Effects of Gallic Acid on In Vitro Ruminal Fermentation, Methane Emission, Microbial Composition, and Metabolic Functions

Abstract

The objective of this study was to assess the effects of gallic acid (GA) on nutrient degradability, gas production, rumen fermentation, and the microbial community and its functions using in vitro fermentation methods. An in vitro experiment was conducted to test GA dose levels (0, 5, 10, 20, and 40 mg/g DM) in the cow's diet. Based on the results of nutrient degradability, gas production, and rumen fermentation, the control group (0 mg/g DM, CON) and the GA group (10 mg/g DM, GA) were selected for metagenomic analysis to further explore the microbial community and its functions. The degradability of dry matter and crude protein, as well as total gas production, CH₄ production, CH₄/total gas, CO₂ production, and CO₂/total gas, decreased quadratically (p < 0.05) with increasing GA doses, reaching their lowest levels at the 10 mg/g DM dose. Total volatile fatty acid (VFA) (p = 0.004), acetate (p = 0.03), and valerate (p = 0.03) exhibited quadratic decreases, while butyrate (p = 0.0006) showed a quadratic increase with increasing GA doses. The 10 mg/g DM dose group had the lowest levels of total VFA, acetate, and valerate, and the highest butyrate level compared to the other groups. The propionate (p = 0.03) and acetate-to-propionate ratio (p = 0.03) linearly decreased with increasing gallic acid inclusion. At the bacterial species level, GA supplementation significantly affected (p < 0.05) a total of 38 bacterial species. Among these, 29 species, such as Prevotellasp.E15-22, bacteriumP3, and Alistipessp.CAG:435, were less abundant in the GA group, while 9 species, including Aristaeella_lactis and Aristaeella_hokkaidonensis, were significantly more abundant in the GA group. At the archaeal species level, the relative abundances of Methanobrevibacter_thaueri, Methanobrevibacter_boviskoreani, and Methanobrevibactersp.AbM4 were significantly reduced (p < 0.05) by GA supplementation. Amino sugar and nucleotide sugar metabolism, Starch and sucrose metabolism, Glycolysis/Gluconeogenesis, and Pyruvate metabolismwere significantly enriched in the GA group (p < 0.05). Additionally, Alanine, aspartate and glutamate metabolism was also significantly enriched in the GA group (p < 0.05). GA use 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.

Keywords: gallic acid; metagenomics; methane emission; rumen fermentation.

Figure 1

Principal coordinate analysis (PCoA) of rumen microbial communities: (a) bacteria; (b) archaea; (c) protozoa; (d) fungi. A(Con) = control group (0 mg/g DM, GA); B(GA) = gallic acid group (10 mg/g DM, GA).

Figure 1

Principal coordinate analysis (PCoA) of rumen microbial communities: (a) bacteria; (b) archaea; (c) protozoa; (d) fungi. A(Con) = control group (0 mg/g DM, GA); B(GA) = gallic acid group (10 mg/g DM, GA).

Figure 2

Comparison of the predominant bacterial community between control and gallic acid groups at the (A) phylum level, (B) genus level, and (C) significantly different species level. Con = control group (0 mg/g DM, GA); GA = gallic acid group (10 mg/g DM, GA).

Figure 2

Comparison of the predominant bacterial community between control and gallic acid groups at the (A) phylum level, (B) genus level, and (C) significantly different species level. Con = control group (0 mg/g DM, GA); GA = gallic acid group (10 mg/g DM, GA).

Figure 3

Comparison of the predominant archaeal community between control and gallic acid groups at the (A) phylum level, (B) genus level, and (C) significantly different species level. Con = control group (0 mg/g DM, GA); GA = gallic acid group (10 mg/g DM, GA).

Figure 3

Comparison of the predominant archaeal community between control and gallic acid groups at the (A) phylum level, (B) genus level, and (C) significantly different species level. Con = control group (0 mg/g DM, GA); GA = gallic acid group (10 mg/g DM, GA).

Figure 4

Significantly different (A) KEGG classifications; (B) KEGG functions at level 2; and (C) significantly different KEGG pathway definitions at the bacterial level. Con = control group (0 mg/g DM, GA); GA = gallic acid group (10 mg/g DM, GA).