The effects of isochlorogenic acid (ICGA) on ewes rumen environment, microbial diversity, and immunity at different physiological stages (estrus, pregnancy and lactation) were studied in this experiment. Twenty healthy female Hu lambs of 1.5 months with similar body weight (17.82 ± 0.98 kg) and body condition were selected and randomly divided into two groups: the control group (CON) and the ICGA group (ICGA). The lambs of CON were fed a basal diet, while the lambs of ICGA were supplemented with 0.1% ICGA based on the basal diet. Lambs rumen fermentation characteristics, microbial diversity and immunity at estrus, pregnancy, and lactation stages were determined and analyzed, respectively. The results showed that the rumen pH in CON increased first and then decreased as lambs grew (p < 0.05). However, it showed the opposite change in ICGA. The content of ammonia nitrogen (NH₃-N) showed the highest at estrus stage in both groups, but it was significantly higher in ICGA than that in CON (p < 0.05). The Acetic acid/propionic acid (A/P) ratio at estrus stage and the volatile fatty acids (VFAs) at pregnancy stage in ICGA were significantly higher than those of the CON (p < 0.05). The 16S rDNA sequencing analysis showed that the Shannon, Chao 1 and ACE indexes of the ICGA were significantly higher than those of the CON both at estrus and lactation stages (p < 0.05), while they showed higher at the pregnancy stage in CON (p > 0.05). Principal component analysis (PCA) showed that there were significant differences in rumen microorganism structure between CON and ICGA at all physiological stages (p < 0.01). At the phylum level, compared with the CON, Firmicutes relative abundance of three physiological stages decreased (p > 0.05) while Bacteroidota increased (p > 0.05). The relative abundance of Synergistota at estrus stage and Patescibacteria at the lactation stage increased significantly too (p < 0.05). At the genus level, compared with the CON, the relative abundance of Prevotella at three stages showed the highest (p > 0.05), while the relative abundance of Succiniclasticum, unclassified_Selenomonadaceae and Rikenellaceae_RC9_gut_group showed different abundances at different physiological stages in ICGA. Compared with the CON, the lambs of the ICGA showed higher blood IgG, IgM, and TNF- α contents at three physiological stages and higher IL-6 contents at pregnancy stage (p < 0.05). Conclusion: Adding ICGA could regulate ewes rumen fermentation mode at different physiological stages by increasing rumen NH₃-N at estrus, VFAs at pregnancy, and the ratio of A/P at lactation. It optimizes rumen microbial flora of different physiological stages by increasing Bacteroidota relative abundance while reducing Firmicutes relative abundance, maintaining rumen microbial homeostasis at pregnant stage, increasing the number of beneficial bacteria in later lactating and ewes blood immunoglobulins content at three physiological stages.

Tibetan sheep are an important livestock breed adapted to the extreme environment of the Qinghai–Tibet Plateau (QTP). Their energy metabolism and environmental adaptability are highly dependent on the rumen microbiome. However, systematic comparisons of the rumen microbiome, its functions, and the resistome between plateau-adapted breeds and lowland breeds remain insufficient. In this study, 6 Tibetan sheep (TS) and 6 Hu sheep (HS) were selected. All the selected sheep had a body weight of 34 kg (±0.5 kg) and an age of 1 year (±1 month) and were all managed under local traditional natural grazing (without supplementary feeding). Using metagenomics and metabolomics techniques, systematic comparative analysis was conducted on the differences in rumen microbial community structure, functions, resistome, and metabolites between the two breeds. Metagenomic analysis showed that at the phylum level, the abundance of Bacteroidetes in the rumen of TS was significantly higher than that in HS (p < 0.05); at the genus level, the abundance of Bacteroides in TS was also significantly higher (p < 0.05). Carbohydrate-active enzymes (CAZy) analysis indicated that the abundance of Glycosyltransferases (GTs) and Carbohydrate-Binding Modules (CBMs) in the rumen of TS were significantly upregulated (p < 0.05), while HS was rich in various Glycoside Hydrolases (GHs). Comprehensive Antibiotic Resistance Database (CARD) analysis revealed that more than 60% of the Antibiotic Resistance Genes (ARGs) in the rumen of HS were present at higher levels than those in TS. Metabolomics identified a large number of differential metabolites, among which metabolites such as 2E,6Z,8Z,12E-hexadecatetraenoic acid, Leukotriene F4, and Tenurin were significantly upregulated in the rumen of TS. Correlation analysis showed that rumen microbial flora and their metabolites jointly act to regulate rumen ARGs. Specifically, microorganisms including Firmicutes and Succiniclasticum had a significantly positive correlation with ARGs such as rpoB2 (p < 0.05), while differential metabolites like endomorphin-1 and Purothionin AII exhibited a significantly negative correlation with ARGs such as rpoB2 (p < 0.05). Therefore, compared with HS, the synergistic effect of the rumen microbial flora, their metabolites, and the resistome in TS is an important characteristic and strategy for their adaptation to the hypoxic environment of the QTP, and also contributes to the formation of their unique rumen resistome. Despite being reared in the same plateau environment, the rumen microbiome of HS still retains low-altitude characteristics, which are manifested as high GHs activity and high ARGs abundance.

