Growth performance results
Table 2 shows the effects of the supplemental sterilized rumen fluid treatments on growth and feed intake of the calf. It can be seen from the table that there was no significant difference in the growth index of the calves between the groups before the test (P>0.05). After the test, the daily weight gain of the S group was significantly higher than that of the C group (P<0.05), and there was no significant difference between the M group and the C group (P>0.05). There was no significant difference in the body length, chest circumference, body height and feed intake among the calves after the test (P>0.05).
Serum index results
Table 3 shows the changes in the calf serum index in each group. Due to differences among the individual test animals, the calf serum index (supplementation 1) between the groups before the test was statistically different, and this difference could not be avoided. Therefore, the change in serum results was statistically analyzed. The numerical magnitude represents the amount of change at two points in time, positive or negative indicating an increase or decrease. The first stage was from 7 days to 15 days of age, indicating tests of serum indicators after treatment; the second stage was from 15 days to 63 days of age, indicating treatment for a period of time until the serum indices change after weaning. It can be seen from the table that the experimental treatment had significant effects for most of the indicators during the two stages, but the effect on TNF-α was not significant. In the two stages, IgA was significantly lower in the M group than in the C group (P<0.05), and the S group was also significantly lower in the second stage (P<0.05). In the first stage, serum IgG of the M and S groups showed increasing trends, whereas the C group showed a downward trend; the treatment groups all showed significant differences with the C group change (P<0.05). Changes occurred in the serum IgG of the treatment group during the second stage. There was no significant difference between the two groups (P>0.05), but there was significant difference between the treatment group and the S group (P<0.05). The serum IL-1β increased in all the experimental groups, but the changes in M group and S group were significantly lower than that in group C (P<0.05). In the second stage, the C group showed an increasing trend, whereas the M and S groups showed significant decreasing trends (P<0.05). Serum TNF-α showed an increasing trend in all the first-stage trials, and the differences were not significant (P>0.05). The second-stage M and S groups showed a decreasing trend, and the change was significantly different from that of the C group (P<0.05). The amounts of serum IL-4 in M and S groups during the first stage were significantly lower than that in the C group. In the second stage, the serum IL-4 decreased In the C group, but the M group remained significantly lower than the C group. Interestingly, there was a significant increase of IL-4 in the S group (P<0.05). The serum IL-6 in the first stage of the C group and the M group showed an increasing trend, while the S group showed a downward trend. The M and S groups had significant differences in IL-6 levels compared with that of the C group (P<0.05). In the second stage, the M group showed a downward trend, whereas the other two groups showed an upward trend; while IL-6 levels in the S group were increased, they remained significantly lower than that in the C group. Serum IFN-γ decreased in all the groups in the first stage, but the decrease in the M group was significantly lower than that in the C group. During the second stage, only IFN-γ in the M group was still decreasing, whereas IFN-γ in the other groups showed an increasing trend; although the differences among the groups increased, the changes were not significant (P>0.05). All the experimental groups in the first stage of serum GH showed a downward trend, but the reduction in group M was significantly higher than that in group C. During the second stage, serum GH of groups C and M still showed a decreasing trend, while group S showed an increasing trend relative to group C; compared with the control group, the changes were not significant (P>0.05). Serum LP showed an increasing trend for all treatment groups in the first stage, while group C showed a downward trend; the amount of change was significantly different (P<0.05). In the second stage, The M group showed a n increase while Serum LP levels in group C decreased; the amount of change in group S was significantly different from that in the C group (P<0.05), and there was no difference in the other groups (P>0.05).
16S rDNA sequencing results
Single sample diversity analysis
From Figure 1, we found that the dilution curves of all samples tended to be flat, indicating that the samples were adequately sequenced and the depth covered almost all species in the sample.
