**Background:** Eggshell quality deterioration in late-laying hens causes economic losses (12–20% cracked/broken eggs) and increased foodborne disease risk. The uterus (shell gland) is responsible for eggshell biomineralization, and age-related changes in gene expression are suspected to impair eggshell ultrastructure. Long non-coding RNAs (lncRNAs) are key regulators of gene expression but had not been systematically studied in the context of shell gland aging. This study aimed to identify differentially expressed lncRNAs and mRNAs in the shell gland of old vs. young laying hens and to characterize their potential regulatory roles.
**Methods:** Eight Hy-Line Brown hens were randomly assigned to old (60-week-old, n=4) and young (31-week-old, n=4) groups, raised under identical conditions. Shell gland tissues were collected 18 hours after oviposition. Total RNA was extracted using TRIzol, rRNA was removed, and strand-specific cDNA libraries were constructed and sequenced on Illumina HiSeq 2500. Clean reads were mapped to the Gallus gallus reference genome (Gallus_gallus-5.0). Transcripts were assembled with StringTie, and lncRNAs were identified using the intersection of CPC, CNCI, and PFAM databases. Differential expression was analyzed using edgeR (p < 0.05, |log2 fold change| > 1). Cis-target genes were identified within 10 kb and 100 kb of lncRNA loci; trans-target genes were predicted using Lnctar. GO and KEGG enrichment analyses were performed. A lncRNA-mRNA co-expression network was constructed using Cytoscape. Four DE-lncRNAs and their target genes were validated by qPCR using β-actin as an internal control.
**Key Results:** Sequencing yielded 80,510,552–85,469,778 clean reads per sample with Q30 > 90.85%. Over 78.77% of clean reads mapped to the reference genome. A total of 5,334 lncRNA transcripts were identified. Compared to young hens, 176 lncRNAs were differentially expressed in old hens (91 up-regulated, 85 down-regulated) and 383 mRNAs were differentially expressed (204 up-regulated, 179 down-regulated). The lncRNA-mRNA co-expression network comprised 37 nodes and 48 connections among 13 DE-lncRNAs and 24 DE-mRNAs. Notably, TCONS_00181492 and TCONS_03123639 showed significant positive correlation with cis-target genes FGF14 and GRXCR1, respectively, both associated with calcium and sodium ion transport. GO enrichment of cis-target genes (<10 kb) included "protein phosphorylation" (p=0.001639), "ATP binding" (p=0.003026), and "ATP-dependent helicase activity" (p=0.003143). For cis-targets (<100 kb), enriched terms included "phosphate-containing compound metabolic process" (p=5.87×10⁻⁵), "extracellular matrix" (p=0.000228), and "mitochondrial proton-transporting ATP synthase complex" (p=0.011813). KEGG pathways for cis-targets (<10 kb) included "Progesterone-mediated oocyte maturation" (p=0.007389) and "Focal adhesion" (p=0.008565). For trans-targets, enriched GO terms included "translation" (p=1.94×10⁻¹⁰), "extracellular matrix structural constituent" (p=0.000281), and "ribosome" (p=7.62×10⁻¹²). KEGG pathways for trans-targets included "Ribosome" (p=6.22×10⁻³⁶), "Focal adhesion" (p=6.26×10⁻⁶), and "ECM-receptor interaction" (p=1.35×10⁻⁵). DE-mRNA GO enrichment included "inorganic anion transport" (p=0.003472), "calcium ion binding" (p=0.021116), and "inorganic anion transmembrane transporter activity" (p=0.000341). DE-mRNA KEGG pathways included "Glycine, serine, and threonine metabolism" (p=0.00205), "ECM-receptor interaction" (p=0.00778), and "Toll-like receptor signaling pathway" (p=0.01567). qPCR validation confirmed RNA-seq expression patterns for TCONS_00181492/FGF14, TCONS_03234147/COL25A1, TCONS_03123639/GRXCR1 (all up-regulated), and TCONS_01464392/GPX8 (down-regulated).
**Clinical Implications:** This is the first systematic genome-wide analysis of lncRNAs and mRNAs in the chicken shell gland during aging. The identified DE-lncRNAs and their target genes—particularly those involved in ion transport (FGF14, GRXCR1, SLC family members), extracellular matrix (COL25A1), and oxidative stress (GPX8)—provide potential molecular targets for improving eggshell quality in late-laying hens. The findings suggest that age-related deterioration of eggshell quality may result from disrupted inorganic ion and amino acid transport, altered matrix protein secretion, and impaired mitochondrial ATP synthesis in the shell gland. These results lay a foundation for future studies on oviductal senescence and for developing nutritional or genetic interventions (e.g., dietary zinc supplementation) to extend the productive laying cycle of hens.