Animals

Nucleated erythroid cells (NECs) have emerged as active participants in immune responses in addition to their canonical oxygen transport function. The subpopulations and immune heterogeneity of chick erythroid cells (ch-ECs) upon infection have not been fully characterized. Single-cell RNA sequencing (scRNA-seq) was used to profile ch-ECs in chicks infected with avian pathogenic Escherichia coli (APEC). Unsupervised clustering uncovered ten distinct ch-EC subpopulations (C1–C10), with significant compositional shifts between infected and control groups. Pseudotime analysis revealed a developmental continuum: C1, C3, C5, and C9 as early progenitors; C2, C4, C6, C7, and C10 as mature erythroid cells; and C8 as a naive population. We revealed 62 immune-related genes, including protein kinases and heat shock proteins, and subpopulation-specific differentially expressed genes (DEGs) linked to immune functions. SCENIC analysis revealed Fos, Srf, and Stat3 as key transcription factors with elevated regulon activity and specificity following infection. Subpopulations C2, C4, C6, and C7, which exhibited marked abundance changes, were scrutinized for immune relevance through integrated multi-omics analysis. Immune-related genes including FOS, AKAP9, HS6ST1, GAB3, TFRC, HSPA8, HSP90AA1, and DNAJB6 were identified. Enrichment analysis indicated activation of the MHC class I antigen presentation pathway, while pathways such as Mitogen-Activated Protein Kinase (MAPK) signaling, NOD-like receptor (NLR) signaling, and the heat shock response were found to be suppressed. In conclusion, this study delineates the immune gene repertoire and signaling networks of ch-ECs during APEC infection, offering new perspectives on NEC immunoregulatory functions.

In intensive farming systems, broilers are exposed to various stressors that trigger immune stress. Reduced glutathione is known to play a crucial role in modulating immune function. This study investigated the effects of different doses of reduced glutathione on the growth performance, intestinal immune function, and gut microbiota of broilers under immune stress. One-day-old broilers were randomly assigned to five groups: group K (control), group L (lipopolysaccharide), and three glutathione supplementation groups, Y1 (50 mg/kg), Y2 (100 mg/kg), and Y3 (200 mg/kg). Each group had four replicates, with 11 broilers per replicate. On days 16, 18, and 20, broilers in groups L and Y1-Y3 received intraperitoneal injections of 0.5 mg/kg of lipopolysaccharide, while group K received an equal volume of saline. On day 16, the average daily weight gain and final body weight of group Y3 were significantly higher than those of groups K and L, whereas on day 21, the average daily weight gain of group Y3 was still significantly higher than that of group L. On day 21 broilers were slaughtered, and samples were collected. In the jejunal mucosa, group Y3 showed significantly reduced levels of IL-2, IL-4, IL-1β, and TNF-α compared to group L. Additionally, group Y3 exhibited reduced relative expression levels of NF-κB, TLR4, IFN-γ, IL-1β, IL-6, IL-4, TNF-α, and IL-2 mRNA; a decreased abundance of Enterococcus, Proteobacteria, and Actinobacteria; and a restored abundance of Ligilactobacillus, Turicibacter, and Firmicutes to normal levels. These findings suggest that 200 mg/kg of reduced glutathione is the optimal dose for improving the gut microbiota composition and mitigating gut damage caused by immune stress by inhibiting the TLR4/NF-κB signaling pathway.

Tibetan sheep, a dominant livestock species on the Qinghai–Tibet Plateau, is characterized by late sexual maturity and low reproductive efficiency. Although long non-coding RNAs (lncRNAs) are known to play critical regulatory roles in mammalian testicular development and spermatogenesis, their expression dynamics and functions in Tibetan sheep remain poorly understood. In this study, we integrated histological and transcriptomic analyses to profile testicular lncRNAs across three developmental stages: pre-pubertal (3 months), sexually mature (1 year), and adult (3 years). Histological examination showed progressive structural maturation of seminiferous tubules, accompanied by significant increases in testicular weight and serum testosterone levels. RNA sequencing identified 10,857 high-confidence lncRNAs and uncovered extensive reprogramming of the lncRNA transcriptome during sexual maturation, with 7784 lncRNAs differentially expressed between pre-pubertal and post-pubertal stages. Functional enrichment analyses of cis- and antisense-target genes indicated that these lncRNAs were involved in key biological processes, including cell cycle regulation, TGF-β and Hippo signaling pathways, extracellular matrix organization, glycolysis, and apoptosis. Co-expression network analysis further linked upregulated lncRNAs to spermatogenesis-related genes involved in processes such as sperm nuclear condensation (e.g., TNP1) and metabolic support (e.g., PFKP). Our findings demonstrated that lncRNAs coordinate testicular development and spermatogenesis in Tibetan sheep by modulating transcriptional networks, remodeling the cellular microenvironment, and reprogramming energy metabolism. This study provides the first comprehensive atlas of testicular lncRNAs in Tibetan sheep and offers novel insights into the epigenetic regulation of male reproduction in high-altitude mammals.