Sequencing of rumen samples
A total of 192 samples of rumen fluid and digesta were sequenced, resulting in 11,657,704 non-chimeric sequences after quality control and the identification of 6,217 unique ASVs from all samples. The minimum number of sequences per sample was 36,270 and the maximum was 95,904. Rarefaction analysis show that sequencing depth was sufficient to capture most of the microbial diversity in the samples with rarefaction curves for all samples approaching an asymptote.
Addition of 3-NOP and OIL reduced microbial diversity
The effect of 3-NOP, OIL and 3-NOP + OIL as a function of time on the alpha diversity of the rumen samples is shown in Table 1. Supplementing diets with 3-NOP resulted in a numerical reduction in observed ASVs at 0 h (P = 0.095), and a significant decrease in ASVs 6 and 12 h after feeding (P ≤ 0.05). 3-NOP decreased the phylogenetic diversity of rumen fluid at all of the sampling time points (P ≤ 0.05). The inclusion of OIL significantly decreased the number of ASVs and phylogenetic diversity of rumen fluid at all of the time points (P ≤ 0.001). No interactions between 3-NOP and OIL were found on the alpha diversity of rumen fluid. 3-NOP decreased alpha diversity of rumen digesta at 12 h (P ≤ 0.05) but there was no significant effect at 0 and 6 h (P > 0.05). The addition of OIL decreased the observed ASVs and phylogenetic diversity of rumen digest at all of the time points (P < 0.001). There was an interaction between 3-NOP and OIL on the phylogenetic diversity in rumen digesta at 0 h (P = 0.024) but not at 6 and 12 h.
Table 1
The impact of feeding diets supplemented with (+) and without (-)3-NOP and OIL on the α-diversity of the rumen microbial community at 0, 6 and 12 h after feeding in beef cattle1.
Item | -3-NOP | | + 3-NOP | SEM | | P-value | |
-OIL | +OIL | | -OIL | +OIL | 3-NOP | OIL | 3-NOP × OIL |
Rumen Fluid | | | | | | | | | |
0 h | | | | | | | | | |
Observed ASVs | 560 | 406 | | 501 | 383 | 23.7 | 0.095 | < 0.001 | 0.46 |
Phylogenetic diversity | 45.5 | 34.9 | | 41.5 | 33.4 | 0.72 | 0.014 | < 0.001 | 0.25 |
6 h | | | | | | | | | |
Observed ASVs | 536 | 379 | | 457 | 335 | 28.8 | 0.021 | < 0.001 | 0.49 |
Phylogenetic diversity | 43.8 | 33.7 | | 38.2 | 30.8 | 1.23 | 0.001 | < 0.001 | 0.24 |
12 h | | | | | | | | | |
Observed ASVs | 528 | 393 | | 429 | 344 | 32.1 | 0.002 | < 0.001 | 0.27 |
Phylogenetic diversity | 43.0 | 34.1 | | 37.7 | 31.4 | 1.28 | 0.013 | 0.001 | 0.37 |
Rumen Digesta | | | | | | | | | |
0 h | | | | | | | | | |
Observed ASVs | 820 | 614 | | 738 | 657 | 32.3 | 0.55 | < 0.001 | 0.062 |
Phylogenetic diversity | 54.8a | 42.6c | | 50.3b | 43.6c | 1.47 | 0.14 | < 0.001 | 0.024 |
6 h | | | | | | | | | |
Observed ASVs | 747 | 592 | | 696 | 603 | 17.7 | 0.46 | < 0.001 | 0.26 |
Phylogenetic diversity | 50.3 | 42.4 | | 48.2 | 42.1 | 1.69 | 0.37 | < 0.001 | 0.49 |
12 h | | | | | | | | | |
Observed ASVs | 810 | 604 | | 686 | 582 | 7.1 | 0.017 | < 0.001 | 0.084 |
Phylogenetic diversity | 52.5 | 42.1 | | 47.5 | 40.3 | 2.44 | 0.006 | < 0.001 | 0.14 |
1OIL = canola oil; 3-NOP = 3-nitrooxypropanol. |
a,b,c Values within a row with different letters differ (P ≤ 0.05) |
Effect of 3-NOP and OIL supplementation on overall microbiome composition
Rumen fluid and rumen digesta samples clustered separately in PCoA plots based on both weighted and unweighted UniFrac calculations (P < 0.001; Fig. 1). Samples also clustered separately based on treatment in PcoA plots based on both weighted and unweighted UniFrac distances. For all plots, the control samples clustered distinctly from 3-NOP, OIL, or 3-NOP + OIL (P < 0.001) samples. The inclusion of OIL alone had the largest effect on the composition of the microbial community and with samples containing OIL significantly separated from control and 3-NOP samples (P < 0.001). Sampling time did not have a significant effect on the clustering of samples (P ≥ 0.26); however, a PERMANOVA analysis revealed that samples collected prior to morning feeding were different from 6 and 12 h samples (P = 0.006). There was no significant difference between 6 and 12 h samples (P = 0.56).
