Briefly, the differences of rumen papilla were observed, and the variations were examined under different diets with combined investigations into rumen metagenome, rumen content metabolomics, and transcriptome profiling of ruminal epithelium.
In the current study, our results on ruminal pH were not different between the SO and SS groups, which ranged between 5.59 and 5.38. This was consistent with previous studies, which reported that rumen pH was not changed by fiber source [23, 24]. As for the low pH value in calf rumen, it may be attributed to the fact that the absorption rate of VFA by the rumen epithelium of calves was lower than the production rate of VFA. VFA acts as the important energy sources which can meet the needs of ruminant growth and development [25]. In this study, there was no significant difference in total VFA concentration between the two groups. The published studies showed that non-forage fiber and forage fiber had no significant effect on the production and composition of total VFA in rumen [4, 5], which was similar with this study.
Rumen epithelium performs many important functions of the rumen, including absorption, transport, short chain fatty acid metabolism and immunity [26]. And rumen papilla length is considered to be the most representative of rumen development, followed by rumen papilla width [27]. It has been reported that compared with feeding alfalfa hay, lambs fed soybean hull had reduced ruminal papilla length, muscle layer thickness, and surface area ratio [4]. It was similar with this study, calves fed with SS diet had lower rumen papillae length and rumen papillae width, which indicated SS feeding was not conducive to rumen development of calves compared with SO feeding. And the rumen development affects the animal production systemically through various hormones [28]. All these facts told us that SS feeding had a negative effect on the rumen development of pre-weaning calves compared with SO feeding.
The diverse microbes play an important role in the rumen development and function [29]. The relative abundance of archaea was decreased under SO feeding. Archaea can exist in many habitats, but those known to exist in the rumen are strictly anaerobic methanogens [30]. In this study, the relative abundances of Euryarchaeota phylum and Methanobrevibacte genus were decreased in the rumen of SO calves. The Methanobrevibacter was found to dominate the total rumen archaea, and it was also the major shareholder to total enteric methane production [30, 31]. This indicated that feeding SO may reduce methane emissions.
Generally, SO feeding decreased the level of Eubacterium sp. CAG:38, Eubacterium sp. CAG:252 and Eubacterium sp. CAG:248, but increased the level of Prevotella sp. CAG:873, Prevotella sp. CAG:279, Prevotella sp. CAG:1031, Prevotella sp. CAG:485, Prevotella sp. P4-76, Bacteroides sp. CAG:443, Bacteroides thetaiotaomicron and Bacteroides sp. CAG:927. The Eubacterium is considered as a fiber degrading bacterium [32–34]. In this study, Eubacterium sp. CAG:252 and Eubacterium sp. CAG:38 were positively correlated with CBM61. Previous study noted that CBM61 can promote the digestion of pectin that was rich in the soybean hull [35], which revealed that Eubacterium sp. CAG:252 and Eubacterium sp. CAG:38 responded to soybean hulls by CBM61, and then increased the relative abundance of Eubacterium sp. CAG:248 through co-occurrence network. The Prevotella can be detected easily in the mammalian hindgut and rumen as generalists, and it commonly has extensive repertoires of polysaccharide utilization loci (PULs) and carbohydrate active enzymes targeting various plant polysaccharides, but the difference is also obvious among the generalists in substrate and PULs [36]. And the Bacteroides belongs to the cellulolytic bacteria [37], which means Bacteroides play important role in degrading forage fiber source. Here, Prevotella sp. CAG:485, Bacteroides sp. CAG:443 and Bacteroides thetaiotaomicron had positive relationships with CBM13. The oat hay contains a large amount of xylan and the primary function of CBM13 is to bind the polysaccharide xylan [38]. Therefore, the three species (Prevotella sp. CAG:485, Bacteroides sp. CAG:443 and Bacteroides thetaiotaomicron) may utilize oat hay to become richer by CBM13. Under microbial interactions, Prevotella sp. CAG:873, Prevotella sp. CAG:1031, Bacteroides sp. CAG:927, Prevotella sp. P4-76 and Prevotella sp. CAG:279 also became greater. Altogether, these results showed the two Eubacterium species (Eubacterium sp. CAG:252 and Eubacterium sp. CAG:38) and the three species (Prevotella sp. CAG:485, Bacteroides sp. CAG:443 and Bacteroides thetaiotaomicron) directly respond to SS diet and SO diet respectively, and caused changes in other bacteria through co-occurrence network.
