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Emission from ruminants has become one of the largest sources of anthropogenic methane emission in China. The structure of the rumen flora has a significant effect on methane production. To establish a more accurate prediction model for methane production, the rumen flora should be one of the most important parameters. The objective of the present study was to investigate the relationship among changes in rumen flora, nutrient levels, and methane production in sheep fed with the diets of different forage-to-concentration ratios, as well as to screen for significantly different dominant genera. Nine rumen-cannulated hybrid sheep were separated into three groups and fed three diets with forage-to-concentration ratios of 50:50, 70:30, and 90:10. Three proportions of the diets were fed according to a 3 × 3 incomplete Latin square, design during three periods of 15d each. The ruminal fluid was collected for real-time polymerase chain reaction (real-time PCR), high-throughput sequencing and in vitro rumen fermentation in a new real-time fermentation system wit. Twenty-two genera were screened, the abundance of which varied linearly with forage-to-concentration ratios and methane production. In addition, during the 12-hour in vitro fermentation, the appearance of peak concentration was delayed by 26-27min with the different structure of rumen bacteria. The fiber-degrading bacteria were positively correlated with this phenomenon, but starch-degrading and protein-degrading bacteria were negative correlated. These results would facilitate macro-control of rumen microorganisms and better management of diets for improved nutrition in ruminants. In addition, our findings would help in screening bacterial genera that are highly correlated with methane production.

The purpose of this study was to investigate the effects of oscillating crude protein (CP) concentration diet on the nitrogen utilization efficiency (NUE) of calves and determine its mechanism. Twelve Holstein calves were assigned randomly into static protein diet (SP, 149 g/kg CP) and oscillating protein diet (OP, 125 and 173 g/kg CP diets oscillated at 2-d intervals) groups. After 60 days of feeding, the weights of total stomach, rumen and omasum tended to increase in calves fed OP. The apparent crude fat digestibility, NUE and energy metabolism also increased. In terms of urea-N kinetics evaluated by urea- 15 N 15 N isotope labeling method, the urea-N production and that entry to gastrointestinal tended to increase, and urea-N reused for anabolism increased significantly in calves fed OP during the low protein phase. These data indicate that urea-N recycling contributed to improving NUE when dietary protein concentration was low. In addition, the differentially expressed genes in rumen epithelium and the rumen bacteria involved in protein and energy metabolism promoted the utilization of dietary protein in calves fed OP.

Antimicrobial peptides (AMPs) possess vaccine adjuvant activity; however, their specific targets and molecular mechanisms remain incompletely understood, which hinders their clinical application. This study aimed to elucidate the key targets and pathways through which the antimicrobial peptide BSN-37 modulates immune responses in macrophages, providing evidence for its potential clinical translation. In this investigation, Balb/c mice were administered BSN-37 for 12 h, after which total RNA was extracted from peritoneal macrophages to assess the mRNA expression levels of cytokines and key molecules on the cell surface, followed by transcriptomic sequencing. The results demonstrated that BSN-37 significantly upregulated the mRNA expression of these molecules and cytokines. A total of 228 differentially expressed long non-coding RNAs (lncRNAs) (121 upregulated, 107 downregulated) and 149 differentially expressed mRNAs (104 upregulated, 45 downregulated) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed significant enrichment of differentially expressed mRNAs in immune response pathways, PI3K-Akt signaling, and NOD-like receptor signaling. Differentially expressed lncRNA target genes were associated with T cell receptor signaling, PD-1 checkpoint regulation, and other immune regulatory pathways. Protein-protein interaction network analysis identified core genes such as CCchemokine receptor 1 (CCR1) and Toll Like Receptor 8 (TLR8). Molecular docking studies confirmed that BSN-37 exhibited strong binding affinity to TLR8 and CCR1, with binding energies less than − 5 kcal/mol. RT-qPCR validation confirmed the reliability of the sequencing data. These findings indicate that BSN-37 activates multiple immune response pathways in macrophages by targeting immune-related genes such as TLR8 and CCR1, offering theoretical support for the development of novel immune adjuvants.