Effect of 3-NOP and OIL on rumen methanogens
The total number of ASVs that were assigned to the phylum Euryarchaeota in rumen digesta samples was significantly higher than in rumen fluid samples (Fig. 2). Methanobrevibacter was the most abundant methanogen in both rumen solids and digesta. Sampling time did not significantly affect methanogen abundance in either rumen fluid or digesta (P ≥ 0.30); however, 3-NOP alone and in combination with OIL had a significant effect on methanogen abundance in both rumen fluid and digesta (P < 0.001). 3-NOP (P < 0.01) and 3-NOP + OIL (P < 0.001) significantly reduced the total number of Euryarchaeota ASVs in rumen fluid and rumen digesta samples (Fig. <link rid="fig2">2</link>A and 2, respectively). The effects of 3-NOP and OIL on microbial abundance were generally independent of one another but there was a significant interaction between 3-NOP and OIL on the relative abundance of Euryarchaeota (P < 0.05, Tables 2 and 3). 3-NOP and 3-NOP + OIL caused significant decreases in the abundance of Methanobrevibacter (P < 0.05), Methanomicrobium (P < 0.001), Methanomethylophilus (P < 0.001), and an uncultured genus of Thermoplasmatales (P < 0.001). The addition of OIL decreased the abundance of Euryarchaeota in rumen fluid (P < 0.01). In contrast, the addition of OIL resulted in a significant increase in the abundance of Euryarchaeota in rumen digesta (P < 0.05). The effects observed for OIL treatment resulted in broad spectrum changes in the methanogen community and could not be attributed to a change in the abundance of a specific methanogen genus.
