DOI: 10.6084/m9.figshare.31374837.v1
title: Additional file 1 of Comparative analysis of rumen metagenomes with dietary supplementation of 3-nitrooxypropanol revealed divergent modes of action in hydrogen metabolism and reductant pathways between beef and dairy cattle

description: Additional file 1: Figure S1. Schematic representation of the four in vivo trials used in the comparative analysis, including short-term and long-term 3-NOP supplementation studies in beef and dairy cattle (Beef1: Romero-Perez et al., 2014 [9]; Beef2: Romero-Perez et al., 2015 [10]; Dairy1: Haisan et al., 2014 [15]; Dairy2: Haisan et al., 2017 [16]). Figure S2. Effect of short-term 3-nitrooxypropanol (3-NOP) supplementation on the abundance of A bacterial, B archaeal, and C protozoal taxa in beef cattle. *3-NOP dose level information: con: 0, low: 53, med: 161, high: 345 mg/kg of DM. Others indicates taxa with less than 5% abundance; UCF: uncultured family-level; UCG: uncultured genus-level; UG: unclassified genus-level. Figure S3. Effect of long-term 3-nitrooxypropanol (3-NOP) supplementation on the abundance of A bacterial, B archaeal, and C protozoal taxa in beef cattle. *3-NOP dose level information: con: 0, high: 280 mg/kg of DM. Others indicates taxa with less than 5% abundance; UCG: uncultured genus-level; UG: unclassified genus-level; recov: recovery period. Figure S4. Effect of 3-nitrooxypropanol (3-NOP) supplementation on the abundance of A bacterial, B archaeal, and C protozoal taxa in dairy cattle. *3-NOP dose level information: con: 0, high: 130 mg/kg of DM. Others indicates taxa with less than 5% abundance; UCG: uncultured genus-level; UG: unclassified genus-level. Figure S5. Dose response effect of 3-nitrooxypropanol (3-NOP) supplementation on the abundance of A bacterial, B archaeal, and C protozoal taxa in dairy cattle. *3-NOP dose level information: con: 0, low: 68, high: 132 mg/kg of DM. Others indicates taxa with less than 5% abundance; UCG: uncultured genus-level; UG: unclassified genus-level. Figure S6. Alpha diversity and beta diversity analysis of rumen microbiota before and after batch correction. Alpha diversity was measured by Shannon index in A bacteria, B archaea, and C protozoa of control and 3-NOP treated groups. P values were calculated using Wilcoxon rank sum test, with significance set at P &lt; 0.05. Principal coordinate analysis (PCoA) plots of D bacteria, E archaea, and F protozoa were based on Bray-Curtis distance. P values were calculated with PERMANOVA (999 permutations). Figure S7. Alpha diversity (Chao1 and evenness) was measured from metataxonomic data (A Beef1, B Beef2, C Dariy1; and D Dairy2). The Kruskal-Wallis test with Dunn’s post-hoc test was applied to Beef1 and Dairy2, while the Wilcoxon test was used for Beef2 and Dairy1. P-values were FDR-adjusted, with &lt; 0.05 considered statistically significant. Figure S8. Beta diversity was measured from metataxonomic data (A Beef1, B Beef2, C Dariy1; and D Dairy2). Principal coordinate analysis (PCoA) plots of bacteria, archaea, and protozoa were based on Weighted Unifrac distance. P values were calculated with PERMANOVA (999 permutations). Figure S9. Effect of short-term 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases and associated terminal reductases in beef cattle. A indicates distributions (in phyla with hydrogenase-encoding genes) of fermentative hydrogenases (group A1, A2, and B FeFe-hydrogenases), bifurcating hydrogenases (group A3 FeFe-hydrogenases), respiratory hydrogenases (group 1i NiFe-hydrogenases), methanogenic hydrogenases (Fe-hydrogenases, group 3a, 3c, 4 h, and 4i NiFe-hydrogenases), sensory hydrogenases (group C FeFe-hydrogenases), and energy-converting hydrogenases (bidirectional; group 4e and 4 g NiFe-hydrogenases). B indicates the H2 uptake pathway includes genes involved in sulfidogenesis (asrA, alternative sulfite reductase; dmsA, DMSO, and TMAO reductase), fumarate reduction (frdA, fumarate reductase), nitrate ammonification (nrfA, ammonia-forming nitrite reductase), and nifH, nitrogenase and methanogenesis (mcrA, methyl-CoM reductase). Only genes with an average relative abundance (CPM: count per million) &gt; 1 are shown. Data with error bars are indicated as mean ± standard error. Asterisks indicate significance: *P &lt; 0.1, **P &lt; 0.05. Figure S10. Effect of long-term 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases and associated terminal reductases in beef cattle. A indicates distributions (in phyla with hydrogenase-encoding