Background The infection of bovine mammary glands by pathogenic microorganisms not only causes animal distress but also greatly limits the development of the dairy industry and animal husbandry. A deeper understanding of the host’s initial response to infection may increase the accuracy of selecting drug-resistant animals or facilitate the development of new preventive or therapeutic intervention strategies. In addition to their functions of milk synthesis and secretion, bovine mammary epithelial cells (BMECs) play an irreplaceable role in the innate immune response. To better understand this process, the current study identified differentially expressed long noncoding lncRNAs (DE lncRNAs) and mRNAs (DE mRNAs) in BMECs exposed to Escherichia coli lipopolysaccharide (LPS) and further explored the functions and interactions of these lncRNAs and mRNAs. Results In this study, transcriptome analysis was performed by RNA sequencing (RNA-seq), and the functions of the DE mRNAs and DE lncRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, we constructed a modulation network to gain a deeper understanding of the interactions and roles of these lncRNAs and mRNAs in the context of LPS-induced inflammation. A total of 231 DE lncRNAs and 892 DE mRNAs were identified. Functional enrichment analysis revealed that pathways related to inflammation and the immune response were markedly enriched in the DE genes. In addition, research results have shown that cell death mechanisms, such as necroptosis and pyroptosis, may play key roles in LPS-induced inflammation. Conclusions In summary, the current study identified DE lncRNAs and mRNAs and predicted the signaling pathways and biological processes involved in the inflammatory response of BMECs that might become candidate therapeutic and prognostic targets for mastitis. This study also revealed several possible pathogenic mechanisms of mastitis.

This study systematically investigated the effects of tea polyphenols on methane (CH4) production and the rumen epithelial cell transport capability in cattle using both in vitro and animal experiments, employing multi-omics techniques. The in vitro results demonstrated that, compared to the control group, tea polyphenols significantly reduced CH4 production and the acetate/propionate ratio (p < 0.05). Tea polyphenols reduced CH4 production by inhibiting the relative abundance of unclassified_d_Archaea methanogens and the protozoa Pseudoentodinium and g__Balantioides. The animal experiments showed that tea polyphenols significantly increased the concentrations of T-AOC and GSH-PX in bovine blood (p < 0.05). In addition, microbial groups such as Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Butyrivibrio_2 were significantly enriched in the ruminal fluid of the tea polyphenol group (p < 0.05). The proteomic results indicated significant upregulation of proteins such as COIII, S100A8, FABP1, SLC2A8, and SLC29A1 (p < 0.05) and downregulation of proteins including HBB, RAB4A, RBP4, LOC107131172, HBA, and ZFYVE19 (p < 0.05), with FABP1 showing a positive correlation with propionate concentration, and RAB4A had a negative correlation (p < 0.05). Overall, tea polyphenols modulate the microbial composition within the rumen, inhibiting CH4 production and enhancing the host’s rumen epithelial cell transport capacity for volatile fatty acids.

Defensins, one of the members of the antimicrobial peptide family, play a vital role in resisting microbial invasion and immune regulation. Porcine β-defensin 2 possesses excellent stability, making it an ideal antibiotic alternative for feed additives. In this study, a total of 15 piglets were used to investigate the effects of supplementing diets with 2.5 mg/kg (LP group) and 5 mg/kg (HP group) of pBD2 to weaned piglets. The results revealed that pBD2 significantly increased the total weight gain and average daily weight gain (p < 0.05), the contents of T-AOC, SOD, IgM, and IL-10 in serum (p < 0.05), the villus-to-crypt ratios, and the expression of tight-junction proteins ZO-1 and claudin-1 (p < 0.05) in the small intestine. Furthermore, pBD2 increased the abundance of beneficial bacteria related to nutrient and energy metabolism while decreasing the abundance of harmful bacteria associated with intestinal inflammation and diarrhea. Alterations in the gut microbiota were closely associated with the levels of T-AOC, SOD, IgM, and IL-10 in serum. pBD2 primarily enhanced the health of weaned piglets by influencing antioxidant capacity, intestinal barrier function, and the intestinal microbiota. Our research provides a novel perspective for addressing the issue of antibiotic residues in feed.

Mastitis is a disease with frequent incidence in dairy cows, causing huge financial losses to the dairy industry globally. The identification of certain biomarkers is crucial for the early diagnosis and management of mastitis. Metabolomics technology is a useful tool to accurately and efficiently analyze the changes of metabolites in biofluids in response to internal and external stimulations. Milk is the secreted by udder, and milk metabolites can directly reflect whether the udder are in the healthy or diseased state. The milk metabolomics analysis of mastitis can reveal the physiological and pathological changes of mammary gland and screen the related biomarkers, so as to offer useful reference for the prediction, diagnosis, and management of mastitis. Therefore, the aim of the present study was to comprehensively summarize milk metabolic change caused by naturally occurring or experimentally induced mastitis in dairy cows. In addition, comparative analysis and enrichment analysis were conducted to further discover potential biomarkers of mastitis and to identify the relevant pathways differentiating the healthy and mastitic cows. Multiple milk metabolites were identified to be altered during mastitis based on different metabolomics platforms. It was noteworthy that there were 28 metabolites not only identified by at least two different studies, but also showed consistent change tendency among the different studies. By comparison with literature, we further identified 12 milk metabolites, including acetate, arginine, β-hydroxybutyrate, carnitine, citrate, isoleucine, lactate, leucine, phenylalanine, proline, riboflavin, and valine that were linked with the occurrence of mastitis, which suggested that these 12 milk metabolites could be potential biomarkers of mastitis in dairy cows. Several pathways were revealed to explain the mechanisms of the variation of milk metabolites caused by mastitis, such as phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, riboflavin metabolism, and tricarboxylic acid (TCA) cycle. These results offer a further understanding for the alteration of milk metabolites caused by mastitis, which have a potential significance in the development of more reliable biomarkers for mastitic diagnosis in dairy cows.