Table 2
Impact of feeding diets supplemented with (+) and without (-) 3-NOP and OIL on the relative abundance of phyla identified in rumen fluid in beef cattle1
Item | -3-NOP | | + 3-NOP | SEM | | P-value | |
-OIL | +OIL | | -OIL | +OIL | 3-NOP | OIL | 3-NOP × OIL |
0 h | | | | | | | | | |
Euryarchaeota | 1.18 | 0.71 | | 0.62 | 0.21 | 0.165 | < 0.001 | 0.002 | 0.80 |
Bacteroidetes | 54.9b | 59.7ab | | 57.4ab | 62.7a | 2.75 | 0.11 | 0.007 | 0.023 |
Firmicutes | 19.3 | 21.8 | | 18.6 | 21.2 | 3.03 | 0.60 | 0.052 | 0.93 |
Fibrobacteres | 10.6 | 0.26 | | 10.2 | 0.14 | 1.16 | 0.81 | < 0.001 | 0.89 |
Actinobacteria | 0.40 | 0.15 | | 0.54 | 0.14 | 0.158 | 0.67 | 0.038 | 0.62 |
Proteobacteria | 4.18 | 12.0 | | 3.48 | 12.0 | 1.55 | 0.81 | < 0.001 | 0.84 |
Spirochaetae | 1.97 | 2.36 | | 3.48 | 1.89 | 0.532 | 0.31 | 0.24 | 0.061 |
Verrucomicrobia | 4.10 | 1.24 | | 3.65 | 0.82 | 0.586 | 0.30 | < 0.001 | 0.97 |
Others (< 0.5%) | 3.31 | 1.79 | | 2.09 | 0.94 | 0.341 | < 0.001 | < 0.001 | 0.43 |
F: B | 0.32 | 0.34 | | 0.29 | 0.31 | 0.009 | 0.17 | 0.43 | 0.95 |
6 h | | | | | | | | | |
Euryarchaeota | 0.89a | 0.54b | | 0.17c | 0.11c | 0.072 | < 0.001 | 0.002 | 0.016 |
Bacteroidetes | 45.0 | 49.5 | | 53.1 | 49.9 | 2.77 | 0.042 | 0.75 | 0.059 |
Firmicutes | 25.5 | 24.6 | | 18.3 | 25.1 | 2.92 | 0.21 | 0.27 | 0.15 |
Fibrobacteres | 7.68 | 0.06 | | 6.04 | 0.02 | 1.018 | 0.39 | < 0.001 | 0.42 |
Actinobacteria | 2.15 | 0.22 | | 0.98 | 0.39 | 0.553 | 0.34 | 0.022 | 0.20 |
Proteobacteria | 12.5 | 22.1 | | 15.2 | 23.1 | 3.79 | 0.58 | 0.016 | 0.79 |
Spirochaetae | 2.36 | 1.25 | | 3.66 | 0.81 | 0.538 | 0.43 | 0.001 | 0.12 |
Verrucomicrobia | 2.22 | 0.66 | | 1.64 | 0.27 | 0.237 | 0.019 | < 0.001 | 0.64 |
Others (< 0.5%) | 1.72 | 1.02 | | 0.89 | 0.34 | 0.192 | < 0.001 | < 0.001 | 0.54 |
F: B | 0.50 | 0.44 | | 0.28 | 0.43 | 0.040 | 0.076 | 0.28 | 0.086 |
12 h | | | | | | | | | |
Euryarchaeota | 0.85 | 0.63 | | 0.26 | 0.12 | 0.086 | < 0.001 | 0.003 | 0.47 |
Bacteroidetes | 50.8 | 49.3 | | 52.0 | 55.6 | 2.24 | 0.070 | 0.60 | 0.20 |
Firmicutes | 17.3 | 20.4 | | 13.6 | 19.5 | 2.20 | 0.21 | 0.023 | 0.46 |
Fibrobacteres | 11.7 | 0.16 | | 11.3 | 0.06 | 1.37 | 0.87 | < 0.001 | 0.93 |
Actinobacteria | 0.86 | 0.28 | | 0.63 | 0.32 | 0.252 | 0.66 | 0.053 | 0.55 |
Proteobacteria | 11.2 | 25.8 | | 15.4 | 22.3 | 3.06 | 0.92 | 0.002 | 0.21 |
Spirochaetae | 2.40 | 1.31 | | 3.75 | 1.19 | 0.564 | 0.21 | 0.001 | 0.14 |
Verrucomicrobia | 2.61 | 0.83 | | 1.96 | 0.44 | 0.286 | 0.041 | < 0.001 | 0.59 |