The experiment was conducted to investigate the potential effects of steviol glycosides on growth performance, rumen fermentation processes, and microbial diversity in Hu sheep. A single-factor design was used for the trial. Twenty healthy weaned Hu lambs, possessing comparable body weights averaging 18.31 ± 1.24 kg, were randomly allocated into two distinct groups: the control group (CON) and the experimental group (STE), with each comprising 10 lambs. The CON was fed the basal diet, and the STE was supplemented with 0.07% steviol glycosides based on the basal diet. During the experimental period, variations in body weight and feed intake were closely monitored and recorded. After feeding for 90 d, blood was collected to determine blood biochemical indices, and rumen fluid samples were gathered for an in-depth analysis of rumen fermentation parameters and microbial diversity. The outcomes revealed no statistically significant differences in growth performance or serum biochemical indices between the two groups (p > 0.05). Rumen pH in STE and CON was within the normal range. The rumen ammonia nitrogen (NH₃-N) and acetic acid (AA) content of STE decreased significantly compared with CON (p < 0.05). No significant variations were observed in the levels of other volatile fatty acids (VFAs) between the two groups (p > 0.05). The rumen microbial OTUs count, as well as the Shannon, Simpson, Chao1, and Ace indices, were notably lower in the STE group compared to the CON group (p < 0.05). Additionally, at the phylum level, the relative abundance of Firmicutes, Bacteroidetes, and Proteobacteria collectively accounted for over 97% of the total phylum composition. In comparison to the CON group, the STE group exhibited an increase in the relative abundance of Proteobacteria (p < 0.05), accompanied by a significant reduction in the relative abundance of Patescibacteria and Desulfobacteria (p < 0.05). At the genus level, there was a notable increase in the relative abundance of Prevotella_7 and Succinivibrionaceae_UCG_001 in the STE group, whereas the relative abundance of Rikenellaceae_RC9_gut_group significantly decreased (p < 0.05). According to the correlation analysis between rumen microflora and VFAs, the relative abundance of Succinivibrionaceae_UCG_001 displayed a significant negative correlation with AA (p < 0.05), whereas Lactobacillus exhibited a notable positive correlation with isobutyric acid (IBA) (p < 0.05). In summary, steviol glycosides had no significant effect on the production performance and blood biochemical indexes of Hu sheep. Steviol glycosides can improve rumen fermentation parameters and rumen microflora structure of Hu sheep and have a certain effect on rumen microbial diversity and composition.

