Alterations in microbiota, transcript and metabolites are critical to intestinal homeostasis and host health. This study used a combination of 16s rRNA, transcriptome sequencing and liquid chromatography–mass spectrometry to investigate intestinal microbiota, genes and metabolic profiles in the ileum of Hu sheep fed broccoli extract. Here, we randomly allocated 14 Hu sheep to two diets: a basal diet without any supplementation (NC) and a basal diet supplemented with 200 mg/kg broccoli tail (BT). After 60 days of treatment, blood and jejunal samples were collected for serum biochemical indicators and multi-omics analysis. In this study, the extract of broccoli tails had a significant effect on the serum biochemical indicators, including white blood cells, red blood cells, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean platelet volume, triglycerides and total protein in Hu sheep (p < 0.05). Transcriptomic analysis showed that the 672 differentially expressed genes between the NC and BT groups were primarily enriched in linoleic acid metabolism, steroid hormone biosynthesis, and cholesterol metabolism. Metabolomics analysis using Kyoto Encyclopedia of Genes and Genomes enrichment showed that the 41 differentially abundant metabolites were mainly enriched in bile secretion, vitamin B6 metabolism, and the mTOR signaling pathway. 16S rRNA sequencing results indicated that the extract of broccoli tails increased the relative abundance of Peptostreptococcaceae and decreased the relative abundance of Lachnospiraceae, Lachnospirales, and Bacteroidaceae. Integrated transcriptome, metabolome, and microbiome analysis showed that the gut microbiota and host transcriptomic changes may participate in systemic metabolic regulation by modulating amino acid metabolism, lipid signal transduction, nucleotide metabolism, and vitamin B6-related metabolic pathways. These findings demonstrate that the extract of broccoli tails modulates intestinal gene expression, systemic metabolism, and gut microbial ecology in Hu sheep, providing new insights into the utilization of agricultural byproducts as a functional feed supplement for ruminants.

Porcine circovirus type 2 (PCV2) represents a principal infectious agent causing considerable economic detriment to swine production. N-acetyltransferase 10 (NAT10), which catalyzes N⁴-acetylcytidine (ac⁴C) deposition, has been implicated in regulating immune responses, RNA stability, and viral replication. However, its role in PCV2 infection remains unclear. In this study, we established a PCV2-infected PK15 cell model and observed a marked downregulation of NAT10 expression following infection. Functional assays demonstrated that NAT10 knockdown significantly suppressed PCV2 replication in PK15 cells. Comparative transcriptomic analysis revealed that NAT10 silencing altered the expression of 81 genes, predominantly involved in immune-related signaling pathways. Notably, integrative omics analysis identified NR1H4 as a potential downstream target of NAT10. Collectively, these findings elucidate the regulatory mechanism of NAT10 in PCV2 replication and provide new insights for identifying NAT10 as a potential biomarker and therapeutic target for PCV2 infection in pigs.

Transmissible gastroenteritis virus (TGEV) represents a critical intestinal pathogen responsible for acute enteritis in pigs, posing significant challenges to global swine production biosecurity. N⁶-methyladenosine (m⁶A), the most abundant epitranscriptomic mark in eukaryotic messenger RNA, has emerged as a regulatory factor in host–virus interactions. Despite its recognized importance, the functional significance of m⁶A modifications during TGEV infection of porcine jejunal epithelial (IPEC-J2) cells remains unexplored. Here, we established a TGEV-infected IPEC-J2 cell model and we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) to comprehensively profile the m⁶A epitranscriptomic landscape and identify N⁶-methyladenosine-bearing transcripts in IPEC-J2 cells following TGEV challenge. A total of 14,813 m⁶A peaks were identified in the IPEC-J2, distributed in 7728 genes, mainly enriched in the CDS and 3′-UTRs. After TGEV infection, we identified 832 m⁶A peaks and 1660 genes with significant changes. Integrative analysis revealed a direct positive relationship between N⁶-methyladenosine modification abundance and transcript expression levels. Through integrated examination of MeRIP-Seq and RNA-Seq datasets, we identified 105 transcripts bearing m⁶A modifications, which were mainly enriched in the mTOR signaling pathway. Protein–protein interaction (PPI) network and RT-qPCR analysis demonstrated that SOS2 probably acts an important moderator in TGEV infection. This work contributes to understanding the m⁶A modification landscape in the TGEV-swine model and suggests SOS2 as potential target for future antiviral strategies.

Free-range and confined feeding are typical farming strategies that produce vastly different outcomes in pig immune development. In this study, we extensively evaluated peripheral blood mononuclear cells (PBMCs) obtained from individually raised Meishan pigs under two distinct conditions: normal captivity (NC) and arch soil free-range (ASF), using single-cell sequencing technology. The purpose of this study was to elucidate the influence of various farming approaches on immune cell development. Our analysis identified 11 immune cell subtypes. Using differential gene analysis, we demonstrated that ASF produced the largest impact on the B cells and gamma delta T cell (γδ T cells) transcriptional profile, with subsequent impact on naïve CD4+ T cells and naïve CD8+ T cells. Based on our IL-16 network central analysis, relative to NC cells, signals obtained by B cells, effector and memory CD4+ T cells, and naïve CD4+ T cells were drastically diminished in the ASF cells. Additionally, we conducted an extensive network analysis using CellChat, and demonstrated a marked augmentation of axes related to cellular signal transduction and immunologic activity, including IL-16, RESISTIN, and MIF, among ASF cells. This indicated that various farming approaches differently impacted pig immune activity, and this process was primarily mediated by cell-cell communication. In conclusion, these findings not only enhance the available knowledge of the association between farming approaches and immune modulation, but also provide essential information for the sustainable establishment of animal husbandry and animal health management.