# Metagenomic Insights into the Effects of Dietary Thymol on the Structure and Function of the Rumen Microbial Community in Beef Steers Consuming Forage

## TL;DR

This study explores how thymol, a compound in essential oils, affects the microbes in the rumen of beef cattle, finding that moderate doses can reduce methane production while supporting digestion.

## Contribution

The study reveals that thymol has dose-dependent effects on rumen microbial function, particularly at 240 mg/kg forage intake, which optimally reduces methane while supporting metabolism.

## Key findings

- Moderate thymol doses (240 mg/kg) reduced methane-related genes and increased energy and protein metabolism genes.
- Thymol's effects on microbial populations followed a quadratic pattern, with higher doses sometimes reducing beneficial microbes.
- 240 mg/kg thymol appeared to balance methane reduction and microbial activity without harming diversity.

## Abstract

Essential oils (EOs) are fed to cattle to improve productivity and decrease methane production. Essential oils contain terpenes and terpenoids, both of which affect microbes in the rumen, the major digestive compartment of the stomach in cattle. It is important to identify how these terpenoids impact microbial populations to help producers maximize the impact of EOs on cattle performance. Some terpenoids, such as thymol, have strong antimicrobial effects, which could impact the animal’s ability to digest feed. To investigate microbial responses to EOs, we fed increasing amounts of thymol to steers and assessed changes in the rumen microbes. Feeding cattle thymol changed the rumen microbiome in dose-dependent ways. Most effects followed a curved (quadratic) pattern rather than a straight-line (linear) response. At moderate levels (240 mg/kg forage intake), thymol reduced genes linked to methane production and increased genes related to energy and protein metabolism, suggesting potential benefits for animal performance. Higher doses affected microbes differently, sometimes reducing beneficial groups. Overall, thymol appears to influence how rumen microbes function beyond just affecting which species are present, with a dose of 240 mg/kg forage intake striking the best balance between reducing methane potential and supporting microbial activity.

While essential oils are gaining momentum as a strategy to modulate rumen function and potentially reduce enteric methane in cattle, little is known about how their bioactive components, terpenes, affect rumen microbes. Our objective was to evaluate how in vivo doses of thymol affect the structure and function of the rumen microbial community via whole genome shotgun sequencing (WGS). Four beef steers were used in a 4 × 4 Latin square with four 28 d periods. Steers consumed ad libitum forage and received one of four thymol doses (0 [CON], 120 [120-T], 240 [240-T], and 480 [480-T] mg/kg forage intake). Rumen contents were separated into liquid and solid fractions, DNA was extracted, analyzed via WGS, and assessed with orthogonal contrasts. After FDR correction, no taxa were affected by thymol; however, raw p-values demonstrated responses to thymol supplementation for solid-associated uncultured Lachnospiraceae bacterium (p = 0.04), uncultured Methanobrevibacter (p = 0.05), and uncultured Coriobacteriaceae bacterium (p = 0.02). Liquid-associated uncultured Prevotellaceae bacterium (p = 0.03), Prevotella sp. (p = 0.04), and Bacteroides sp. (p = 0.02) also responded to thymol, with the highest abundances observed at various thymol doses. Genes involved in energy production and amino acid metabolism transport were observed at the highest abundances at 240-T, while genes associated with cell cycle control, cell division, and chromosome partitioning were present in the highest abundances at 120-T. The findings suggest that thymol exerts dose-dependent effects on rumen microbial abundances and functional pathways, with 240 mg/kg forage intake appearing to be the most effective dose to downregulate methanogenic enzymes while also enhancing the enzymes associated with metabolism without negatively impacting microbial diversity.