Neocallimastix. Previous studies have shown that flavonoids are usually present in their glycoside form, which is further deglycosylated by glycosidases secreted by microbial communities, increasing aglycone concentrations (Murota et al., 2018); bacteria have evolved diverse metabolic lifestyles and strategies to harvest energy from glycosides (Wang et al., 2022). Moreover, compared to the Proteobacteria phylum, microorganisms within the Bacteroidetes phylum are selectively inhibited by flavonoids (Yu et al., 2023). This explains the increased relative abundances of Succiniclasticum and NK4A214_group and the decreased relative abundance of Prevotella after adding a high dose of silibinin. Furthermore, Orpinomyces and Neocallimastix ferment CO₂ and H₂ as end products (Williams et al., 2020), and their relative abundances significantly decrease, reducing not only the proportions of CO₂ and H₂ but also indirectly lowering methane concentrations. In summary, we speculate that high doses of silibinin regulate the relative abundances and metabolic products of bacteria and fungi in the rumen, affecting the concentration of methane precursors and promoting propionate synthesis through competitive utilization of H₂, ultimately leading to a reduction in methane production (Zhou et al., 2021).

The direct effect of silibinin in reducing methane production involves modulating the activity of rumen methanogenic bacteria. Ruminal methanogenic bacteria utilize the reducing power generated through rumen fermentation to reduce carbon dioxide, formic acid, methanol or methylamine to CH₄. This study showed that compared to the control group, the relative abundance of Methanosphaera was significantly reduced after adding a high dose of silibinin. It is worth noting that Methanosphaera accounts for a relatively small proportion and may have a limited effect on reducing methane concentration. Previous reports have also found that flavonoids can inhibit the activity of Methanosphaera and other methanogenic bacteria and thus reduce CH₄ emissions (Seradj et al., 2014; Hassan et al., 2020). Furthermore, epicatechin, quercetin, isoquercetin, and luteolin-7-glucoside reduced the ratio of CH₄ production to total gas production (Sinz et al., 2018). In this study, the percentage of methane in the total gas production also significantly decreased with increasing doses of silibinin, consistent with previous research findings.

Furthermore, after analyzing the metabolites, we found that flavin mononucleotide adenine dinucleotide reduced form (FMNH), Pyridoxine 5′-phosphate, and 5,6,7,8-Tetrahydromethanopterin exhibited upregulation. FMNH plays a role in the electron transfer during methane production (Thauer et al., 2008), ultimately being reduced to FMNH₂. Pyridoxine 5′-phosphate can be converted into pyridoxal 5′-phosphate (Garrido-Franco, 2003), which inhibits the activity of the final enzyme involved in methane synthesis, namely coenzyme M methyltransferase (Van Der Meijden et al., 1983). During methane generation, 5,6,7,8-Tetrahydromethanopterin (H₄MPT) acts as a carrier for the formyl group through the methyl-reduced C1 unit (Van Beelen et al., 1984), forming 10-formyl-H₄MPT in methylotrophic organisms (Keltjens et al., 1990). We speculate that it may be that Methanosphaera, a methylotrophic pathway archaea (Wolin, 1985), whose relative abundance significantly decreased, lead to the obstruction of CH₄ synthesis and resulting in the upregulation of intermediates such as FMNH, Pyridoxine 5′-phosphate, and 5,6,7,8-Tetrahydromethanopterin. However, further research is needed to validate this. In addition to impacting the production of metabolites, the addition of 0.60 g/L silibinin also had an effect on the phenylalanine metabolic pathway. We presume that the high dose of silibinin affecting the phenylalanine metabolism resulted in the upregulation of differential metabolites 2-Phenylacetamide, Phlorizin, and Dalspinin. In summary, we hypothesize that silibinin modulates the relevant pathways and metabolites in the rumen in a way that affects methane production in the rumen and reduces the proportion of CH₄.