OTU analysis
The tags were clustered at the 97% similarity level to obtain OTU, and OTU was classified based on the Silva (bacteria) taxonomy database to obtain the OTU number of each sample. A total of 355 OTUs were obtained from 12 samples. There were 337 OTUs in the control group, with 315 OTUs in groups M and S. It can be seen from the Venn diagram that 280 of the OTUs were shared between the three groups; 19 OTUs were only in group C, 1 OTUs was only in group M, and 3 OTUs were only in group S (Fig. 2).
Species annotation and taxonomic analysis
The OTU representative sequence was compared with the microbial reference database to obtain species classification information corresponding to each OTU. Furthermore, the composition of each sample community was counted at each level (phylum, class, order, family, genus, species), and the abundance for each species at different classification levels was obtained. Only the top ten species in abundance levels are shown, and other species are combined into the “Others” category. In the figure, the “Unclassified” category represents species that were not taxonomically annotated.
At the phylum level, 11, 11, and 10 phyla were detected in rumen fluid of groups C, M, and S, respectively (Figure 3). Group C contained 49.95% of Bacteroidetes, 32.06% of Firmicutes, 13.19% of Proteobacteria, 1.64% of Fibrobacteres, and 2.31% of Teneriquets. Group M contained 38.07% of Bacteroidetes, 48.04% of Firmicutes, 11.98% of Proteobacteria, 0.50% of Fibrobacteres, 0.22% of Teneriquets, and 0.60% of Cyanobacteria. Group S mainly contained 52.41% of Bacteroidetes, 34.37% of Firmicutes, 6.80% of Proteobacteria, 4.46% of Fibrobacteres, and 0.61% of Teneriquets.
In Fig. 4, at the genus level, 122, 124 and 124 genera were detected in the rumen fluid of groups C, M and S, respectively. Group C mainly contained 15.40% of Prevotella_7, 24.22% of Prevotella_1, 10.93% of Succinivibrionaceae_UCG-001, 9.80% of Roseburia. Group M mainly contained 24.79% of Prevotella_7, 6.14% of Prevotella_1, 10.70% of Succinivibrionaceae_UCG-001, 9.51% of Xiaoster, 7.27% of Ruminococcaceae_UCG-014 and 7.26% of Megasphaera. Group S mainly contained 38.55% of Prevotella_7, 1.77% of Prevotella_1, 4.14% of Succinivibrionaceae_UCG-001, 6.22% of Roseburia, 7.28% of Dialister and 6.08% of Ruminococcaceae_UCG-014.
Ternary phase analysis
From Figure 5, we can see the proportion and relationship of different species among the three groups. Proteobacteria had the largest proportion in group C and the smallest proportion in group S. Fibrobacteres have the largest proportion in group S and extremely small in group M. Bacteroidetes have the largest proportion in group S and the smallest proportion in group M. Tenericutes has the largest proportion in group C and the smallest proportion in group M, whereas Firmicutes have the largest proportion in group M and the smallest proportion in group C.
Significant difference analysis between sample groups
Table 4 shows the taxonomic statistics of bacteria within rumen fluid with contents greater than 0.01% in groups C and M, among which, there are significant differences between Firmicutes, Ruminococcaceae, uncultured_bacterium_o_Gastranaerophilales, p-2534-18B5_gut_group, Oxalobacteraceae, uncultured_bacterium_o_Gastranaerophilales, [Eubacterium]_coprostanoligenes_group, Oxalobacter, Dialister. There are different trends in Selenomonadales, Veillonellaceae, Porphyromonadaceae, Selenomonas_1, [Eubacterium]_xylanophilum_group and Moryell.
Table 5 shows the taxonomic statistics of bacteria within rumen fluid with contents greater than 0.01% in groups C and S, among which, there were significant differences between Deltaproteobacteria, Desulfovibrionales, uncultured_bacterium_o_Gastranaerophilales, Desulfovibrionaceae, Oxalobacteraceae, Ruminococcaceae_NK4A214_group, uncultured_bacterium_o_Gastranaerophilales, uncultured_bacterium_f_Erysipelotrichaceae, Desulfovibrio, Dialister, [Ruminococcus]_gauvreauii_group, Mitsuokella, Prevotella_7. There were different trends in Selenomonas, Ruminococcaceae_UCG-014, Selenomonas_1, Veillonellaceae_UCG-001, uncultured_rumen_bacterium, Schwartzia.
