Effect of Inulin Supplementation on Lactation Performance
The milk yield and compositions are showed in Table 1. With the supplementation of inulin, the dry matter intake (DMI) (P = 0.003), milk yield (P = 0.001), energy corrected milk (ECM), fat corrected milk (FCM) (P < 0.001), milk protein (P = 0.042) and lactose (P = 0.004) were significantly increased, while milk fat was showed a tendency of increase (P = 0.075). Whereas, the MUN (P = 0.023), SCC (P = 0.036) and the fat to protein ratio (F/P) was significantly decreased (P = 0.027), but the F/P was still in the normal rage (1.12-1.36).
The effect of adding inulin on the level of milk FAs is listed in Table 2. Compared with control group, the proportion of saturated fatty acid (SFA) in inulin group was significantly increased (P < 0.001), mainly including the significant increase of C6:0 (P = 0.034), C8:0 (P = 0.006), C10:0 (P = 0.038), C12:0 (P = 0.001) and C16:0 (P = 0.001). While, the proportion of C18:0 (P = 0.002) and C22:0 (P = 0.031) were significantly reduced. On the other hand, the proportion of unsaturated fatty acid (UFA) in inulin group was significantly declined (P = 0.041), which were mainly attributed to the decrease of polyunsaturated fatty acids (PUFA) (P = 0.013). Among them, the proportion of C18:2 cis-6 (P = 0.011) and C18:3n3 (P < 0.001) were significantly reduced. Additionally, the level of short and medium-chain fatty acids (SMCFA) in inulin group was significant increase (P < 0.001).
Effect of Inulin Supplementation on Serum Indexes
As shown in Table 3, compared with control group, the concentration of TC (P = 0.008) and TG (P = 0.01) were significantly declined in the inulin group, while the level of TP, ALB, GLO and BUN had no significant difference (P > 0.05).
Effects of Inulin Supplementation on Rumen Fermentation Characteristics
The rumen fermentation parameters were listed in Table 4. The pH value in rumen was significantly declined (P = 0.040) with the addition of inulin, which accompanied by the significant increase of the concentration of acetate (P < 0.001), propionate (P = 0.003), butyrate (P < 0.001), isobutyrate (P = 0.002), valetate (P = 0.001), isovaletate (P < 0.001) and LA (P = 0.043). Meanwhile, the significant decrease of the concentration of NH3-N (P = 0.024) was also observed in inulin group.
Effect of Dietary Supplementation of Inulin on the Richness, Diversity and Composition of Ruminal Bacteria
A total of 1,349,120 effective 16S rRNA sequences were detected in 16 rumen fluid samples and 2,337 OTUs were obtained by performing OTU clustering on non-repetitive sequences according to 97% similarity. Rarefaction curves (Additional file 1:Figure S1) showed that the current sequencing depth and sample size were sufficient to assess the microbial diversity, total species richness and core species number of rumen fluid samples. The α-diversity analysis revealed that the ACE (P = 0.031), Chao (P = 0.017) and Shannon (P = 0.026) indexes in inulin group were significantly increased, the Simpson index also showed a tendency of rise (P = 0.071), which illustrated that the addition of inulin increased the ruminal microbial community richness and diversity (Table 5).
The RDP classifier Bayesian algorithm was used to perform taxonomic analysis on the OTU representative sequences, and 21 bacteria phyla and 304 genera were obtained from 16 samples (Additional file 1:Table S2 and S3). At the phylum level, Bacteroidota (48.4 ± 0.47% and 51.7 ± 0.36%), Firmicutes (46.3 ± 0.41% and 42.3 ± 0.38%), Patescibacteria (1.81 ± 0.13 % and 1.85 ± 0.11%) and Actinobacteriota (1.43 ± 0.25% and 1.82 ± 0.17%) were the dominant bacteria in control and inulin groups, respectively (Figure 1A and Additional file 1: Figure S2 A and B). At the genus level, Prevotella (36.8 ± 0.41% and 39.5 ± 0.37%), Oscillospirales_NK4A214_group (4.70 ± 0.11% and 5.39 ± 0.24%), Succiniclasticum (3.89 ± 0.24% and 3.78 ± 0.20%), Ruminococcus (4.15 ± 0.32% and 3.90 ± 0.17%) and Muribaculaceae (2.18 ± 0.10% and 5.59 ± 0.15%) were the predominant genera in control and inulin groups, respectively (Figure 1B and Additional file 1:Figure S2 C and D).