genes) of fermentative hydrogenases (group A1, A2, and B FeFe-hydrogenases), bifurcating hydrogenases (group A3 FeFe-hydrogenases), respiratory hydrogenases (group 1i NiFe-hydrogenases), methanogenic hydrogenases (Fe-hydrogenases, group 3a, 3c, 4 h, and 4i NiFe-hydrogenases), sensory hydrogenases (group C FeFe-hydrogenases), HydB, hydrogenase-associated diaphorase, energy-converting hydrogenases (bidirectional; group 4e and 4g NiFe-hydrogenases). B indicates the H2 uptake pathway includes genes involved in sulfidogenesis (asrA, alternative sulfite reductase; dmsA, DMSO, and TMAO reductase), fumarate reduction (frdA, fumarate reductase), nifH, nitrogenase, nitrate ammonification (nrfA, ammonia-forming nitrite reductase), methanogenesis (mcrA, methyl-CoM reductase). Only genes with a relative abundance (CPM: count per million) &gt; 1 are shown. Data with error bars are indicated as mean ± standard error. Asterisks indicate significance: *P &lt; 0.1, **P &lt; 0.05. Figure S11. Effect of 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases and associated terminal reductases in dairy cattle. A indicates distributions (in phyla with hydrogenase-encoding genes) of fermentative hydrogenases (group A1, A2, and B FeFe-hydrogenases), bifurcating hydrogenases (group A3 FeFe-hydrogenases), respiratory hydrogenases (group 1d and 1i NiFe-hydrogenases), methanogenic hydrogenases (Fe-hydrogenases, group 3a, 3c, 4 h, and 4i NiFe-hydrogenases), sensory hydrogenases (group C FeFe-hydrogenases), HydB, hydrogenase-associated diaphorase, and energy-converting hydrogenases (bidirectional; group 4e and 4 g NiFe-hydrogenases). B indicates the H2 uptake pathway includes genes involved in sulfidogenesis (asrA, alternative sulfite reductase), aerobic respiration (cydA, cytochrome bd oxidase), fumarate reduction (frdA, fumarate reductase), nifH, nitrogenase, nitrate ammonification (narG, dissimilatory nitrate reductase; nrfA, ammonia-forming nitrite reductase), and methanogenesis (mcrA, methyl-CoM reductase). Only genes with a relative abundance (CPM: count per million) &gt; 1 are shown. Data with error bars are indicated as mean ± standard error. Asterisks indicate significance: *P &lt; 0.1, **P &lt; 0.05. Figure S12. Dose response effect of 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases and associated terminal reductases in dairy cattle. A indicates distributions (in phyla with hydrogenase-encoding genes) of fermentative hydrogenases (group A1, A2, and B FeFe-hydrogenases), bifurcating hydrogenases (group A3 FeFe-hydrogenases), respiratory hydrogenases (group 1i NiFe-hydrogenases), methanogenic hydrogenases (Fe-hydrogenases, group 3a, 3c, 4 h, and 4i NiFe-hydrogenases), sensory hydrogenases (group C FeFe-hydrogenases), HydB, hydrogenase-associated diaphorase, and energy-converting hydrogenases (bidirectional; group 4e and 4g NiFe-hydrogenases). B indicates the H2 uptake pathway includes genes involved in sulfidogenesis (asrA, alternative sulfite reductase; dsrA, dissimilatory sulfite reductase), fumarate reduction (frdA, fumarate reductase), and nifH, nitrogenase, nitrate ammonification (nrfA, ammonia-forming nitrite reductase), and methanogenesis (mcrA, methyl-CoM reductase). Only genes with an average relative abundance (CPM: count per million) &gt; 1 are shown. Data with error bars are indicated as mean ± standard error. Asterisks indicate significance: *P &lt; 0.1, **P &lt; 0.05. Figure S13. Effect of 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases, associated terminal reductases, and electron transferases in beef cattle. A results were obtained from a comparative analysis using MMUPHin and further validated by MaAsLin2 analysis. The gray, yellow, and skyblue strips represent hydrogenases, associated terminal reductases, and electron transferases, respectively. The comparative analysis was conducted with a threshold of Q &lt; 0.05 and cross-verified by MaAsLin2 with Q &lt; 0.05. CPM: count per million; DM: dry matter. Figure S14. Effect of 3-nitrooxypropanol (3-NOP) supplementation on the distributions of hydrogenases and associated terminal reductases in dairy cattle. A results were obtained from a comparative analysis using MMUPHin and further validated by MaAsLin2 analysis. The gray and skyblue strips represent hydrogenases and associated terminal reductases, respectively. The comparative analysis was conducted with a threshold of Q &lt; 0.05 and cross-verified by MaAsLin2 with Q &lt; 0.05. CPM: count per million; DM: dry matter.