The rumen is the most efficient bioreactor. Besides, an indivisible relationship exists between rumen microbes and the host. The rumen microbiota comprises bacteria, protozoa, fungi, and archaea. These microorganisms exist in symbiotic, competitive, predation, or antagonistic relationships and are jointly responsible for the microbial fermentation in the rumen (Morgavi et al., 2013). Research on rumen microbiota and the host mainly focuses on how nutrition strategies influence the rumen microbiome and how alteration of rumen microbiota affects the host.
Dietary energy is an important factor affecting nutrient intake, digestion, metabolic efficiency, and production performance. This research belongs to part of the previous experiment, in our previous research, we found that high dietary energy levels increase the final weight, live weight, average daily gain and feed rewards. The increase of dietary energy levels significantly increased (p < 0.05) the mRNA expression of fat deposition-related genes in subcutaneous fat (HSL), tail fat (FASN) andlongissimus dorsi(FABP4) (Zhang et al., 2021c). As rumen fermentation and microbiota play a key role in nutrient digestion and utilization of ruminants, therefore, the influence of dietary energy on rumen fermentation and microbiota was further investigated in current research.
4.1. High -energy diet affects ruminal fermentation
VFA, NH3-N, and MCP are the critical rumen fermentation pattern indices. Feed in ruminants first undergoes microbial fermentation in the rumen. Ruminal microbiota breaks down carbohydrates into VFA, the main metabolite of microbial fermentation (Tan et al., 2014). Microbes first break down carbohydrates (crude fiber, starch, and soluble sugar) to pyruvate. The pyruvate is then metabolized to different VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) through different metabolic pathways. Proteins and fats can also be degraded to VFA (mostly brunch chain fatty acids). Thus, ruminants utilize VFA which degrade from carbohydrates by ruminal microbes rather than glucose, which account for 70%~80% metabolizable energy of ruminant energy needs(Na and Guan, 2022). ATP for microbial growth is released during the VFA synthesis processes.
Rumen fermentation patterns usually change under different nutrition conditions. VFA concentration increases with energy level or a high concentrate diet. Also, high metabolizable energy (ME) significantly increases the proportions of butyrate and valerate (Wang et al., 2020a). Furthermore, high acetate and butyrate concentrations have been observed after intake of high grain diet (Xie et al., 2021). In the present study, butyric acid levels in the rumen fluid increased with diet energy concentration. A significant increase in MCP with energy intake was also observed, consistent with previous findings (Lu et al., 2019). These findings show that feed energy level is directly proportional to the rumen MCP yield. However, a positive correlation between acetic acid and propionic acid and daily feed energy was not observed in this study, which may be due to a strong correlation between rumen VFA concentration and feed type, and nutrient level (Flint et al., 2007). The LE group diet contained more crude fibers as the substrate for microbial fermentation to produce VFA. Our result indicates that a high-energy diet had limited influences on the rumen fermentation pattern, and provided sufficient energy for microorganisms to synthesize MCP, giving sheep more rumen-protected proteins to metabolize.
4.2. High-energy diet changes the composition of ruminal microbiota
Bacteria are the most abundant and diverse microbiota in the rumen, attributable to diet (Henderson et al., 2015). On the other hand, the microbiota affects animal health. Therefore, understanding the effect of dietary energy on microflora is of great significance to animal husbandry. A more diverse microbial community increases the resilience, resistance, and stability of the rumen ecosystem (Konopka, 2009). Diet energy affects the overall rumen multi-kingdom microbiota (Park et al., 2020). Mostly, ruminal microbiota diversity decreases with dietary energy (Lv et al., 2020; Wang et al., 2021). In the present study, ACE and Chao indices were significantly higher in the LE group than in the CON and HE groups, consistent with previous studies (Plaizier et al., 2017; Zhang et al., 2021d), which indicates that a high-energy diet reduces richness of the microbiome.
Despite the complexity of the rumen ecosystem, the (composition and abundance of) rumen microbiota are relatively stable at the phylum level. Firmicutes, Bacteroidetes, Fibrobacteres, and Proteobacteria are the four most abundant phyla in the sheep rumen, whereas the dominant genera include unidentified Prevotellaceae, Fibrobacter, unidentified Lachnospiraceae, Saccharofermentans, and Succinivibrio (Zhang et al., 2021d). In the present study, the dominant gut microbial phyla were Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria, whereas the dominant genera were Lentisphaera, Prevotella, unclassified Lachnospiraceae, unclassified Bacteroidales, unclassified Clostridiales, and unclassified Prevotellaceae.