In addition, this experiment found that the concentration of NH₃-N did not show a linear decrease with increasing dosage of silibinin, and only the group with 0.60 g/L silibinin showed a significant reduction in NH₃-N concentration. Previous reports have also shown that supplementation of citrus flavonoids, flavones, myricetin, naringin, catechin, rutin, quercetin, and kaempferol does not affect rumen NH₃-N concentration (Oskoueian et al., 2013; Amin et al., 2021). Additionally, Prevotella is involved in the degradation of plant-derived semi-fibrous materials and participates in the process of protein degradation (Betancur-Murillo et al., 2022). We speculate that the decrease in Prevotella relative abundance, possibly due to the addition of 0.60 g/L silibinin, leads to a reduction in protein degradation and subsequently decreases NH₃-N concentration (Zhan et al., 2017). However, the optimal range for rumen microbial growth is between 10–50 mg/dL of NH₃-N concentration (Firkins et al., 2007). In this experiment, the rumen NH₃-N concentration was within this range, indicating that adding a high dose of silibinin may not have a negative impact on microbial biomass protein synthesis. Moreover, related studies have shown a negative correlation between the relative abundance of rumen family NK4A214_group and NH₃-N concentration (Liu et al., 2022), as well as a positive correlation between protozoa and NH₃-N concentration (Wanapat and Pimpa, 1999), which is consistent with the results of this study. These findings provide new insights into the role of silibinin in regulating the rumen microbial community and methane production mechanisms. On the other hand, from the experimental results, the ingestion of a high dose of silibinin caused a decrease in dry matter degradation rate, which is likely the main reason for the decrease in total volatile fatty acid concentration, and, consequently, the pH value would correspondingly increase. Relevant literature has also indicated that supplementing mulberry leaf flavonoids did not affect the apparent digestibility of dry matter and organic matter (Chen et al., 2015). These results suggest that different plant-derived flavonoid compounds may produce different effects at varying concentrations. In ruminant animal production, volatile fatty acids are important sources of energy, and changes in VFA concentration can affect milk production in cows (Morvay et al., 2011). Therefore, the aim of our study is to reduce methane production while ensuring cow productivity. In this experiment, we selected the group supplemented with 0.60 g/L of silibinin for analysis to better observe the effect of silibinin on rumen methane production. Compared to the group supplemented with 0.00 g/L of silibinin, the group supplemented with 0.075 g/L of silibinin showed no significant difference in total VFA concentration but a significant decrease in methane concentration. Based on this, it can be preliminarily concluded that adding 0.075 g/L of silibinin in vitro experiments can reduce methane concentration without affecting rumen fermentation. Therefore, this group can serve as an additive for in vivo experiments to evaluate the methane-inhibiting effect of silibinin in living organisms.

This study provides a theoretical basis for the application of a novel rumen methane regulator. In an in vitro fermentation experiment, silibinin modulates rumen microbial community diversity and community structure, leading to changes in rumen metabolic pathways and metabolites to reduce methane emissions. Notably, silibinin demonstrated the ability to lower methane emissions at reasonable doses without adversely impacting rumen fermentation. Nevertheless, further verification through long-term in vivo experiments is crucial in light of the possible adaptation of rumen microbes to metabolites and differences between in vitro and in vivo findings.

The data presented in the study are deposited in the NCBI Sequence Read Archive (SRA) repository, accession number PRJNA975466 and PRJNA976941. https://www.ncbi.nlm.nih.gov/sra.

The animal use protocol was approved following the Animal Care and Use Committee of Northeast Agricultural University (protocol number: NEAUEC20230268).

RL and YS: methodology, investigation, and writing—original draft. HM, ML, BD, WS, and YQ: investigation and data curation. YL and BD: conceptualization, methodology, resources, funding acquisition, and writing—review and editing. YZ: writing—review and Editing. All authors contributed to the article and approved the submitted version.

This study was financially supported by the Natural Science Foundation of Heilongjiang Province (YQ2023C011), Key Research and Development Program of Heilongjiang Province of China (grant no. 2022ZX01A24), “Academic Backbone” Project of Northeast Agricultural University (22XG35) and Key Laboratory of Low-carbon Green Agriculture in Northeastern China, Ministry of Agriculture and Rural Affairs P. R. China (grant no. LCGANE14).

YS was employed by Beijing Sunlon Livestock Development Company Limited.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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