Significantly Different Ruminal Bacteria between the Control and Inulin Groups
The β‐diversity analysis was performed to explore differences in rumen microbial community between the two groups (Figure 2). The PCoA and NMDS plots based on the Bray-Curtis distance matrix showed that the points representing rumen microorganisms in control and inulin groups respectively were significantly separated in different quadrants on the coordinate axis, which indicated inulin intake had a distinct effects on rumen microbial species and abundance. Venn diagram performed on the samples of OTU with 97% identity identified 87 and 117 unique OTU in control and inulin groups, respectively (Additional file 1: Figure S3). Further, the significantly different abundant ruminal bacteria between the two groups were identified through LEfSe analysis and LDA (Figure 3 A and B). The relative abundance of Muribaculaceae (FDR-adjusted P < 0.01), Butyrivibrio (FDR-adjusted P = 0.036), Prevotellaceae_NK3B31_group (FDR-adjusted P = 0.032), Eubacterium_coprostanoligenes_group (P = 0.043), Lachnospiraceae_XPB1014_group (FDR-adjusted P = 0.035), Acetitomaculum (FDR-adjusted P = 0.043), Eubacterium_hallii_group (FDR-adjusted P = 0.031), Lachnospiraceae (FDR-adjusted P = 0.026) and Veillonellaceae_UCG-001 (FDR-adjusted P = 0.036) were significantly increased in inulin groups compared with control group. In contrast, Erysipelotrichaceae__UCG-004 (FDR-adjusted P < 0.01), Erysipelotrichaceae__UCG-008 (FDR-adjusted P < 0.01), Clostridia_UCG-014 (FDR-adjusted P = 0.031), Selenomonas (FDR-adjusted P = 0.040), Bacteroidales_BS11_gut_group (FDR-adjusted P = 0.037), Escherichia-Shigella (FDR-adjusted P = 0.022), Anaerobiospirillum (FDR-adjusted P = 0.041), Succinivibrionaceae_UCG_001 (FDR-adjusted P = 0.042), Syntrophococcus (FDR-adjusted P = 0.037) and RF39 (FDR-adjusted P = 0.042) were significantly decreased in rumen with the inulin addition (Additional file 1:Table S3).
Effect of Dietary Supplementation of Inulin on Ruminal Metabolites
The rumen metabolites were analyzed through untargeted metabolomics techniques. The total ion chromatograms (TIC) plot of QC samples in positive and negative ion mode are shown in Additional file 1: Figure S4 A and B. The overlap of QC samples revealed the well repeatability and high-accuracy of the data. The unsupervised multivariate statistical analysis, PCA, generally reflected that a distinct difference existed in the ruminal metabolites between the two groups and a less degree of variation among samples within a group in positive and negative ion modes (Additional file 1:Figure S5 A and B). Further OPLS-DA provided a supervised discriminant analysis method, which could further distinguish the differences of ruminal metabolites between control and inulin groups and improve the effectiveness and analytical capabilities of the model (Figure 4). In OPLS-DA plots, R2Y and Q2 were used to evaluate the modeling and prediction ability of OPLS-DA model respectively. The cumulative value of R2Y and Q2 in positive (0.995 and 0.806) and negative (0.907 and 0.839) ion model were all above 0.80, which illustrated the stability and reliability of the model (Figure 4 A and C). Response permutation testing (RPT) was a randomized sequencing method to evaluate the accuracy of OPLS-DA models. As shown in Figure 4 B and D, the value of R2 (0.953 and 0.931) and Q2 (-0.0067 and -0.082 < 0) in positive and negative ion models revealed a well accuracy of OPLS-DA models.
