241. Vaginal Gene Expression Profiles in Pre-pubertal Replacement Gilts Associated with Variation in Age at Puberty.

Abstract Reproduction is one of the most important factors affecting the efficiency and sustainability of livestock production, particularly in the swine industry. Delayed onset of puberty in replacement gilts significantly increases non-productive days and decreases lifetime performance. Early detection of females with high reproductive potential before boar exposure or breeding eligibility is not currently feasible. The goal of this project was to identify differential vaginal transcriptomes that may have relevance for downstream prediction of age at puberty onset in gilts while they remain in the prepubertal stage. 300 prepubertal gilts between 140 to 150 days of age were sampled at each of two commercial swine farms prior to boar exposure. Vaginal swabs were collected using a standardized protocol by inserting the swab approximately 4 cm into the vagina, rotating three times, and placing into 2 mL of Trizol reagent for RNA stabilization. Gilt identification and sampling dates were recorded. Barn staff monitored animals daily for detection of first standing estrus and gilts were weighed just prior to sampling and again at first estrus (i.e. puberty) for calculation of daily gain. Based on observed age at puberty, gilts were classified as early 164 to 191 days of age or late/anestrus 230 to 260 days of age. Representative individuals from each group were selected for gene-expression profiling via RNA sequencing. Differential gene expression was analyzed using linear modeling and empirical Bayes moderation, with false discovery rate (FDR) correction to control multiple comparisons. To ensure robustness across different production systems, analyses were performed separately within each farm, and genes with consistent directionality and meaningful effect sizes (absolute log2 fold change ≥1.0 and FDR ≤0.05) across farms were prioritized as discovery-stage candidates. Differential gene expression in an independent population from South Dakota State University sow farm was used to evaluate cross-population trends and provide support for candidate selection. This integrated statistical approach identified a set of immune and inflammation-related genes (interferon-stimulated gene 15 [ISG15], C–C motif chemokine ligand 5 [CCL5], C–C motif chemokine ligand 4 [CCL4], purinergic receptor P2Y14 [P2RY14], swine leukocyte antigen class II [SLA-2], and extended synaptotagmin 2 [ESYT2] that reliably distinguished early and late puberty phenotypes across farms. These genes showed reproducible regulation patterns between puberty groups and demonstrated consistent expression levels to support the development of downstream quantitative PCR assays. These results establish a robust pipeline for identifying transcriptomic biomarkers associated with age at puberty in gilts. The identified gene panel provides a focused set of biologically plausible candidates for future validation via targeted qPCR in independent populations. Ultimately, developing practical molecular tools based on these markers could help producers make earlier selection decisions, reduce non-productive days, and enhance sow herd efficiency.