Alterations in the composition of rumen microbiome lead to parallel changes in rumen metabolites. Interestingly, “vitamin B6 metabolism” pathway was enriched based on the increased metabolites in the rumen of SO calves. The vitamin B group plays an important role in some essential metabolic processes such as fatty acid synthesis and gluconeogenesis [39]. Vitamin B6 can suppress apoptosis of bovine endothelial cells [40], revealing that enriched “vitamin B6 metabolism” pathway may have positive effect on the rumen development of SO calves. Besides, some microbiota such as Prevotella bacteria can possess a vitamin B6 biosynthesis pathway [41]. Thus, this inspires us that the increased Prevotella species may improve the rumen development by vitamin B6 biosynthesis pathway under SO feeding. Another interesting finding was the increased levels of the three metabolites including Pyridoxine, 4a-Carbinolamine tetrahydrobiopterin and Gentisic acid. Pyridoxine belongs to vitamin B6. The previous study reported that feeding dairy cattle with Pyridoxine can improve the dry matter intake and milk yield ratio [42], indicating that a higher Pyridoxine level has positive effects on productive and reproductive efficiency. The 4a-Carbinolamine tetrahydrobiopterin is an intermediate product of polyphenol degradation such as condensed tannin and hydrolysable tannin, which is normal in the rumen[43]. So et al. demonstrated that 4a-Carbinolamine tetrahydrobiopterin can reduce the oxidative stress induced by peroxyl radicals, NO, superoxide anions, and 4a-Carbinolamine tetrahydrobiopterin also can increase the cell viability and decreased lipid peroxidation [44]. Kang et al. also found that 4a-Carbinolamine tetrahydrobiopterin can against oxidative stress induced cell damage in Chinese hamster lung fibroblast [45]. Taken together, we can infer that SO feeding may take oxidative stress away from rumen through 4a-Carbinolamine tetrahydrobiopterin. Moreover, the competition can be made for H2 between methanogen and phloroglucinol-degrading bacteria when 4a-Carbinolamine tetrahydrobiopterin was supplied, which might be related to methane inhibition [46]. This finding means the higher 4a-Carbinolamine tetrahydrobiopterin can inhibit the methanogen to reduce methane production, which may explains SO feeding decreased the methanogens. The published study revealed that Gentisic acid ccould increase proliferation in the cell [47]. It was not difficult to infer that higher Gentisic acid may promote proliferation of rumen epithelial cells rumen development under SO feeding.
In lambs, the published study revealed that feeding alfalfa hay caused an accelerated cell proliferation and a facilitated rumen morphological development compared with feeding soybean hull [4]. In this study, the result of rumen epithelial transcriptome revealed that the DEGs were signifificantly enriched in the GO biological processes of energy metabolism (D-gluconate catabolic process, gluconokinase activity and D-gluconate metabolic process), the transcription and translation (3'-5' DNA helicase activity, regulation of RNA export from nucleus, RNA polymerase II activating transcription factor binding, histone H2A acetylation, DNA helicase complex, positive regulation of cytoplasmic translation and activating transcription factor binding) and immunity (positive regulation of interferon-beta secretion and positive regulation of interferon-alpha secretion) as well as the KEGG pathway of signaling molecules (PI3K-Akt signaling pathway and B cell receptor signaling). There were three classical GO terms about energy metabolism, such as D-gluconate catabolic process, gluconokinase activity and D-gluconate metabolic process. According to the former study, it demonstrated that gluconatecatabolism was computed to take place through gluconokinase catalyzes gluconate to generate 6-phosphogluconate which could then be further degraded through the hexose monophosphate shunt (HMS) via 6-phosphogluconate dehydrogenase [48]. Rohatgi et al. [49] evaluated changes of cellular core metabolism induced by gluconate degradation, which found that an increase in the flux range of all reactions in the HMS upon uptake of gluconate. More specifically, gluconate degradation effected metabolic pathways involving metabolites or reactions derived from the HMS, and these pathways included nucleotide, carbohydrate, amino acid and lipid metabolic pathways due to increased levels of ribose-5-phosphate, xylulose-5-phosphate, erythrose-4-phosphate and NADPH, respectively[49]. Thus, the three GO biological processes may promote the degradation of gluconate, thereby affecting the metabolic pathway derived from the HMS, providing energy (NADPH) and raw materials (ribose-5-phosphate) for rumen epithelial development of SO calves. Another interesting finding was the GO biological process named positive regulation of interferon-alpha secretion. Interferon-alpha has been studied for treatment of various viral diseases such as bovine respiratory diseases, and it also showed a positive effect to reduced the mortality rate and enhance average weight gain in calves [50, 51]. It may tell us that SO feeding may regulate interferon-alpha secretion to promote rumen development. It must be emphasized that almost all corresponding DEGs were upregulated under SO feeding. Taken together, we inferred that these enrichments reflect improved energy metabolism, transcription and translation levels, and immune levels in the rumen epithelium of SO calves. The observations of rumen epithelial papilla correspond to the speculation, which showed lengthened and widened rumen papilla under SO feeding.
Further WGCNA suggested that MEroyalblue was positively correlated with rumen papillae length. Subsequently, enrichment analysis of the genes in the MEroyalblue revealed that functions are mainly focused on mRNA metabolic process, RNA 3'-end processing, response to virus, RNA processing, endoplasmic reticulum lumen, endoplasmic reticulum, RNA binding, nucleotidyltransferase activity, mRNA binding and cysteine-type peptidase activity, which may imply these enriched functions reflected an accelerated cell cycle of the rumen epithelial cells.