Significantly Different Ruminal Metabolites between the Control and Inulin Groups
A total of 99 differential metabolites in rumen (64 in positive and 35 in negative ion models) between the control and inulin groups were detected with VIP > 1 and FDR-adjusted P < 0.05 (Additional file 1: Table S4). Among them, lipids and lipid-like molecules, organic acids and derivatives, organic oxygen compounds and organoheterocyclic compounds were accounted for 37.0 ± 0.48%, 22.2 ± 0.32%, 16.7 ± 0.11% and 14.8 ± 0.24%, respectively (Additional file 1:Figure S6). The top 70 ruminal metabolites were selected for HCA analysis (Additional file 1: Figure S7). The differential metabolites between the two groups were divided into two cluster. The relative expression of PC (16:0/0:0), LysoPC (16:0), palmitoyl glucuronide, R-palmitoyl-(2-methyl) ethanolamide, 2-O-Protocatechuoylalphitolic acid, sphinganine, LysoPC (18:2(9Z, 12Z)) and LysoPC (18:1(9Z)) etc. were higher in control group than in inulin group. While, the relative expression of deltonin, 3,4,5-trihydroxy-6-(2-oxoethoxy) oxane-2-carboxylic acid, L-Proline, 2-Hydroxycinnamic acid, L-Tyrosine, L-Phenylalanine, L-Tyrosine, daidzein and L-Lysine etc., were elevated in inulin group compared with control group.
Furthermore, several significantly differential ruminal metabolites with VIP ≥ 2 and FDR-adjusted P < 0.05 between the two groups were screened out. Compared with control group, LysoPC (18:1(9Z)) (FDR-adjusted P = 1.03×10-3), LysoPC (16:0) (FDR-adjusted P = 0.0108), LysoPC (18:2(9Z, 12Z)) (FDR-adjusted P = 1.65×10-3), Phenylmethylglycidic ester (FDR-adjusted P = 1.19×10-3), N-Acetylcadaverine (FDR-adjusted P = 0.0353) and 8-Methylnonenoate (FDR-adjusted P = 1.60×10-3) were significantly decreased in the inulin group. However, the L-Lysine (FDR-adjusted P = 4.24×10-3), L-Proline (FDR-adjusted P = 0.0158), L-Phenylalanine (FDR-adjusted P = 0.027), daidzein (FDR-adjusted P = 0.0130), uracil (FDR-adjusted P = 0.0396), deltonin (FDR-adjusted P = 0.0393) and L-Tyrosine (FDR-adjusted P = 0.0353) were significantly increased in the rumen with the supplementation of inulin (Additional file 1: Figure S8 and Table S4).
Receiver operator characteristic curve was performed to evaluate whether the significantly differential metabolites screened were critical to the intergroup differentiation. As shown in Figure 5, the AUC of 8-Methylnonenoate, LysoPC (18:1(9Z)), LysoPC (18:2(9Z, 12Z)), L-Lysine, LysoPC (16:0) and L-Proline were all above 0.95, which meant these significantly differential ruminal metabolites were representative to illustrate the effect of inulin on the shift of rumen metabolism.
Metabolic pathway enrichment analysis of differentially abundant metabolites
KEGG pathway enrichment analysis showed that the supplementation of inulin mainly affected the lipid and amino acid metabolism, vitamin metabolism, biosynthesis of plant secondary metabolites and protein metabolism in the rumen of dairy cows (Table 6).
Correlation analysis among differential ruminal bacteria, metabolites, lactation and rumen fermentation performance.