Table 6 shows the taxonomic statistics of bacteria with rumen fluid bacteria content greater than 0.01% in groups M and S, among which, there were significant differences between Syntrophococcus, Rikenellaceae_RC9_gut_group, p-2534-18B5_gut_group, [Ruminococcus]_gauvreauii_group, uncultured_bacterium_f_p-2534-18B5_gut_group, Pyramidobacter、Prevotella_7. There were different trends in Bacteroidales.
Metabolomics analysis
Metabolite volcano map analysis
The volcano map can be used to quickly view the differences in metabolite expression levels between the two groups, as well as the statistical significances of these differences. The differential expression volcano is as follows: Compared with group C, group M had 42 significant metabolites in positive ion mode, of which, levels of 3 were significantly down and levels of 39 were significantly up; There were 5 significant metabolites in negative ion mode, of which the level of 1 was significantly down and levels of 4 were significantly up. Compared with group C, group S had 38 significant metabolites in positive ion mode, of which, levels of 9 were significantly down and levels of 29 were significantly up; There were 27 significant metabolites in negative ion mode, of which, levels of 10 were significantly down and levels of 17 were significantly up (Figure 7).
Analysis of differential metabolites and metabolic pathways
As can be seen from table 7, compared with group C, levels of some metabolites in group M were significantly increased, including 5-methylcytosine, xanthine, L-citrulline, myo-inositol, 2-isopropylmalic acid, 3-methoxy-4-hydroxyphenylethyleneglycol, indoleacetic acid, deoxyadenosine, erucic acid, UDP-D-galactose, uracil, and homovanillic acid. The main metabolic pathways involved include: pyrimidine metabolism (ko00240), purine metabolism (ko00230), biosynthesis of amino acids (ko01230), arginine biosynthesis (ko00220), ascorbate and aldarate metabolism (ko00053), inositol phosphate metabolism (ko00562), galactose metabolism (ko00052), 2-oxocarboxylic acid metabolism (ko01210), valine, leucine and isoleucine biosynthesis (ko00290), pyruvate metabolism (ko00620), tyrosine metabolism (ko00350), tryptophan metabolism (ko00380), unsaturated fatty acid biosynthesis (ko01040), galactose metabolism (ko00052), amino sugar and nucleotide sugar metabolism (ko00520), β-alanine metabolism (ko00410); pantothenic acid and CoA biosynthesis (ko00770). Significantly down metabolites include: myristic acid, thiamine, and the major metabolic pathways involved: fatty acid biosynthesis (ko00061), thiamine metabolism (ko00730).
As can be seen from table 8, compared with group C, levels of some metabolites in group S were significantly increased, including cytidine, D-ribulose 5-phosphate, ergothioneine, thymidine 5'-monophosphate, cytidine 5'-monophosphate, UDP-D-galactose, uracil, urocanic acid, xanthine, N- acetyl-L-glutamate, salidroside. The main metabolic pathways involved include: pyrimidine metabolism (ko00240), pentose phosphate pathway (ko00030), vitamin B6 metabolism (ko00750), riboflavin metabolism (ko00740), carbon metabolism (ko01200), amino acid biosynthesis (ko01230), pentose and glucuronic acid (ko00040), histidine metabolism (ko00340), amino sugar and nucleoside sugar metabolism (ko00520), galactose metabolism (ko00052), pantothenate and CoA biosynthesis (ko00770), β-alanine metabolism (ko00410), purine metabolism (ko00230), 2-oxocarboxylic acid metabolism (ko01210), arginine biosynthesis (ko00220), tyrosine metabolism (ko00350). Metabolites with significantly decreased levels include: 5-Aminopentanoic acid, and the major metabolic pathways involved: arginine and proline metabolism (ko00330), lysine degradation (ko00310).