A randomized complete block design was used to determine the effects of 4 concentrations of 4 essential oils (EO) on in vitro ruminal fermentation variables. In vitro fermentation consisted of 0.7 g of high concentrate substrate (86.7% DM) and 50 mL of 2:1 buffer:ruminal fluid inoculum incubated for 24 h for each batch (n = 3; separate days) Treatments were arranged as a 4 × 5 factorial. Factors included 4 EO (eugenol, cinnamic aldehyde, anethole, and garlic oil) at 5 concentrations (0, 10, 75, 200, and 400 mg/L of inoculum). Data were analyzed using the MIXED procedure of SAS with the fixed effects of EO, concentration, and their interaction, and random effect of day (block). Batch was considered the experimental unit. There was an interaction (P < 0.001) for total gas production, where a cubic effect (P ≤ 0.041) was observed for eugenol, cinnamic aldehyde, and anethole, and a quadratic effect (P = 0.001) was observed for garlic oil. No interactions (P > 0.05) were observed for in vitro OM digestibility (IVOMD) or CH4 production. There was an effect of EO (P < 0.001) on IVOMD, where eugenol reduced (P ≤ 0.007) digestibility compared with anethole and garlic oil, which promoted the greatest (P ≤ 0.029) IVOMD. Methane production (mmol/g OM fermented) was affected by EO (P < 0.001), where it was decreased (P ≤ 0.001) by garlic oil compared with all other EO. There was an interaction (P < 0.001) for H2S production (µmol/g OM fermented), where it was linearly decreased (P = 0.003) and linearly increased (P < 0.001) as concentrations of eugenol and garlic oil increased, respectively. These EO had contradictory impacts on in vitro ruminal fermentation, thus combining them could potentially improve multiple aspects of in vitro and in vivo fermentation.
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