The sensory quality and flavor of lamb meat, critical to market competitiveness, are influenced by rumen microbial fermentation and dietary management strategies. Coated betaine (CBet), a rumen-protected methyl donor, exerts systemic nutritional regulation in ruminants. This study explored the effects of CBet supplementation on lamb meat quality using 18 Dorset ♂ × Hu sheep ♀ F1 crossbred lambs, randomly assigned to either a control group (basal diet) or a 0.20% CBet-supplemented diet for 60 days (n = 9 per group). The results demonstrated that CBet significantly increased ruminal concentrations of total volatile fatty acids (TVFAs), acetic acid, propionic acid, and butyric acid (p < 0.05). Additionally, CBet supplementation enhanced muscle redness (a*), crude fat, crude ash, heptadecanoic acid (C17:0), and tricosanoic acid (C23:0) (p < 0.05) while decreasing shear force and the concentration of cis-13,16-docosadienoic acid (C22:2) (p < 0.05). Furthermore, CBet elevated characteristic flavor compounds (e.g., nonanal) and their relative odor activity values (ROAVs), and decreased undesirable odors (e.g., dodecanal) (p < 0.05). As illustrated in the graphical abstract, these improvements were mediated through regulatory effects of CBet on rumen microbiota composition, muscle fatty acids, amino acids, and volatile flavor compounds. Specifically, CBet significantly increased the relative abundances of Firmicutes, Proteobacteria, Prevotella, and Bifidobacterium in the rumen (p < 0.05) and altered the Firmicutes/Bacteroidota ratio. In conclusion, dietary supplementation with 0.20% CBet effectively enhances lamb meat quality and flavor, effects closely associated with changes in the abundance of key ruminal microbial taxa.

Background Lambs in intensive farming face susceptibility to environmental and physiological stress, which can adversely affect their health and growth. Although selenium and vitamin E are recognized for their strong antioxidant and stress-alleviating properties, the combined effects of these substances in ruminants, especially within the regulatory framework of the "rumen-liver" axis, remain inadequately elucidated. This study sought to explore the regulatory pathways within this axis by evaluating lamb growth, immunity, antioxidative capacity, rumen function, liver transcriptome, and their interrelationships. Results The average daily weight gain, growth hormone levels, and immunoglobulin concentrations in the MIX group of lambs were significantly elevated, accompanied by an enhancement in serum antioxidant capacity ( P < 0.05). Concurrently, the rumen environment was notably improved, as evidenced by a reduction in pH and ammonia nitrogen levels, an increase in volatile fatty acid production, and a rise in the abundance of beneficial bacteria, including Firmicutes and Prevotella. Further analysis of the liver transcriptome indicated that differentially expressed genes were enriched in immune and antioxidant-related pathways, such as arachidonic acid metabolism. Correlation analysis suggested that rumen microorganisms may influence liver gene expression via volatile fatty acid metabolism. Conclusion The combined addition of selenium yeast and Vitamin E to the diet enhances antioxidant and immune capabilities through the "microbiotic-VFA-liver" axis, thereby synergistically improving the growth and health of lambs.

This study aimed to explore the generational succession rules of rectal microbes and metabolites in crossbred dairy cows, and clarify the regulatory mechanism underlying generational differentiation of milk quality traits, so as to provide a theoretical basis for improving milk quality and realizing precision breeding of dairy cows based on hindgut microecology. In this experiment, 10 healthy cows in mid-lactation were selected from each of three consecutive generations (F₁, F₂, and F₃) of Holstein–Norwegian Red crossbred cattle, totaling 30 cows. High-throughput 16S rRNA sequencing combined with UPLC-MS/MS-based untargeted metabolomics was applied to systematically analyze the correlations between the rectal microbial community structure and metabolic profiles and milk quality indicators. The results indicated that milk quality traits exhibited obvious generational differentiation: daily milk yield increased gradually with generation and was significantly higher in F₃ cows than in F₁ cows (p < 0.05), while milk fat rate and milk protein rate showed an upward-then-downward trend and reached the highest level in the F₂ generation. The rectal microbial community underwent directional generational succession, with the relative abundance of Firmicutes continuously increased and Actinobacteriota gradually decreased, and the microbial functions were mainly specialized in carbohydrate and amino acid metabolism. Significant generational differences were also observed in rectal metabolic profiles, and the differential metabolites screened between F₁ and F₃ generations were primarily enriched in steroid hormone biosynthesis, nucleotide sugar metabolism, bile acid metabolism, and amino acid metabolism pathways. Multi-omics correlation analysis further verified that the core rectal microbial genera could regulate the lactation performance of the mammary gland by affecting the abundance of nutritional metabolic substrates and signaling molecules, with metabolites acting as critical mediators in the regulatory relationship between rectal microbes and milk quality traits. This study systematically characterizes the generational succession characteristics of rectal microbes and metabolites in crossbred dairy cows and identifies potential correlative mediating links of rectal metabolites between microbes and milk-quality traits, providing a novel theoretical reference for exploring hindgut microecology to regulate dairy cow milk quality and assist elite breeding.