Abstract
High-grain diets can reduce rumen pH, which may damage the process of keratinization of the ruminal stratified squamous epithelium, leading to areas of abnormal keratinization (AK). However, the comprehension of molecular biological processes leading to the development of an AK area in the rumen is limited. A total of 48 wethers (2-month-old) were fed a diet containing 48% barley and 24% starch (DM basis) to induce AK development. Rumen fluid samples were collected via an oral stomach tube at 0, 2.5, and 6 h post-morning feeding on three consecutive days (d 58, 59, and 60) to measure pH. Following a 63-day feeding period, all lambs were slaughtered, and based on macroscopic pathological observation of the rumen, they were retrospectively classified into two categories of individuals: individuals with normal rumen (INR; n = 38) and individuals with abnormal keratinization rumen (IAKR; n = 10). Tissue samples from the AK and adjacent morphologically normal (MN) areas of IAKR were analyzed histologically and via RNA sequencing and quantitative real-time PCR (qRT-PCR). Statistical analysis used Shapiro-Wilk tests for histological and qRT-PCR data and linear mixed models for pH comparisons. Key genes and pathways were identified through Differential Expression Analysis (DEA), Weighted Gene Co-expression Network Analysis (WGCNA), and Gene Set Enrichment Analysis (GSEA). Compared to the INR, the IAKR had a lower rumen pH at 2.5 h post-morning feeding (5.352 vs 6.035, P < 0.01). Histological examination showed significant reductions in papillae length, width, and stratum corneum thickness in the AK area compared to the MN area (P < 0.01). Transcriptomic analysis revealed the gene expression downregulation of ACTR2, which play a role in tight junctions; Downregulation of SLC38A2, a gene involved in the protein digestion and absorption pathway, was also observed; The upregulation of MMP9 was observed in the IL-17 signaling pathway, contributing to tissue damage; The expression of ITGB7 was also upregulated, which intensified the local immune response. The expression patterns of these genes were further confirmed by qRT-PCR analysis. In conclusion, the current study revealed molecular processes involved in the disruption of intercellular connections and structural support in tissue cells and impaired ability of cells to absorb nutrients and capture signaling molecules within these AK areas caused by high grain feeding.