PSI-6 Early weaning stress alters the ileal mucosal transcriptome in a sex-specific manner in pigs.

Abstract In commercial pig production systems, weaning is a stressful event that usually occurs between 15 and 28 days of age and is associated with significant stressors, including maternal separation, dietary changes, transportation, and exposure to unfamiliar piglets. Early weaning (< 21 days) is often implemented for economic reasons and has been linked to long-term negative consequences for intestinal health, including chronic inflammation, intestinal barrier dysfunction, and heightened susceptibility to infectious diseases. However, the underlying molecular mechanisms driving long-term effects remain poorly understood. To address this gap, we aimed to analyze transcriptomic changes in the ileal mucosa following a later-life systemic inflammatory challenge. Moreover, given that biological sex influences immune responses, we examined the transcriptome of females (n=23), intact males (n=24), and castrated males (n=24), weaned at either 15 days (early-wean, EW) or 28 days (later-weaned, LW) of age and then injected intramuscularly with 25 µg/kg of lipopolysaccharide (LPS) or saline at 70 d of age. Samples were collected 4h post-challenge for RNA sequencing. Enrichment analysis was performed using gene set enrichment analysis (GSEA); genes were ranked based on signal-to-noise ratio, and all gene sets with FDR≤0.05 were considered significant. In female pigs, 69 gene sets were enriched in the LW group and 321 in the EW group. Regardless of weaning age, LPS exposure in female pigs was associated with negative enrichment of cell cycle gene sets, including S phase (105 genes) and DNA synthesis (85 genes). In EW females, gene sets involved in the extracellular matrix dynamics were positively enriched, including organization of the extracellular matrix (160 genes) and degradation of the extracellular matrix (62 genes), suggesting altered tissue remodeling responses. Intact males had 54 and 67 enriched gene sets in the LW and EW groups. Positive enrichment of RNA metabolism gene sets was predominant in the LW group. In contrast, cytokine signaling gene sets, including cytokine signaling in the immune system (271 genes) and signaling by interleukins (190 genes) were the most significant gene sets in the EW intact male group. Castrated males had 59 gene sets enriched in the LW group and 84 in the EW group. Gene sets involved in translation processes, such as eukaryotic translation initiation (89 genes) and cap-dependent translation initiation (89 genes), were positively enriched in castrated males independent of weaning age. Notably, gene sets linked to heat stress response, such as attenuation phase (10 genes) and HSF1-dependent transactivation (16 genes), were positively enriched across all wean ages and sexes, suggesting a conserved mucosal response to LPS-induced immune activation. These results suggest that early weaning alters ileal mucosal programming in a sex-dependent manner, offering insights into its long-term impact on gut immunity and informing strategies to reduce immune dysfunction in swine production.