Others (< 0.5%) | 2.26 | 1.19 | | 1.08 | 0.50 | 0.188 | < 0.001 | < 0.001 | 0.21 |
F: B | 0.32 | 0.38 | | 0.23 | 0.31 | 0.026 | 0.037 | 0.042 | 0.39 |
1 OIL = canola oil; 3-NOP = 3-nitrooxypropanol. |
a,b,c Values within a row with different letters differ (P ≤ 0.05) |
F: B = Firmicutes: Bacteroidetes |
Table 3
Impact of feeding diets supplemented with (+) and without (-) 3-NOP and OIL on the relative abundance of phyla identified in rumen digesta in beef cattle at 0, 6 and 12 h after feeding1
Item | -3-NOP | | + 3-NOP | SEM | | P-value | |
-OIL | +OIL | | -OIL | +OIL | 3-NOP | OIL | 3-NOP × OIL |
0 h1 | | | | | | | | | |
Euryarchaeota | 3.43b | 6.04a | | 2.18b | 1.69b | 0.631 | < 0.001 | 0.031 | 0.003 |
Bacteroidetes | 24.4 | 36.9 | | 30.0 | 39.0 | 1.56 | 0.005 | < 0.001 | 0.15 |
Firmicutes | 48.4 | 51.3 | | 45.0 | 52.7 | 1.55 | 0.49 | 0.001 | 0.096 |
Fibrobacteres | 14.6 | 0.06 | | 11.8 | 0.05 | 1.45 | 0.33 | < 0.001 | 0.34 |
Actinobacteria | 1.22 | 0.74 | | 1.58 | 1.17 | 0.368 | 0.25 | 0.19 | 0.91 |
Proteobacteria | 1.15 | 2.28 | | 0.90 | 2.53 | 0.293 | 0.98 | < 0.001 | 0.40 |
Spirochaetae | 4.43 | 1.54 | | 6.73 | 1.81 | 0.707 | 0.078 | < 0.001 | 0.16 |
Verrucomicrobia | 0.72 | 0.29 | | 0.67 | 0.30 | 0.080 | 0.71 | < 0.001 | 0.67 |
F: B | 1.92 | 1.37 | | 1.47 | 1.33 | 0.118 | 0.026 | 0.002 | 0.063 |
Others (< 0.5%) | 1.59 | 0.85 | | 1.14 | 0.74 | 0.171 | 0.11 | 0.002 | 0.32 |
6 h | | | | | | | | | |
Euryarchaeota | 3.24a | 4.11a | | 1.39b | 0.96b | 0.466 | < 0.001 | 0.44 | 0.030 |
Bacteroidetes | 23.8 | 33.2 | | 30.5 | 35.5 | 1.34 | 0.001 | < 0.001 | 0.056 |
Firmicutes | 52.1a | 51.6ab | | 46.6b | 51.9a | 1.43 | 0.052 | 0.068 | 0.032 |
Fibrobacteres | 9.97 | 0.05 | | 9.27 | 0.03 | 1.18 | 0.75 | < 0.001 | 0.77 |
Actinobacteria | 4.00 | 0.83 | | 2.62 | 1.49 | 1.17 | 0.74 | 0.061 | 0.36 |
Proteobacteria | 2.69 | 8.23 | | 3.95 | 8.31 | 1.122 | 0.52 | < 0.001 | 0.58 |
Spirochaetae | 2.47 | 1.11 | | 4.46 | 1.14 | 0.525 | 0.065 | < 0.001 | 0.073 |
Verrucomicrobia | 0.53 | 0.23 | | 0.51 | 0.19 | 0.067 | 0.62 | < 0.001 | 0.90 |
F: B | 2.17b | 1.54a | | 1.52a | 1.45a | 0.145 | 0.002 | 0.004 | 0.020 |
Others (< 0.5%) | 1.18 | 0.60 | | 0.73 | 0.50 | 0.170 | 0.10 | 0.023 | 0.29 |
12 h | | | | | | | | | |
Euryarchaeota | 3.11b | 4.51a | | 1.48c | 0.84c | 0.498 | < 0.001 | 0.25 | 0.005 |
Bacteroidetes | 26.7 | 35.9 | | 32.7 | 39.2 | 1.20 | < 0.001 | < 0.001 | 0.22 |
Firmicutes | 50.0 | 49.2 | | 44.9 | 50.1 | 1.52 | 0.14 | 0.13 | 0.044 |
