Precision dietary interventions (e.g., altering proportions of dietary protein fractions) has significant implications for the efficiency of nutrient use in ruminants, as well as lowering their environmental footprint, specifically nitrogen (N) emissions. Soluble protein (SP) is defined as the protein fraction that is rapidly degraded in the rumen (e.g., non-protein N and true protein), and our previous study found that regulating SP levels could improve N efficiency in Hu sheep. Thus, the present study was conducted to explore in vitro how protein fractions with different SP levels modulate the rumen microbial community and its association with N metabolism. Four dietary treatments with different SP proportions and similar crude protein (CP) content (~14%) were formulated (% of CP): 20 (S20), 30 (S30), 40 (S40) and 50 (S50). Results showed that NH₃-N content increased with increasing SP levels at 4, 12 and 24 h; TVFA, acetate, propionate and valerate were higher in S30 and S40 (p < 0.05) and had quadratic effects (p < 0.05). Moreover, dry matter digestibility (DMD) and N digestibility (ND) were all decreased with S20 and S50 (p < 0.05). The S30 and S40 treatments increased the abundance of Bacteroidetes and Prevotella (Prevotella_ruminicola) but decreased the abundance of Firmicutes and Proteobacteria (p < 0.05). Bacterial pathways related to amino acid and fatty acid metabolism also were enriched with S30 and S40. The abundance of Entodinium was increased with S30 and S40 and had a positive correlation with Prevotella, and these two genera also played an important role in N metabolism and VFA synthesis of this study. In conclusion, bacterial and protozoal communities were altered by the level of SP (% of CP), with higher SP levels (~50% of CP) increasing the microbial diversity but being detrimental to rumen N metabolism.

Ruminants account for a relatively large share of global nitrogen (N) emissions. It has been reported that nutrition control and precise feeding can improve the N efficiency of ruminants. The objective of the study was to determine the effects of soluble protein (SP) levels in low-protein diets on growth performance, nutrient digestibility, rumen microbiota, and metabolites, as well as their associations of N metabolism in fatteningHusheep. Approximately 6-month-old, 32 healthy fattening maleHusheep with similar genetic merit and an initial body weight of 40.37 ± 1.18 kg were selected, and divided into four groups (n= 8) using the following completely randomized design: the control diet (CON) with a 16.7% crude protein (CP) content was prepared to meet the nutritional requirements of fattening sheep [body weight (BW): 40 kg, average daily gain (ADG): 200–250 g/d] according to the NRC recommendations; other three include low protein diets (LPA, LPB, and LPC) of CP decreased by ~10%, with SP proportion (%CP) of 21.2, 25.9, and 29.4 respectively. The feeding trial lasted for 5 weeks including the first week of adaptation. The results showed no difference in the growth performance (P> 0.05); DM and CP digestibility were higher in LPB and LPC, with maximum organic matter digestibility in LPB (P< 0.05). Low-protein diets decreased serum urea-N whereas urinary urea-N was lower in LPB and LPC (P< 0.05), while N retention and the biological value of N were higher in LPB and LPC (P< 0.05). Ruminal NH₃-N concentration in LPA and LPB was low than CON (P< 0.05), while total volatile fatty acid (TVFA), acetate, propionate, and butanoate were all lowest in LPA (P< 0.05). In the rumen microbiome, LPB increased the community richness in Prevotellaceae andPrevotella_1(P< 0.05); Metabolomics analysis revealed low-protein diets downregulated the amino acid metabolism pathways, while the biosynthesis of unsaturated fatty acids along with vitamin B6 metabolism were upregulated with increased SP. These findings could help us understand the role of different SP levels in the regulation of rumen microbial metabolism and N efficiency. Overall, low-protein diets (CP decreased by ~10%) can reduce serum urea-N and ruminal NH₃-N without affecting the growth performance of fatteningHusheep. Additionally higher N efficiency was obtained with an SP proportion of ~25–30%.

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal ${\text{NH}}_4^{+}$–N and ${\text{NO}}_2^{-}$–N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

Studies have shown that exogenous thiamine (THI) supplementation can alleviate inflammation and promote rumen epithelial development in goats and cows. This research aimed to evaluate the effect of THI supplementation on LPS-induced inflammation and energy metabolic dysregulation in RECs of goats. Cells were stimulated with either 5 μg/mL THI for 18 h (THI group) or with 5 μg/mL LPS for 6 h (LPS group). The CON group was stimulated with DMEM/F-12 medium without THI for 18 h. The LPTH group was pretreated with THI for 18 h, followed by LPS stimulation for 6 h. THI supplementation decreased the ROS content (p < 0.05), as well as the ratios of phosphorylated (p)-p65 to p65 (p < 0.05) and p-AMPKα to AMPKα (p < 0.05). Interestingly, when the p38 gene was overexpressed in the LPTH group, the ratio of p-p65 to p65 and p-AMPKα to AMPKα proteins significantly increased, and ATP content decreased (p < 0.05). Our results suggest that THI possesses anti-inflammatory and metabolic-modulatory effects in RECs. The mechanism is largely related to the suppression of the NF-κB/p38 MAPK/AMPK signaling pathway. Additionally, we also revealed that THI supplementation can inhibit LPS-induced oxidative damage and apoptosis to protect mitochondrial function in RECs.