Spearman correlation coefficient was used to calculate the correlation among several indicators (Figure 6). Correlation analysis between significantly differential bacteria and milk components showed that the milk yield was positively associated with Muribaculaceae (r = 0.521, FDR-adjusted P = 0.042), Butyrivibrio (r = 0.602, FDR-adjusted P = 0.028) and Prevotellaceae_NK3B31_group (r = 0.607, FDR-adjusted P = 0.027), while negatively associated with Escherichia-Shigella (r = -0.613, FDR-adjusted P = 0.007). Milk protein was positively associated with Muribaculaceae (r = 0.595, FDR-adjusted P = 0.015), Eubacterium_hallii_group (r = 0.569, FDR-adjusted P = 0.037) and Prevotellaceae_NK3B31_group (r = 0.579, FDR-adjusted P = 0.039), but negatively associated with Erysipelotrichaceae_UCG_008 (r = 0.488, FDR-adjusted P = 0.044) and Erysipelotrichaceae_UCG_004 (r = 0.510, FDR-adjusted P = 0.044). The lactose was positively associated with Muribaculaceae (r = 0.585, FDR-adjusted P = 0.025), Eubacterium_coprostanoligenes_group (r = 0.565, FDR-adjusted P = 0.047), Eubacterium_hallii_group (r = 0.602, FDR-adjusted P = 0.023), Treponema (r = 0.573, FDR-adjusted P = 0.037) and Saccharofermentans (r = 0.582, FDR-adjusted P = 0.034), but negatively associated with Escherichia-Shigella (r = -0.481, FDR-adjusted P = 0.048). Milk fat was positively associated with Muribaculaceae (r = 0.543, FDR-adjusted P = 0.041), Butyrivibrio (r = 0.476, FDR-adjusted P = 0.042), Prevotellaceae_NK3B31_group (r = 0.468, FDR-adjusted P = 0.046) and Acetitomaculum (r = 0.584, FDR-adjusted P = 0.038). Milk SCC was negatively associated with Muribaculaceae (r = -0.759, FDR-adjusted P = 0.011), Butyrivibrio (r = -0.678, FDR-adjusted P = 0.014), Eubacterium_hallii_group and Eubacterium_coprostanoligenes_group (r = -0.743, FDR-adjusted P = 0.010), while positively associated with Erysipelotrichaceae_UCG_008 (r = 0.575, FDR-adjusted P = 0.031), Escherichia-Shigella (r = 0.594, FDR-adjusted P =0.022) and Anaerobiospirillum (r = 0.594, FDR-adjusted P = 0.031) (Figure 6A).
In addition, the correlation analysis between significantly differential bacteria and rumen fermentation parameters revealed that, acetate was positively associated with Muribaculaceaec(r = 0.712, FDR-adjusted P = 0.002), Eubacterium_coprostanoligenes_group (r = 0.629, FDR-adjusted P = 0.014) and Acetitomaculum (r = 0.700, FDR-adjusted P = 0.003). Propionate was positively associated with Muribaculaceae (r = 0.649, FDR-adjusted P = 0.007), Eubacterium_coprostanoligenes_group (r = 0.622, FDR-adjusted P = 0.010), Eubacterium_hallii_group (r = 0.670, FDR-adjusted P = 0.021), Treponema (r = 0.608, FDR-adjusted P = 0.023) and Saccharofermentans (r = 0.626, FDR-adjusted P = 0.031). Butyrate was positively associated with Muribaculaceae (r = 0.739, FDR-adjusted P = 0.001), Eubacterium_coprostanoligenes_group (r = 0.719, FDR-adjusted P = 0.002) and Butyrivibrio (r =0.700, FDR-adjusted P = 0.003), while negatively associated with Escherichia-Shigella (r = -0.681, FDR-adjusted P = 0.032). Prevotellaceae_NK3B31_group was positively associated with isovaletate (r = 0.644, FDR-adjusted P = 0.030) and isobutyrate (r = 0.602, FDR-adjusted P = 0.041). The LA was positively associated with Butyrivibrio (r = 0.639, FDR-adjusted P = 0.034), Prevotellaceae_NK3B31_group (r = 0.799, FDR-adjusted P < 0.001), Acetitomaculum (r = 0.678, FDR-adjusted P = 0.019) and Eubacterium_hallii_group (r = 0.653, FDR-adjusted P = 0.017). However, the NH3-N was negatively associated with Muribaculaceae (r = -0.735, FDR-adjusted P = 0.001), Eubacterium_coprostanoligenes_group (r = -0.685, FDR-adjusted P = 0.030), Butyrivibrio (r = -0.648, FDR-adjusted P = 0.028), Eubacterium_hallii_group (r = -0.650, FDR-adjusted P = 0.036) and Treponema (r = -0.653, FDR-adjusted P = 0.021) (Figure 6B).