Fibrobacteres | 9.38 | 0.09 | | 8.18 | 0.06 | 1.052 | 0.55 | < 0.001 | 0.57 |
Actinobacteria | 2.73 | 0.69 | | 2.17 | 1.28 | 0.848 | 0.98 | 0.052 | 0.43 |
Proteobacteria | 2.90 | 7.51 | | 4.06 | 6.71 | 0.959 | 0.85 | 0.001 | 0.31 |
Spirochaetae | 2.96b | 1.14c | | 5.18a | 1.19c | 0.531 | 0.041 | < 0.001 | 0.051 |
Verrucomicrobia | 0.56 | 0.28 | | 0.51 | 0.20 | 0.069 | 0.27 | < 0.001 | 0.76 |
F: B | 1.85 | 1.35 | | 1.35 | 1.27 | 0.115 | 0.005 | 0.004 | 0.064 |
Others (< 0.5%) | 1.69 | 0.69 | | 0.90 | 0.46 | 0.342 | 0.14 | 0.046 | 0.42 |
1OIL = canola oil; 3-NOP = 3-nitrooxypropanol. |
a,b,c Values within a row with different letters differ (P ≤ 0.05) |
F: B = Firmicutes: Bacteroidetes |
Effect of 3-NOP and OIL on the bacterial community of the rumen
The addition of 3-NOP, OIL and 3-NOP + OIL resulted in significant changes to the composition of the rumen bacterial community. The relative abundance of bacterial taxa in rumen fluid and digesta samples as a function of treatment and time are shown in Tables 2 and 3, respectively. The most abundant phyla in all samples were Bacteroidetes and Firmicutes regardless of time and treatment. Together these two phyla made up between 68–92% of the sequences identified. There was no effect of 3-NOP addition on the abundance of Bacteroidetes in rumen fluid samples before feeding (P = 0.11) but there was a significant increase after 6 h after feeding (P = 0.042) and a tendency for greater abundance in samples 12 h after feeding (P = 0.070). The addition of 3-NOP significantly increased the abundance of Bacteroidetes in rumen digesta at all of the time points (P ≤ 0.05). This effect was primarily due to an increase in the relative abundance of Prevotella_1 (Supplementary Tables 1 and 2). The addition of OIL to the diet also resulted in an increase in the abundance of Bacteriodetes in the rumen fluid before morning feeding (P = 0.007), but not 6 h (P = 0.75) or 12 h later (P = 0.60). The effect of OIL on rumen digesta samples was similar, with a significant increase in the abundance of Bacteroidetes at all of the time points due to an increase in the relative abundance of Prevotella_1 (P < 0.001). Conversely, there were decreases in the abundance of other genera including RC9 gut group (P ≤ 0.01), S24-7 (P ≤ 0.02), and RF16 (0 h and 6 h P ≤ 0.002). A significant interaction between treatments was observed in rumen fluid prior to feeding but not at other time points. There was no significant interaction between OIL and 3-NOP in rumen digesta.
