Assessment of the Pinus koraiensis cone essential oil on methane production and microbial abundance using in vitro evaluation system

Abstract

The objective of this study was to evaluate the potential of Pinus koraiensis cone essential oil (PEO) on in vitro fermentation, methane (CH4) production and microbiota quantification in three different in vitro experiments. First, the main active compounds present in PEO were identified and quantified. Subsequently, the in vitro fermentation characteristics and CH4 production after PEO supplementation (0.20 g/L) at different ratios of forage-to-concentrate (F:C, 100:0, 70:30, 50:50, 30:70, and 0:100; Experiment 1) were evaluated. At a F:C ratios of 50:50, control (CON) and PEO supplementation led to similar total volatile fatty acid concentrations, while CH4 production (13.7%) significantly reduced. Based on this result, we determined that 50:50 F:C ratio can serve as an appropriate diet ratio to evaluate dose-response PEO supplementation. The effects of three levels of PEO supplementation (0.20, 0.50, and 1.0 g/L) on CH4 production, fermentation, and microbiota were then determined in vitro (Experiment 2). Methane production decreased (P < 0.001) with increasing PEO supplementation, with a reduction of up to 65% at 1.0 g/L levels at 24 h of incubation compared with that in the CON. However, PEO supplementation exhibited adverse effects (P < 0.05) on the abundance of most ciliate protozoa, fungi, and fibrolytic bacteria, including Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens. In contrast, the abundance of CH4-producing archaea (total methanogens and Methanobrevibacter sp. AbM4) was greater (P < 0.05) than that in the CON. Furthermore, the effects of PEO supplementation using in vitro continuous culture system for a total of 16 d (including 7 d for adaptation and 9 d for fermentation; Experiment 3) were examined. Methane production was reduced (P < 0.001) (47.1%) upon PEO supplementation compared with that in the CON. Similar to Experiment 2, in vitro fermentation and microbiota were affected (P < 0.05) in response to PEO supplementation. In all three experiments, butyrate was greater (P < 0.001) under PEO supplementation than that in the CON. In addition, butyrate-producing bacteria (Butyrivibrio proteoclasticus) were less sensitive to PEO and contributed to the increased butyrate in the continuous cultures. However, all three in vitro results showed that reduction in CH4 production was accompanied by decreased fermentation parameters. These results suggest that although PEO supplementation can reduce CH4 production, it can alter in vitro fermentation and microbiota, such that the supplementation of a great concentrations of PEO may not be beneficial to ruminants.