More research continues to reveal the functions of ruminal microbiota in ruminants. For instance, Bacteroidetes degrade carbohydrates and polysaccharides more efficiently than Proteobacteria (Pitta et al., 2014). At the genus level, Butyrivibrio, Fibrobacter, Olsenella, and Prevotella are critical in degrading cellulose (Huws et al., 2016). Overall, studies (Cui et al., 2019; Wang et al., 2019; Lin et al., 2021) demonstrate that a high-energy diet reduces the relative abundance of ruminal microbiota that participate in crude fiber fermentation. A former study revealed that Bacteroidetes utilize crude fiber in the form of glycans (oligomeric and polymeric glycans), and the abundance of this bacteria is influenced by the dietary intake of these indigestible carbohydrates (Patnode et al., 2019). We found that the relative abundance of Bacteroides in the HE group was relatively low (feed of sheep in the HE group contained little crude fiber). Furthermore, at the species level, the relative abundance of Rikenellaceae_bacterium and Bacteroidales_bacterium decreased with an increase in the dietary energy level. Rikenellaceae is one of the main producers of VFA. Previous studies have found that a high-fat diet drastically decreased the Rikenellaceae composition in the rumen (Matsushita et al., 2021). Accordingly, our research demonstrated that the relative abundance of crude fiber metabolizing bacteria in rumen decreased with an increase in energy diet.
The rumen microbiota produces numerous metabolites, including VFA and polyamines, through anaerobic fermentation. Notably, 10% of metabolites in the mammalian blood are derived from microbes or microbial activities (Cummings and Macfarlane, 1997). The blood metabolites participate in developing and regulating host physiology and immunity and can be modified to other novel metabolites (Wikoff et al., 2009). Relationships between some microorganisms and rumen metabolites have been well established. Members of the Lachnospiraceae family are among the main producers of short-chain fatty acids (Vacca et al., 2020). Bacteroidetes produce enzymes that degrade plant cell wall compounds (e.g., cellulose and pectin) to release VFA (mainly acetate, propionate, and butyrate) (Thomas et al., 2011). The phylum Lentisphaerae is associated with changes in feed efficiency (Mao et al., 2013). Succiniclasticum degrades starch into acetic and succinic acids and further converts succinic acid to propionic acid (Ferrario et al., 2017). The abundance of Prevotellaceae UCG-003 positively correlated with the rumen VFA content (Ahmad et al., 2020). In the present study, correlation analysis revealed that at the species level, only the relative abundance of Lachnospiraceae_bacterium, Prevotella_brevis, Prevotellaceae_bacterium, and Succiniclasticum_ruminis was significantly different among the groups. Further analyses revealed that Prevotella_brevis promotes acetic acid synthesis, Succiniclasticum_ruminis promotes synthesis, Prevotellaceae_bacterium promotes propionic acid, isobutyric acid, and isovaleric acid synthesis, whereas Lachnospiraceae_bacterium inhibit ammonia nitrogen and propionic acid synthesis.
4.3. High-energy diet alters rumen microbial function
Changes in the KEGG pathway reflect an alteration in rumen microbial function under specific conditions, and the changes in enzyme function modify microbial function. Rumen microbial function changes with dietary energy concentration. Zhang et.al (Zhang et al., 2021b) investigated the effect of feeding sheep with caragana, corn straw and alfalfa, they found that sheep with caragana (this group had a higher digestible energy) mainly enhanced the microbial function about starch and sucrose metabolism, fructose and mannose metabolism, photosynthesis, and D-alanine metabolism in the rumen. A separate study (Wang et al., 2020a) further confirmed that higher energy digestible diet increased the abundance of bacteria related to carbohydrate metabolism. In the current study, we found that the expression of genes related to lipid metabolism increased with dietary energy, but a reverse trend was observed for genes related to glycan biosynthesis and metabolism. Microbial CAZy regulates carbohydrate metabolism and, thus, is critical to the energy available to the host. In the present study, we found that a high-energy diet increased the expression of these genes involved in energy metabolism. However, the expression of genes that encode enzymes that catalyze plant cell wall degradation decreased.
KEGG and CAZy enrichment data further revealed that a high-energy diet improved lipid metabolism in sheep by promoting the expression of related genes. Meanwhile, low-energy diets enhance glycan biosynthesis and metabolism by promoting the expression of enzymes involved in plant cell wall degradation to meet the animals' energy needs.