The addition of 3-NOP did not significantly change the relative abundance of Firmicutes in rumen fluid at any of the time points (Table 2). However, at the genus level, 3-NOP supplementation significantly altered the abundance of several uncharacterized genera within this phylum (Supplementary Table 1). In contrast, the abundance of Firmicutes in rumen digesta was reduced at 6 h after feeding (P = 0.052) but not in the 0 h samples (P = 0.49) or 12 h after feeding (P = 0.13) (Table 3). OIL increased the abundance of Firmicutes before feeding (P = 0.052) and 12 h after feeding (P = 0.023) but not 6 h after feeding (P = 0.27) in rumen fluid samples (Table 2). There was a significant increase in the abundance of Firmicutes in the rumen digesta as a result of OIL addition before feeding (P = 0.001) and a tendency to be higher at 6 h after feeding (P = 0.068), but no significant difference at 12 h after feeding (P = 0.14) (Table 3). At the genus level, there were significant decreases in the abundance in Christensenellaceae R-7 group at all of the time points in rumen digesta but not in rumen fluid (Supplementary Tables 1 and 2). OIL supplementation resulted in an increase in the relative abundance of Ruminococcus_1 (P ≤ 0.001) and Succinoclasticum (P ≤ 0.001) (Supplementary Tables 1 and 2). The majority of other genera within Firmicutes that showed significant change in abundance due to the addition of OIL were unknown and/or uncultured taxa. There was a significant interaction between treatments on the relative abundance of Firmicutes in rumen digesta 6 h (P = 0.032) and 12 h (P = 0.044) after feed was consumed. All treatments resulted in a significant decrease in the Firmicutes:Bacteroidetes ratio in rumen digesta samples primarily due to the increase in the abundance of Prevotella_1 (Supplementary Tables 1 and 2).
The relative abundance of Proteobacteria in both rumen fluid and digesta was not affected by the addition of 3-NOP (Tables 2 and 3). In contrast, OIL supplementation resulted in a significant increase in Proteobacteria in both rumen fluid and digesta at all of the time points (P < 0.001). The impact of OIL on Proteobacteria was primarily due to increases in the abundance of Ruminobacter and an uncultured group of Succinivibrionaceae (Supplementary Tables 1 and 2). The addition of 3-NOP did not affect the abundance of Fibrobacteres, Spirochaetae, or Verrucomicrobia in any of the samples but the addition of OIL reduced the abundance of all three of these phyla. The most dramatic effect of OIL was observed for the genus Fibrobacter. The addition of OIL to the diet resulted in a large decrease in the number sequences attributable to Fibrobacter (41–243 fold decrease; P < 0.001) in both rumen fluid and digesta at all of the time points (Tables 2 and 3). There was also an interaction between OIL and 3-NOP that resulted in a larger decrease in the abundance of Fibrobacter (75–382 fold decrease; P < 0.001) in animals receiving 3-NOP + OIL. The relative abundance of Spirochatae in the rumen digesta was reduced at all of the time points in animals receiving OIL. Similarly, OIL decreased the abundance of Spirochatae in rumen fluid 6 and 12 h after feeding but not in samples taken prior to morning feeding. All of these sequences were attributed to the genus Treponema_2 (Supplementary Tables 1 and 2). The abundance of Verrucomicrobia was lowered by the addition of OIL relative to control at all of the time points in both rumen fluid and rumen digesta.
Addition of Oil altered protozoal community in rumen fluid
The impact that the addition of OIL and 3-NOP had on the composition of rumen protozoa was assessed by microscopic analysis of rumen fluid samples (Table 4). The addition of 3-NOP did not alter the total number of protozoa or the composition of the protozoal community at any time point. In contrast, the addition of OIL resulted in a significant decrease of up to 16-fold (P < 0.001) in the total number of protozoa at all of the time points. OIL altered the composition of the protozoal community significantly decreasing the abundance of Dasytricha spp., Entodinium spp., Ostracodinium spp., and Osphyoscolex spp. in all samples and Metadinium spp. at 0 h and 12 h. No interaction effects between 3-NOP and OIL were observed for protozoa.
Table 4
Impact of feeding diets supplemented with (+) and without (-) 3-NOP and OIL on the protozoal populations (cell/mL) in the rumen fluid of beef at 0, 6 and 12 h after feeding1.
