Buccal Swab Samples from Japanese Brown Cattle Fed with Limonite Reveal Altered Rumen Microbiome

Abstract

The areas of the Mount Aso grasslands in Kumamoto, Japan, are the primary location for the breeding of the Kumamoto strain of Japanese Brown cattle (JBRK). Although Aso limonite, deposited by volcanic ash and magma, has been commonly fed to pregnant JBRK in this area, the mechanisms of its salutary effects on pregnant JBRK have not yet been elucidated. Approximately 100 days before the expected day of calf delivery, seven JBRK (four supplemented with limonite and three controls without limonite) were assigned to this study, from which a buccal swab was collected at the highest rumination every 30 days for 90 days. DNA extracted from these swabs was then analyzed using a 16S rRNA gene amplicon sequence analysis. Statistically significant differences between the two groups were discovered through beta-diversity analysis, though results from alpha-diversity analysis were inconclusive. The microbiota identified were classified into six clusters, and three of the main clusters were core-rumen bacteria, primarily cellulose digestion in cluster 1, oral bacteria in cluster 2, and non-core-rumen bacteria in cluster 3. In the limonite group, core-rumen bacteria decreased while non-core-rumen bacteria increased, suggesting that limonite feeding alters rumen microbiota, particularly activation of non-core-rumen microbiota.

Keywords: Aso limonite; Kumamoto strain of Japanese brown cattle (JBRK); correlation analysis; microbiota; rumen metagenome.

Figure 1

Beta diversity of buccal swab microbiome. Beta diversity is shown using 3D plots of Bray–Curtis distances. ○ 30 days, △ 60 days, □ 90 days following the initiation of limonite (blue) or not limonite (control, red) feeding.

Figure 2

Beta diversity of buccal swab microbiome. Statistical differences in the structure between groups were detected by ANOSIM. Differences in groups of samples from one another were calculated using a permutation-based statical test. The ‘n’ corresponds to the calculation times that were performed at each group in the statistical analysis. Distance to Control is shown on the left and distance to limonite is presented on the right. * denotes statistical significance at p < 0.001.

Figure 3

Relative abundance of bacterial taxa at the phylum levels. Bacterial taxa in the rumen microbe composition of each JBRK without (control) or with limonite feeding are shown.

Figure 4

Relative abundance of bacterial taxa at the genus levels. Bacterial taxa in the rumen microbe composition of each JBRK without (control) or with limonite feeding are shown.

Figure 5

A heatmap chart of taxa abundance. Clusters 1 and 2 correspond to the abundance of taxa in the control and limonite groups, respectively.

Figure 6

Correlation network analysis of rumen microbiota. A structural representation of the entire microbial community in the rumen. The structure is divided into 6 main clusters denoted by color (cluster 1, pale orange; 2, pale blue; 3, green; 4, yellow; 5, deep blue; 6, orange). In cluster 1, the three “Hub nodes” (denoted as squares) (Pyramidobacter species, and unidentified species of order Bacteroidales, and Desulfuromonadales) are significantly more connected within the network than other nodes.

Figure 7

Correlation network analysis of rumen microbiota in the control or limonite group. A structure of the whole microbial community in the rumen of JBRK cattle fed (B) limonite or (A) not (control): (A) Among 261 taxa in the control group and 405 taxa in the limonite group, “hub” node was not found in this separate analysis. In the control group, two major and eight minor clusters were found: 127 taxa in cluster 1 (green), most of which belong to core-rumen bacteria; 129 taxa in pale orange, consisting of those in clusters 2–10; and 5 taxa in pale blue, consisting of clusters 1, 2, and 6. (B) In the limonite group, there are four major and six minor clusters: 94 taxa in cluster 1, related to the core-rumen bacteria (deep blue); 150 taxa in cluster 2 with oral bacteria (orange); 118 taxa in cluster 3 with non-core-rumen bacteria (yellow); 28 taxa in cluster 2 and 6, having oral bacteria (pale blue); 150 taxa, consisting of oral bacteria (orange); 9 taxa in cluster 4 (pale orange); 6 taxa in clusters 1, 2, and 5 (green).