The correlation between significantly differential metabolites and microbacteria is showed in Figure 6C. LysoPC (16:0), LysoPC (18:2(9Z, 12Z)) and LysoPC (18:1(9Z)) were negatively associated with Muribaculaceae (r = -0.806, FDR-adjusted P < 0.001; r = -0.740, FDR-adjusted P = 0.001; r = -0.717, FDR-adjusted P = 0.022) and Prevotellaceae_NK3B31_group (r = -0.730, FDR-adjusted P = 0.043; r = -0.676, FDR-adjusted P = 0.024; r = -0.649, FDR-adjusted P = 0.031), but positive associated with Escherichia-Shigella (r = -0.643, FDR-adjusted P = 0.030; r = -0.713, FDR-adjusted P = 0.024; r = -0.717, FDR-adjusted P = 0.036) and Anaerobiospirillu (r = -0.735, FDR-adjusted P = 0.038; r = -0.675, FDR-adjusted P = 0.042; r = -0.694, FDR-adjusted P = 0.043). N-Acetylcadaverine were positively correlated with Escherichia-Shigella (r = -0.572, FDR-adjusted P = 0.042), Erysipelotrichaceae_UCG_008 (r = -0.566, FDR-adjusted P = 0.045) and Erysipelotrichaceae__UCG-004 (r = -0.562, FDR-adjusted P = 0.046). Uracil was positively correlated to Eubacterium_hallii_group (r = 0.673, FDR-adjusted P = 0.025). L-Lysine, L-Proline and L-Tyrosine were positively associated with Muribaculaceae (r = 0.602, FDR-adjusted P = 0.031; r = 0.585, FDR-adjusted P = 0.035; r = 0.607, FDR-adjusted P = 0.032), Eubacterium_hallii_group (r = 0.624, FDR-adjusted P = 0.042; r = 0.580, FDR-adjusted P = 0.024; r = 0.613, FDR-adjusted P = 0.031) and Prevotellaceae_NK3B31_group (r = 0.705, FDR-adjusted P = 0.032; r = 0.584, FDR-adjusted P = 0.047; r = 0.631, FDR-adjusted P = 0.040).
As shown in Figure 6D, the relevance between significantly differential metabolites and milk compositions showed that, the milk SCC was positively associated with LysoPC (16:0) (r = 0.772, FDR-adjusted P = 0.021), LysoPC (18:2(9Z, 12Z)) (r = 0.752, FDR-adjusted P = 0.035), LysoPC (18:1(9Z)) (r = 0.798, FDR-adjusted P = 0.023) and N-Acetylcadaverine (r = 0.683, FDR-adjusted P = 0.041), while negatively associated with L-Tyrosine (r = -0.677, FDR-adjusted P = 0.033) and L-Lysine (r = -0.680, FDR-adjusted P = 0.037). Milk fat and protein was positively associated with L-Proline (r = -0.741, FDR-adjusted P = 0.040; r = -0.769, FDR-adjusted P = 0.030) and L-Lysine (r = -0.785, FDR-adjusted P = 0.036; r = -0.744, FDR-adjusted P = 0.040). Additionally, milk protein was also positively associated with L-Tyrosine (r = -0.750, FDR-adjusted P = 0.035). Moreover, uracil was positively related with lactose (r = 0.679, FDR-adjusted P = 0.045).