Item | -3-NOP | | + 3-NOP | SEM | P-value |
-OIL | +OIL | | -OIL | +OIL | 3-NOP | OIL | 3-NOP × OIL |
0 h | | | | | | | | | |
Isotricha spp. (×102) | 1.25 | ND | | 8.75 | ND | 4.69 | 0.40 | 0.26 | 0.40 |
Dasytricha spp. (×102) | 11.3 | ND | | 25.0 | ND | 14.1 | 0.42 | 0.037 | 0.42 |
Entodiniomorphs | | | | | | | | | |
Entodinium spp. (×105) | 4.56 | 0.29 | | 4.93 | 0.67 | 0.77 | 0.32 | < 0.01 | 0.97 |
Ostracodinium spp. (×102) | 10.0 | ND | | 18.8 | ND | 9.88 | 0.47 | 0.020 | 0.47 |
Metadinium spp. (×102) | 17.5 | ND | | 16.3 | 1.25 | 10.2 | 0.99 | 0.002 | 0.80 |
Osphyoscolex spp. (×102) | 10.0 | ND | | 17.5 | ND | 10.2 | 0.52 | 0.022 | 0.52 |
Total, 105 cell/mL | 4.61 | 0.29 | | 5.01 | 0.67 | 0.75 | 0.30 | < 0.01 | 0.98 |
6 h | | | | | | | | | |
Isotricha spp. (×102) | 2.50 | ND | | 3.75 | ND | 2.69 | 0.78 | 0.16 | 0.78 |
Dasytricha spp. (×102) | 10.0 | ND | | 8.75 | ND | 7.18 | 0.87 | 0.021 | 0.87 |
Entodiniomorphs | | | | | | | | | |
Entodinium spp. (×105) | 3.89 | 0.25 | | 4.41 | 0.58 | 0.74 | 0.27 | < 0.01 | 0.81 |
Ostracodinium spp. (×102) | 3.75 | ND | | 8.75 | ND | 4.11 | 0.36 | 0.024 | 0.36 |
Metadinium spp. (×102) | 1.25 | ND | | 2.50 | ND | 1.63 | 0.65 | 0.17 | 0.65 |
Osphyoscolex spp. (×102) | 11.3 | ND | | 12.5 | ND | 8.60 | 0.92 | 0.046 | 0.92 |
Total, 105 cell/mL | 3.92 | 0.25 | | 4.45 | 0.58 | 0.73 | 0.26 | < 0.01 | 0.80 |
12 h | | | | | | | | | |
Isotricha spp. (×102) | ND | ND | | 2.50 | ND | 1.25 | 0.32 | 0.32 | 0.32 |
Dasytricha spp. (×102) | 5.00 | ND | | 6.25 | ND | 4.53 | 0.84 | 0.07 | 0.84 |
Entodiniomorphs | | | | | | | | | |
Entodinium spp. (×105) | 3.47 | 0.28 | | 3.90 | 0.59 | 0.61 | 0.18 | < 0.01 | 0.84 |
Ostracodinium spp. (×102) | 8.75 | ND | | 5.00 | ND | 4.59 | 0.54 | 0.026 | 0.54 |
Metadinium spp. (×102) | 3.75 | ND | | 5.00 | ND | 2.79 | 0.76 | 0.033 | 0.76 |
Osphyoscolex spp. (×102) | 5.00 | ND | | 11.3 | ND | 6.28 | 0.39 | 0.027 | 0.39 |
Total, 105 cell/mL | 3.50 | 0.28 | | 3.93 | 0.59 | 0.60 | 0.17 | < 0.01 | 0.83 |
1OIL = canola oil, 3-NOP = 3-nitrooxypropanol. ND = Not detected |
CH4 and H2 emissions
A detailed analysis of the treatment effects on daily gaseous emissions is presented by Zhang et al., 2021 [27]. The present study examined the impact of 3-NOP and OIL on CH4 and H2 emissions, and concentration of dH2 over the day. A typical diurnal pattern of CH4 emissions was observed for control cattle, with a rapid increase in CH4 emissions peaking at 11.7 g/h 3 h after feeding followed by a slow decrease to pre-feeding baseline levels of approximately 5 g/h (Fig. 3A). Compared to control diets, supplementation with 3-NOP or OIL decreased the rate of CH4 emission by 28.2% and 23.9%, respectively. There was a 51.4% decrease for the combined treatment which indicates that the effects of these mitigation strategies were additive (Fig. 3A). Feeding OIL or 3-NOP alone delayed the peak CH4 emission rate from 3 h in the control to 6 h after feeding, while emission rate for 3-NOP + OIL peaked 12 h after feeding. The greatest reduction in CH4 occurred in the first 6 h after feed consumption (Fig. 3A).
