With the growth of young ruminants from birth to adulthood, their rumen morphology and functions undergone huge changes, which also results in a remarkable shift in nutrient digestion patterns. Our previous study and others confirmed that feeding solid feed, including concentrate (high grain diet) and forages, has a beneficial effect on rumen development [19, 20]. With alteration of rumen microbiome and increased concentration of volatile fatty acids (VFA) by solid diet intervention [21], the growth of the rumen epithelium and its ability of nutrition transport and metabolism was improved [22]. In the current study, greater rumen epithelial morphology in solid diet groups was identified. Using the proteomic analysis, our results suggested that the DEPs of rumen epithelium driven by solid diet were mostly associated with cell division, immunity, metabolism and signal transduction, which indicates rumen in goats consumed solid diet had a more mature function.
Rumen morphological changes
Rumen is a significant digestive organ in ruminants [23]. The papillae on the rumen wall can promote the contact area of the rumen wall with the digesta, and enhance the ability to absorb nutrients. The length, width and thickness of the rumen papilla are professional indicators for evaluating rumen development. In this study, significant improvement of rumen tissue morphology in solid diet group, especially the rumen papilla and stratum corneum, were observed. it was supposed that solid diet can improve rumen development and functions. Many studies [24–27] have shown that the rumen tissue morphology has undergone tremendous changes with the feeding of solid feed, including increased rumen papilla width, appropriate stratum corneum thickness and better integrity. therefore, it is not surprising that our proteomic results revealed the DEPs between treatment. Additionally, increased function including muscle protein synthesis and cell framework construction, were observed in solid diet rumen which was corresponded to the results of tissue sections.
Cell synthesis, protein construction and immune
Solid feed can stimulate rumen growth through increasing rumen VFA concentration [3, 28]. In this research, due to the feeding of solid feed, the processes of cell synthesis and protein binding had more active performance, including extracellular matrix, DNA packaging complex, extracellular matrix organization, extracellular structure organization, nucleosome organization, protein complex binding, actin binding, growth factor binding, and actin filament binding. Notably, in previous studies, the increases of rumen VFA concentration can synchronously rise the rate of cell mitosis [29], which was exactly the first step in cell synthesis. In addition, 6 up-regulated proteins participated in cell differentiation, gene expression and immune regulation were enriched in the pathway of focal adhesion, and complement and coagulation cascades. In contrast, disease pathways were enriched by down-regulated proteins, which may imply that the intake of solid feed can improve the immune ability and disease resistance of the body. In fact, body's health influenced by diet has been reported in many studies [30–32]. Recently, researchers discovered that the timely introduction of solid foods for infants can effectively reduce the incidence of allergic diseases [33], which could be due to the characteristics of dietary metabolites that shape and improve the intestinal immune system of young animals [34]. The results of our analysis also support this view. Surprisingly, the proteins enriched in the pathway of protein digestion and absorption were all down-regulated, this phenomenon might be related to the stress response caused by the introduction of solid feed. To sum up, we can confirm that supplementing solid feed accelerated the proliferation of rumen epithelial cells and increased the resistance of the gut to disease.
Rumen metabolism and signal transduction
With the changes of rumen tissue morphology, the material metabolism functions of rumen also undergo essential shifts. The energy metabolisms a critical metabolic pathway that has a decisive effect on the growth and development of organisms. However, the rumen is a unique organ of ruminants, and the energy consumes for growth does not depend on sugars, ketone bodies or glutamines, but on the utilization of microbial terminal fermentation products, e.g. VFA [35–37]. Thus, lipid metabolism is more prominent during the process of rumen epithelial growth. VFA can also be used as signaling molecules and intermediate mediators to indirectly regulate the expression levels of some genes and proteins in the rumen epithelium. For example, butyrate in VFA can effectively regulate histone deacetylation, affect the expression of associated genes, and mediate multiple ion transport channels.
Our data suggested that many proteins associated with lipid metabolism are mostly up-regulated by solid diet. In our analysis, we can also recognize over half of the active upregulated proteins gathered by pathways of arachidonic acid metabolism, steroid hormone biosynthesis and folate biosynthesis. Among them, arachidonic acid is an indispensable polyunsaturated fatty acid for the development of animals, especially for young animals. In fact, arachidonic acid can be involved in cell membrane synthesis as a structural lipid in combination with phospholipids. It also acts as a direct precursor of prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane A2 (TXA2) and leukotrienes and C4 (LTC4), which have important regulatory effects on the biological metabolism. Notably, in the steroid hormone biosynthesis pathway, the synthesis process of glucuronosyltransferase and 3α-Hydroxy-Steroid Dehydrogenase were enriched by most upregulated proteins. Coincidentally, both enzymes can effectively promote the conversion of multiple steroid substances into bile [38, 39], thereby improving the ability of rumen to absorb fat-soluble vitamins and fatty acids, including arachidonic acid [40]. Recent studies also showed that dietary and microbial factors can control the synthesis of intestinal bile and its metabolites and further regulate the growth and function of related cells [41]. In another our study, the introduction of solid feed caused dual changes in the rumen microbiota and its digestion end products. Obviously, the metabolism of bile acids was also affected by the complex changes, and its mechanism deserves further study. Folic acid, converted by reductase into physiologically active tetrahydrofolate (THFA or FH4), is involved in base synthesis and plays a critical role in protein synthesis and cell division.
In addition to lipid metabolism-related pathways, the lysosome and PI3K-Akt signaling pathways were also enriched with the DEPs. Lysosomes have a strong substance degradation function. In the case of tissue nutrient deficiency, cells initiate autophagy, and macromolecules such as proteins in the cytoplasm are moved into lysosomes to degrade into small molecules, thus maintaining the internal environment of the cells [42]. In this study, the way of feeding solid feed provided abundant nutrients for the rumen, which might have some inhibitory effect on lysosome activity. The PI3K-Akt (protein kinase B) signaling pathway can be mediated by a variety of signaling molecules, the lipid molecule PIP3 is involved in the activation of Akt as a signaling molecule. Once Akt is activated, it can regulate critical cellular responses such as apoptosis, protein synthesis, and metabolism through substrate phosphorylation. Unsurprisingly, A0A452FAR7 regulated by PIK3CB gene was significantly up-regulated in many other pathways, such as cellular processes, transport and catabolism, carbohydrate metabolism. In previous study, PIK3CB was confirmed to be closely related to the development of cells [43]. In this study, the introduction of solid feed activated the expression of PIK3CB and, as previously reported [44], PIK3CB was involved in multiple pathways related to the growth of rumen epithelium. The above analysis may provide some insights into the underlying causes of rumen development.