PSV-16 Investigating the long-term effects of neonatal castration on gut development in pigs: Insights from the gut transcriptome.

Abstract Biological sex influences immune function and disease susceptibility across species, with males often exhibiting weaker immune responses and lower survival rates. In the swine industry, male castrates experience nearly twice the mortality rate of females, contributing to substantial financial losses. While this male-biased vulnerability is well-established, the underlying mechanisms remain unclear. A potential factor in male immune development is the perinatal androgen surge which coincides with a critical window for postnatal development of the gut, the body’s largest immune organ. These hormones shape sex-specific immune responses and may influence long-term gut immune programming. Castration, a routine practice in swine production, removes perinatal androgen exposure during this key developmental period, potentially altering long-term gut immune function. Despite extensive research on castration’s effects on growth and behavior, its impact on long-term gut immune programming remains unexplored. The objective of this study was to investigate whether neonatal castration influences gut immune development leading to lasting changes in mucosal gene expression. We hypothesized that castrated males would exhibit differences in baseline intestinal mucosal gene expression and an exaggerated inflammatory response following immune challenge later in life. To test this, we compared intact male pigs (Yorkshire-Duroc cross) to littermates castrated at 9 days. A total of 24 pigs (6 pigs per group) were used. Pigs were weaned at 15 days and housed in an environmentally controlled nursery. At 10-11 weeks, pigs were exposed to either saline or 25 μg/kg lipopolysaccharide (LPS) to induce a systemic inflammatory response. Ileal mucosa samples were collected 4 hours post-injection to assess early changes in gene expression before visible tissue damage. RNA was extracted, mRNA sequencing libraries were prepared, and gene counts were normalized using DESeq2. Data were analyzed using Gene Set Enrichment Analysis (GSEA) to identify pathways and biological functions. Differentially expressed genes were significant at P-value ≤ 0.05, with False Discovery Rate (FDR) ≤ 0.1 for pathway analysis. Under basal saline conditions, castrated males showed enrichment in pathways related to cell-extracellular matrix interactions, cytokine and chemokine signaling, and inflammation, while intact males showed enrichment in pathways related to translation and protein synthesis (FDR ≤ 0.1). Under LPS-challenged conditions, castrated males demonstrated significant enrichment in genes and pathways related to cell cycle regulation, DNA repair, and antibody diversification (FDR ≤ 0.1), compared to intact males. These findings indicate that neonatal castration leads to long-lasting developmental reprogramming in the gut, resulting in heightened inflammatory and reactive immune responses, as well as increased cellular stress when challenged later in life. Collectively, these results point to a critical role for perinatal gonadal androgens in shaping gut immune development, emphasizing the need to reassess castration practices and consider the impact perinatal androgens have on lifelong health and development in swine and other species.