Ejaculate fraction-driven changes in porcine uterine gene expression and histological profiles

Seminal plasma contains bioactive components capable of modulating the uterine environment. Although the boar ejaculate is fractionated, with each portion differing in composition, the cumulative effect of these fractions on the female reproductive tract remains poorly understood. This study evaluated how stepwise inclusion of ejaculate fractions affects uterine gene expression, cell proliferation, and immune response after artificial insemination. Semen doses were prepared using three combinations: FR1 (sperm-rich fraction), FR2 (FR1 + intermediate fraction), and FR3 (FR2 + post-sperm fraction). Each dose was used for intrauterine artificial insemination in sows (n = 5 per group), with a non-inseminated control group (n = 5). Uterine tissues (U) were collected 6 days post-estrus onset for transcriptomic and histological analysis. RNA-sequencing analysis identified the highest number of differentially expressed genes (DEGs; false discovery rate < 0.05 and |log2 FC|≥1) in U-FR2 group (n = 602), followed by U-FR3 (n = 465) and U-FR1 (n = 414), relative to the control. Most DEGs were down-regulated in inseminated sows, including immune-related genes such as VNN2. Gene ontology analysis revealed biological processes conserved across groups, including metabolism, immune modulation, and epithelial remodeling. Histological assessment showed reduced Ki-67 positivity and decreased CD3+ T-cell infiltration in all the inseminated groups (p < 0.05) compared to the control. Notably, uteri from U-FR2- and U-FR3-treated sows showed a faster transition toward a secretory phenotype (percentage of glandular tissue; p < 0.05). These findings demonstrate that the inclusion of different ejaculate fractions triggers distinct uterine transcriptomic and histological responses, highlighting the role of seminal plasma (SP) composition on the uterine environment and suggesting that fraction-specific SP components may influence early pregnancy outcomes.