3-Nitrooxypropanol (3-NOP) is a nitrooxy compound that specifically targets methyl-coenzyme M reductase (MCR), ultimately resulting in a reduction in methane production. In this study, we undertook an in vitro investigation of the effects of different dosages of 3-NOP on ruminal fermentation parameters, methane production, and the microbial community. A single-factor completely randomized design was adopted, comprising a control treatment (C), where no 3-NOP was added to the fermentation substrate, and three 3-NOP treatments, where 0.025 mg (low-dose treatment, LD), 0.05 mg (medium-dose treatment, MD), or 0.1 mg (high-dose treatment, HD) was added to 1 g of fermentation substrate (DM basis), followed by incubation for 24 h in vitro. The results showed that, compared with the control treatment, the three dosages of 3-NOP reduced total gas production, methane production, and acetate production (all p < 0.01). In contrast, 3-NOP treatment increased H₂ production and the molar proportions of propionate and butyrate (all p ≤ 0.02), resulting in a decrease in the acetate-propionate ratio (p < 0.01). Meanwhile, the microbial profiles were not altered by the treatments, but the relative abundances of Prevotella, Methanobrevibacter, and Ophryoscolex were increased by the MD and HD treatments (all p < 0.01), whereas those of Methanosarcina, Methanosaeta, Sphaerochaeta, RFN20, Entodinium, and Diplodinium were decreased by the HD treatment (all p ≤ 0.03). Moreover, the results of a correlation analysis showed that there was a certain correlation between these microorganisms and total gas production, methane production, H₂ production, acetate, propionate, and butyrate. In summary, under in vitro conditions, the addition of 3-NOP to the diet affected the microbial community structure, thereby altering the ruminal fermentation pattern and reducing methane production. Our results indicated that 0.05 mg per g of dietary DM is the recommended inclusion ratio for 3-NOP in the diet of lambs.

Alfalfa is conserved in silo-type bunkers in the cold and humid regions of Inner Mongolia, China. Its quality is essential to ensure a healthy and sustainable dairy production. However, the impact of environmental factors on the microbiota and fermentation products of alfalfa silage remains unclear. The present research examined changes in the microbiota and fermentation products and their association with environmental parameters in 72 samples collected from 12 farms located at 4 different latitudes and longitudes across four regions. The samples were labeled with distinct codes, A, B, and C, from the cold–rainy region, D, E, and F, from the warm–rainy region, G, H, and I from the cold–dry region, and J, K, and L from the warm–dry region. The lactic acid levels ranged from 14.25 to 24.27 g/kg of DM across all samples. The pH and concentrations of NH₃-N and butyric acid in samples A, B, and H were higher (p < 0.01) than in the other samples. Samples D and E had higher acetic acid concentrations and 1, 2-propanediol content (p < 0.01). The fresh material was dominated by Pantoea and Pseudomonas, whereas Lactobacillus was the most dominant genus in all silages, except for the B silage. The A, B, and H silages contained more Clostridium but less Lactobacillus than the other silages. The lactic acid levels were strongly associated with Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus acetotolerans, Pedobacter borvungensis, and Lactobacillus brevis (p < 0.01). In contrast, the pH and the NH₃-N and butyric acid concentrations were strongly associated (p < 0.01) with the presence of Clostridium estertheticum. A correlation analysis revealed that precipitation, temperature, longitude, and latitude were the most critical factors influencing epiphytic microbes in the fresh material. After silage fermentation, low-temperature conditions significantly affected the fermentation products and microbial community composition. In conclusion, the microbial community of silages is distinctive in cold and humid regions, and climatic parameters ultimately affect the microbiota and fermentation products. Furthermore, the findings of this study demonstrate that Illumina MiSeq sequencing combined with environmental factor assessment might provide new information about the microbiota composition and fermentation quality of silages, facilitating the achievement of high-quality silage.

Immune stress under intensive high density production conditions impairs broiler health and performance, highlighting the need for effective nutritional strategies. This study established an immune stress model by intraperitoneal lipopolysaccharide (LPS) injection to investigate the mechanism by which Glycyrrhiza polysaccharides (GPs) alleviate LPS-induced immune stress. A total of 240 AA broilers were randomly assigned to four groups: control (CON), GP, LPS, and GP + LPS, with six replicates per group and ten birds per replicate. During the stress phase (28 d), LPS challenge increased serum ACTH and CORT concentrations and altered several immune-related parameters; whereas, GP supplementation improved ileal morphology and reduced serum IL-1β, NF-κB, and TNF-α concentrations. Significant GP × LPS interactions were observed for IL-4, IgA, IgG, and iNOS. During the recovery phase (35 d), GP supplementation increased serum IgA and decreased iNOS, while significant GP × LPS interactions were detected for IL-1β, NF-κB, and sCD4. Notably, GP attenuated the LPS-associated increase in NF-κB. Integrated multi-omics analysis indicated coordinated regulation at transcriptional and proteomic levels. GP upregulated FOS, JUN, and CATH3 and enriched NF-κB and Wnt signaling at the transcript level, while increasing IRF1 and CATH3 and decreasing CTNNBIP1 at the protein level, primarily involving Wnt and MAPK pathways. Joint analysis identified CATH3 as a central regulatory target. Collectively, GP alleviates immune stress by restraining excessive inflammation and modulating Wnt/MAPK signaling, thereby promoting intestinal structural recovery.