Thymol is an antimicrobial terpene with potential as a feed additive for cattle; however, in vivo data describing the response of forage-fed beef cattle to the increasing provision of thymol is limited. As thymol may affect palatability and exert antimicrobial effects on rumen microbes, defining its effects in vivo is critical to inform adoption. Accordingly, the objectives of this study were to evaluate cattle acceptance of thymol and characterize the effects of increasing thymol doses on diet utilization and ruminal fermentation, with the overarching goal of identifying a maximum tolerable dose for beef cattle. Two 4 × 4 Latin Square experiments were conducted using beef steers consuming forage and providing thymol dosed on alfalfa. Experiment 1 assessed acceptance of thymol at increasing concentrations (0, 110, 220, and 330 mg/kg intake), and experiment 2 assessed diet utilization and fermentation in response to one of four thymol doses: 0, 120, 240, and 480 mg/kg intake. For experiment 1, thymol dose did not affect treatment or forage intake (p ≥ 0.17). For experiment 2, thymol did not linearly or quadratically affect (p ≥ 0.28) forage organic matter (OM) intake or OM digestibility. There were also no linear or quadratic effects (p ≥ 0.09) on ruminal ammonia-N, volatile fatty acids, or pH. These data indicate that thymol can be provided to beef cattle at up to 330 mg/kg intake and 480 mg/kg intake without negatively impacting acceptance and diet utilization, respectively.

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.

Previous research has established distinctions between microbial communities that associate with either the solid or liquid rumen microenvironments utilizing 16S rRNA sequencing. Whole genome metagenomic sequencing (WGS) facilitates comprehensive analysis of a microorganism’s entire genome, producing strain level data and providing insights into microbial functionality. The objective of our study was to utilize WGS to characterize microbial species and enzyme profiles associated with the liquid versus solid rumen microenvironments in beef steers consuming forage. Four steers consuming forage were utilized in a 4 × 4 Latin Square experiment; on d 14 of each period, rumen contents were separated into solid and liquid samples and DNA was extracted then sequenced using WGS. Alpha and beta diversity measures revealed differences between solid and liquid microenvironments. There were also differences in enzyme profiles: carbohydrate-active enzyme classes were different across microenvironments, except glycosyl transferase. Further, the solid microenvironment was enriched in enzymes related to energy production/conversion and nutrient transport and metabolism, while the liquid microenvironment was enriched in coenzyme transport and metabolism enzymes. Species associated with cellulolytic activities were generally more abundant in the solid microenvironment while those associated with amylolytic activity were more abundant in the liquid. Genes related to methanogenesis were also different across microenvironments. Our findings offer valuable insights into the stratification of microorganisms across rumen microenvironments, expanding on existing literature with findings from next-generation sequencing and application to forage-based beef systems.