Oral administration of Pinus koraiensis cone essential oil reduces rumen methane emission by altering the rumen microbial composition and functions in Korean native goat (Capra hircus coreanae)

Abstract

This study aimed to investigate Pinus koraiensis cone essential oil (PEO) as a methane (CH₄) inhibitor and determine its impact on the taxonomic and functional characteristics of the rumen microbiota in goats. A total of 10 growing Korean native goats (Capra hircus coreanae, 29.9 ± 1.58 kg, male) were assigned to different dietary treatments: control (CON; basal diet without additive) and PEO (basal diet +1 g/d of PEO) by a 2 × 2 crossover design. Methane measurements were conducted every 4 consecutive days for 17-20 days using a laser CH₄ detector. Samples of rumen fluid and feces were collected during each experimental period to evaluate the biological effects and dry matter (DM) digestibility after PEO oral administration. The rumen microbiota was analyzed via 16S rRNA gene amplicon sequencing. The PEO oral administration resulted in reduced CH₄ emission (eructation CH₄/body weight^0.75, p = 0.079) without affecting DM intake; however, it lowered the total volatile fatty acids (p = 0.041), molar proportion of propionate (p = 0.075), and ammonia nitrogen (p = 0.087) in the rumen. Blood metabolites (i.e., albumin, alanine transaminase/serum glutamic pyruvate transaminase, creatinine, and triglyceride) were significantly affected (p < 0.05) by PEO oral administration. The absolute fungal abundance (p = 0.009) was reduced by PEO oral administration, whereas ciliate protozoa, total bacteria, and methanogen abundance were not affected. The composition of rumen prokaryotic microbiota was altered by PEO oral administration with lower evenness (p = 0.054) observed for the PEO group than the CON group. Moreover, PICRUSt2 analysis revealed that the metabolic pathways of prokaryotic bacteria, such as pyruvate metabolism, were enriched in the PEO group. We also identified the Rikenellaceae RC9 gut group as the taxa potentially contributing to the enriched KEGG modules for histidine biosynthesis and pyruvate oxidation in the rumen of the PEO group using the FishTaco analysis. The entire co-occurrence networks showed that more nodes and edges were detected in the PEO group. Overall, these findings provide an understanding of how PEO oral administration affects CH₄ emission and rumen prokaryotic microbiota composition and function. This study may help develop potential manipulation strategies to find new essential oils to mitigate enteric CH₄ emissions from ruminants.

Keywords: enteric methane emission; essential oil; feed additives; metataxonomic; rumen microbiota.

Figure 1

Principal coordinate analysis (PCoA) of the rumen microbiota based on the matrices of (A) Bray–Curtis dissimilarity and (B) Weighted UniFrac distance. CON, without PEO; PEO, Pinus koraiensis cone essential oil.

Figure 2

Rumen bacterial compositional profiles of goats. (A) Relative abundance of major bacteria phyla (relative abundance ≥ 0.01% in more than 50% animals) for all individuals. (B) Relative abundance of major bacteria families (relative abundance ≥ 0.01% in more than 50% animals) for all individuals. (C) Cladogram and (D) LDA effect size of significantly different taxa identified in the rumen microbiota data sets of goats based on the cutoff LDA ≥ 2.0 and p < 0.05. Only major classified taxa (each representing ≥ 0.01% in at least one of the dietary treatments) were visualized. CON, without PEO; PEO, Pinus koraiensis cone essential oil; LDA, linear discriminant analysis.

Figure 3

Quantification of selected bacterial species in the samples using real-time PCR and 16S rRNA analysis. The box in the box plots indicates the interquartile range (IQR; middle 50% of the data), and the middle line represents the median value. Data points represent individual animals. CON, without PEO; PEO, Pinus koraiensis cone essential oil.

Figure 4

Differentially abundant KEGG modules and pathways affected by (A) CON and (B) PEO oral administration were detected using LEfSe with an LDA ≥ 2.0. Only the functional parameters accounting for ≥0.01% average relative abundance in at least one of the treatments were statistically analyzed by LEfSe. The box in the box plots indicates the interquartile range (IQR; middle 50% of the data), and the middle line represents the median value. Data points represent individual animals. CON, without PEO; PEO, Pinus koraiensis cone essential oil; KEGG, Kyoto Encyclopedia of Genes and Genomes; LDA, linear discriminant analysis; LEfSe, LDA effect size; PRPP, phosphoribosyl diphosphate.

Figure 5

FishTaco analysis of genera contribution to KEGG modules. The x-axis represents the Wilcoxon test statistic scores, and the y-axis represents the related functions. The driving factors for each differential function transformation were divided into four parts, represented by a histogram in two directions. Rumen microbiota in the PEO group drove the increase in the corresponding functional abundance (top right). Rumen microbiota in the PEO group inhibited the proliferation in the related practical quantity (top left). Rumen microbiota in the CON group drove the increase in the related functional mass (bottom right). Rumen microbiota in the CON group inhibited proliferation in the corresponding available abundance (bottom left). Different color bars represent the related genera. The longer the bar, the greater the driving or inhibitory effect of the species on the corresponding function. Only genera accounting for ≥0.01% average relative abundance in at least one of the treatments were used. CON, without PEO; PEO, Pinus koraiensis cone essential oil; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

Exclusive co-occurrence and mutual exclusion microbial network in (A) CON and (B) PEO oral administration. The node color represents exclusive microbial nodes (blue) and keystone genera (green) selected based on the authority and eigenvector centrality measurements within each exclusive network. The edge color represents co-occurrence (blue) or mutual exclusive (red) interactions. Edge thickness was adjusted based on the absolute value of the correlation coefficients of each interaction. Only genera accounting for ≥0.01% average relative abundance in at least one of the treatments were used. CON, without PEO; PEO, Pinus koraiensis cone essential oil.

Figure 7

Spearman’s correlation coefficients between measurements (rumen fermentation and methane parameters) with differentially abundant prokaryotic taxa (including absolute abundance of methanogens and fungi). (|r| ≥ 0.5, P ≤ 0.05). DM, dry matter; NH₃-N, ammonia nitrogen; AP, acetate to propionate; BW, body weight; CH₄, methane; DMI, dry matter intake; DDMI, digestible dry matter intake.