A commercial concentrate mix for fattening beef was purchased from a feed company (Easybio, Inc., Seoul, Korea). The detailed chemical composition was as follows: dry matter (DM), 900 g/kg; crude protein, 255 g/kg; neutral detergent fiber (NDF), 246 g/kg; acid detergent fiber (ADF), 116 g/kg; lignin, 35.3 g/kg; ether extracts, 48.1 g/kg; ash, 87.3 g/kg; Ca, 4.3 g/kg; P, 7.8 g/kg. The experimental substrates were dried at 60°C for 96 h, and ground in a cyclone mill (Foss Tecator Cyclotec 1093, Foss, Hillerød, Denmark) fitted with a 1 mm screen, for later use. A commercial EOM (NOVATAN, Techna, France) was used in this study as a feed additive. According to the manufacturer, the main constituents in the EOM were cinnamaldehyde, thymol, and eugenol.

A complete randomized block design was used for the experiment, with treatment as the main effect. Three experimental treatments were used, i.e., control (CON, no additive), EOM 0.1 (1 g EOM/kg of experimental substrate), EOM 0.2 (2 g EOM/kg of experimental substrate). The amount of EOM added was determined based on the manufacturer’s recommendation.

In vitro batch culture fermentation was carried out using rumen fluid, which was collected before the morning feeding from two cannulated Korean native cattle heifers (body weight 450±30 kg) at Center for Animal Science Research, Gyeongsang National University, Korea. Animals were fed a diet consisting of 600 g/kg timothy hay and 400 g/kg of a commercial concentrate mix. The rumen contents were mixed, transferred into a thermos bottle, and immediately transported to the laboratory. Rumen contents were strained through 4 layers of cheesecloth and mixed with 3× volumes of in vitro rumen buffer solution [13] under strictly anaerobic conditions. Approximately 0.9 g DM of the ground experimental substrate and a corresponding quantity of EOM for each treatment were moved into pre-weighed filter bags (F57, Ankom Technology, Macedon, NY, USA). All bags were heat-sealed and transferred into empty 125 mL serum bottles, to which 50 mL of rumen fluid and buffer mixture were transferred accompanied by continuously flushing with O₂-free CO₂ gas. The bottles were sealed with butyl rubber stoppers and aluminum caps and incubated on a rotary shaker (JSSI-300T, JS Research Inc., Gongju, Korea) at 125 rpm, for 12 and 24 h at 39°C. Three replicates were prepared for each treatment and each incubation time. After each incubation period, the three replicates of each treatment were removed from the incubator, and total gas production was measured using a pressure transducer (Sun Bee Instrument Inc., Seoul, Korea) as described by Theodorou et al [14]. Subsequently, the bottles were immediately placed on ice and the caps were opened to stop the fermentation; the filter bags were removed from bottles and rinsed with flowing water until the water ran clear. The pH of the culture fluid was measured using a pH meter (FP20, Mettler Toledo, Columbus, OH, USA). Sample fluid (5 mL) was stored at −80°C until rumen microbial population analysis. The remaining culture fluid was then centrifuged at 15,000×g for 10 min at 4°C and stored at −20°C, until the analysis of volatile fatty acids (VFA) and ammonia nitrogen (NH₃-N) concentrations. The washed bags were dried at 55°C for 48 h and weighed to measure in vitro DM degradability (IVDMD). The NDF content was assessed for the weighed bags using a modified version of the micro-NDF method proposed by Pell and Schofield [15] to evaluate in vitro NDF degradability (IVNDFD).

Collected rumen fluid samples were thawed in a refrigerator at 4°C, and centrifuged at 16,000×g and 4°C for 5 min. The supernatant was discarded, and the remainder was resuspended in 800 μL of 1× phosphate-buffered saline solution to wash the pellet. This was recentrifuged (16,000×g, 4°C, 5 min) and the supernatant was discarded. Total DNA was extracted from the washed pellet using a NucleoSpin DNA Stool kit (MACHEREY-NAGEL GmbH Co. KG, Düren, Germany), according to the manufacturer’s instructions. The concentration and purity of total DNA were measured using a NanoDrop (ND-1000, Thermo Fisher, Waltham, MA, USA).

The information on primer sequences of rumen microbes was collected from previous studies [16,17] and presented in Table 1. Conventional polymerase chain reaction (PCR) was performed to validate the specificity of the primer in 50 μL reaction volumes using AccuPower PCR PreMix K-2013 (Bioneer, Daejeon, Korea), by adding 2 μL each of forward and reverse primers, 2 μL of genomic DNA and 44 μL of PCR grade water. Reactions were performed using a C1000 Thermal cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initialization for one cycle at 95°C for 10 min; 35 cycles each for denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s; and final elongation at 72°C for 5 min. The PCR products were analyzed by electrophoresis using 1.5% agarose gel impregnated with Staining STAR (DYNEBIO INC, Seongnam, Korea), to confirm a single specific band and the absence of primer dimer products.

Real-time quantitative PCR assays were performed on a CFX 96 Touch system (Bio-Rad Laboratories, lnc. USA). Reactions were performed in triplicate with reaction volumes of 20 μL using optical reaction plates sealed with optical adhesive film. Each reaction mixture contained 0.5 μL 10 mM dNTP Mix (BioFACT, Daejeon, Korea), 2 μL 10× buffer (BioFACT, Korea), 1 μL 40 ng genomic DNA, 1 μL forward primer (10 pmol), 1 μL reverse primer (10 pmol), 0.1 μL Taq polymerase (BioFACT, Korea), 1 μL Evagreen (SolGent Co., Ltd., Daejeon, Korea) and 13.4 μL PCR grade water. Real-time PCR was carried out according to the manufacturer’s instructions, as follows: initialization for one cycle at 95°C for 10 min; 40 cycles each for denaturation at 95°C for 30 s, annealing at 60°C for 30 s and elongation at 72°C for 30 s; and final elongation at 72°C for 5 min. Fluorescence was recorded at the end of each denaturation and extension step, and specificity of amplicon was confirmed via dissociation curve analysis of PCR end products by increasing the temperature at a rate of 1°C after every 30 s, from 60°C to 95°C. The threshold cycle (Ct) was analyzed and transformed using a standard curve with CFX manager software (Bio-Rad, USA).

To evaluate the efficiency (E) of amplification of each primer set, a 6-point standard curve and a nontemplate control (NTC) were run for each sample to test the relative expression levels. The standard curve was generated using genomic DNA (40 μg/reaction mixture), which was serially diluted. Amplification efficiency was calculated from the slope of the standard curve generated by plotting the threshold cycle (Ct) against logarithmic values of different DNA concentrations using the equation, E = 10^−1/slope. The gene ‘general bacteria’ was used as a reference gene. Relative quantification of microbes was carried out using the 2^−ΔΔCt method as follows [18,19]:

Statistical analysis of the data was performed using the MIXED procedure of SAS 9.3 (SAS institute Inc., NC, USA). The fixed effect in this model was a feed treatment. Orthogonal contrast was used to analyze linear and quadratic effect when the level of EOM supplementation increased. Differences among treatments were compared using Tukey’s range test if a significant effect was observed. Statistical significance was declared at p<0.05 and a trend was speculated at 0.05≤p<0.10.