Compost-bedded pack barns (CBP) are getting huge attention as an alternative housing system for dairy cows due to their beneficial impact on animal welfare. Effective microorganisms (EM) inoculums are believed to enhance compost quality, improve soil structure and benefit the environment. However, little information is available on the impact of incubation with external EM combinations on the barn environment, compost quality and microbial diversity in CBP. This experiment was carried out to investigate the effect of inoculating different combinations of EM [Lactobacillus plantarum (L), Compound Bacillus (B) and Saccharomyces cerevisiae (S)] on compost quality and microbial communities of CBP products, as well as the relationship with the heifers’ barn environment. CBP barns were subjected to the following four treatments: CON with no EM inoculum, LB/LS/LBS were Incubated with weight ratios of 1:2 (L: B), 1:2 (L: S), 1:1:1 (L: B: S), respectively. The EM inoculation (LB, LS, LBS) reduced the concentration of respirable particulate matter (PM10 and PM2.5) in the CBP, and decreased the serum total protein and total cholesterol levels in heifers. Notably, LBS achieved the highest content of high-density lipoprotein compared to other treatments. Microbiome results revealed that EM inoculation reduced the bacterial abundance (Chao1 index) and fungal diversity (Shannon & Simpson indexes), while increasing the relative abundance of various bacterial genera (Pseudomonas, Paracoccus, Aequorivita) and fungi (Pestalotiopsis), which are associated with cellulose decomposition that ultimately resulted in accelerating organic matter degradation and humification. Furthermore, high nutrient elements (TK&TP) and low mycotoxin content were obtained with EM inoculation, with LBS showing a particularly pronounced effect. Meanwhile, LBS contributed to a decline in the proportion of fungal pathogen categories but also led to an increase in fungal saprotroph categories. Generally, EM inoculation positively impacted compost product quality as organic fertilizer and barn environment by modifying the abundance of cellulolytic bacteria and fungi, while inhibiting the reproduction of pathogenic microbes, especially co-supplementing with L, B and S achieved an amplifying effect.

Manipulating dietary nutrients, especially protein fractions, holds significance in enhancing the antioxidant capacity and immunity function of ruminants. This study investigated the impact of dietary adjustments in soluble protein (SP) levels, in conjunction with a reduction in crude protein (CP) content, on the antioxidant capacity, inflammatory response, carcass characteristics, and meat quality of sheep. This study had four dietary treatments, including a control diet (CON) adhering to NRC standards with a CP content of 16.7% on a dry matter basis and three diets with an approximately 10% reduction in CP content compared to CON with SP levels (% of CP) of 21.2 (SPA), 25.9 (SPB) and 29.4% (SPC), respectively. Thirty-two healthy male Hu sheep, with an initial live weight of 40.37 ± 1.18 kg and age of 6 months, were randomly divided into four groups to receive these respective diets. Our data revealed no significant differences in slaughter performance among treatments (p > 0.05), although low-protein treatments decreased the stomachus compositus index (p < 0.05). Compared with CON, as SP was adjusted to 21.2%, total antioxidant capacity (T-AOC) and catalase (CAT) concentrations were decreased in the serum (p < 0.05), glutathione peroxidase (GSH-Px) content was decreased in jejunum and ileum (p < 0.05), superoxide dismutase (SOD) concentration was reduced in the duodenum (p < 0.05), and malondialdehyde (MDA) content was increased in spleen and ileum (p < 0.05). On the other hand, pro-inflammatory cytokine (IL-1β, IL-6 and IL-8) contents were upregulated in the serum (p < 0.05), while immunoglobulin (IgA and IgM) contents were reduced in the duodenum (p < 0.05) with SP adjustments. Additionally, the SPB and SPC diets reduced the content of saturated fatty acids and increased the content of polyunsaturated fatty acids compared with CON (p < 0.05), along with retention in the tenderness and water-holding capacity of the longissimus lumborum muscle. In summary, reducing CP by 10% with an SP proportion of ~25–30% improved meat quality without compromising antioxidant capacity and immunity function, while lower SP levels had adverse effects.