The aim of this study was to improve the utilization of peanut vines as forage material for ruminants by investigating the degradation pattern of peanut vines in the dairy cow rumen. Samples of peanut vine incubated in cow rumens were collected at various time points. Bacterial diversity was investigated by scanning electron microscopy (SEM) and 16S rRNA gene sequencing. Carbohydrate-active enzymes (CAZymes) were analyzed by metagenomics. The peanut vines degraded rapidly from 2 to 24 h, before slowing from 24 to 72 h. SEM images confirmed dynamic peanut vine colonization. Firmicutes and Bacteroidetes were the two most dominant bacterial phyla throughout. Principal coordinates analysis indicated significant microbial composition changes at 6 and 24 h. This may be because, in the early stage, soluble carbohydrates that are easily degradable were degraded, while in the later stage, fibrous substances that are difficult to degrade were mainly degraded. Glycoside hydrolases (GHs) were the most abundant CAZymes, with peak relative abundance at 6 h (56.7 trans per million, TPM), and reducing at 24 (55.9 TPM) and 72 h (55.3 TPM). Spearman correlation analysis showed that Alistipes_sp._CAG:435, Alistipes_sp._CAG:514, Bacteroides_sp._CAG:1060, Bacteroides_sp._CAG:545, Bacteroides_sp._CAG:709, Bacteroides_sp._CAG:770, bacterium_F082, bacterium_F083, GH29, GH78, and GH92 were important for plant fiber degradation. These findings provide fundamental knowledge about forage degradation in the cow rumen, and will be important for the targeted improvement of ruminant plant biomass utilization efficiency.

Dietary crude protein concentration oscillation can improve the nitrogen utilization efficiency of ruminants. However, little is known about the dynamic changes in microbiota and fermentation in the rumen of calves during the oscillation period. In this study, six calves were fed an oscillating diet at 2-day intervals, including a high-protein diet (HP) and a low-protein diet (LP). The rumen fermentation parameters, plasma urea-N concentration, and rumen bacterial diversity were characterized throughout the oscillation period. The concentrations of volatile fatty acids, NH3-N, and plasma urea-N in rumen changed significantly with an oscillating diet. The abundance of Prevotella_1, Selenomonadales, Succiniclasticum, Clostridiales, Ruminococcaceae, Lachnospiraceae, and Rikenellaceae_RC9_gut_group showed significant changes with diet. Prevotella_1 was positively correlated, and Lachnospiraceae_AC2044_group and Saccharofermentans were negatively correlated with NH3-N. The abundance of Amino Acid Metabolism, Metabolism of Other Amino Acids, and Glycan Biosynthesis and Metabolism pathways, annotated by bacterial functional genes, decreased when the diet changed from HP to LP. The abundance of the Carbohydrate Metabolism pathway increased after the two dietary changes. In conclusion, the plasma urea-N concentration was not as sensitive and quick to adapt to diet changes as the rumen fermentation parameters. Rumen bacteria were responsible for increasing the nitrogen utilization efficiency of calves fed an oscillating diet.

To develop and validate dual detection platforms integrating recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) for the rapid and specific identification of serotype 2. The conserved gene was selected as the molecular target. Key RPA parameters and PfAgo reaction conditions were systematically optimized, including temperature, reaction time, MnCl concentration, gDNA design and probe concentration. Specificity and sensitivity were evaluated using plasmid dilutions and multiple serotypes together with other common swine pathogens. A total of 41 clinical samples were also tested and compared with the national standard PCR assay (GB/T 19915.3-2005). Two assay formats were established: real-time fluorescence system (RPA-PfAgo-RTF) and lateral flow dipstick system (RPA-PfAgo-LFD). The RPA-PfAgo-RTF assay achieved a detection limit of 10 copies/μL, while the RPA-PfAgo-LFD assay detected 10 copies/μL. Both formats showed high specificity without cross-reactivity. Among 41 field samples, six were SS2-positive, and results showed 100% agreement with the reference PCR method. Total detection time for either assay was < 1 h. Both assay formats provide rapid, sensitive, and accurate tools for SS2 detection suitable for laboratory use and on-farm point-of-care testing.