Enteric H2 emissions were minimal in cattle consuming the control or OIL supplemented diets (Fig. 3B). However, in cattle consuming 3-NOP supplemented diets, enteric H2 emissions rapidly increased 37-fold relative to control diets, peaking at 0.17 g/h, 3 h after feeding. Co-administering 3-NOP and OIL increased levels of H2 in the rumen 20-fold relative to control diets, peaking at 0.094 g/h 3–6 h after feeding. There was also an increase in dH2 concentration in rumen fluid (P < 0.05). The inclusion of 3-NOP resulted in a significant increase in the peak concentration of dH2 in rumen fluid (Fig. 3C). The peak concentration of dH2 was observed 3 h after feeding and reached a concentration of 20.1 µmol/L in control diet. Inclusion of 3-NOP resulted in a 9.7-fold increase in the concentration of dH2 to 195.6 µmol/L compared to control (P < 0.05). The addition of both 3-NOP and OIL also resulted in an increase in dH2 concentration of 7.5-fold relative to 151.0 µmol/L compared to control (P < 0.05).
Changes in rumen fermentation and gas production were associated with the shifts in composition of the rumen microbiome
Both CH4 mitigation strategies influenced a number of rumen fermentation parameters, gas production and dissolved H2 concentration. The influence of the treatments on animal metabolism is described in a separate manuscript [27]. Many of the observed changes in the rumen fermentation and gas production were significantly associated with the observed shifts in the rumen microbiome (Fig. 4). Samples from animals fed 3-NOP did not cluster separately in an NMDS ordination based on Bray-Curtis dissimilarity but pH (R2 = 0.67; P = 0.002) and g CH4/kg DMI (R2 = 0.38; P = 0.06) were significantly associated with the microbiome composition in rumen digesta. Rumen pH (R2 = 0.45; P = 0.034), acetate to propionate ratio (R2 = 0.45; P = 0.04), g H2/kg of DMI (R2 = 0.38; P = 0.06) and g CH4/kg DMI (R2 = 0.84; P < 0.0001) were significantly associated with the microbiome composition of rumen fluid. The inclusion of OIL resulted in large changes in the microbial community of rumen fluid and digesta and these shifts were significantly associated with total VFA concentration (R2 ≥ 0.48; P < 0.05), the molar proportion of acetate (R2 ≥ 0.47; P < 0.05), isobutyrate (R2 ≥ 0.53; P < 0.05), and butyrate (R2 ≥ 0.55; P < 0.01), total protozoa (R2 ≥ 0.45; P < 0.01), and g CH4/kg DMI (R2 0.61; P < 0.01). In addition to these factors, pH (R2 ≥ 0.55; P ≤ 0.01) and valerate proportion (R2 ≥ 0.44; P ≤ 0.05) were associated with the observed changes in the microbial composition of rumen digesta due to OIL. The combination of NOP and OIL also resulted in distinct clustering of samples. Acetate (R2 ≥ 0.57; P < 0.01), isobutyrate (R2 ≥ 0.42; P ≤ 0.05), total VFA (R2 ≥ 0.44; P ≤ 0.05), NH3 (R2 ≥ 0.45; P < 0.05), pH (R2 ≥ 0.47; P < 0.05), protozoa count (R2 ≥ 0.59; P < 0.01), g CH4/kg DMI (R2 0.87; P < 0.001), and g H2/kg DMI (R2 0.45; P < 0.05) were significantly associated with the changes in microbiome composition observed for the combined treatment.