N-carbamylglutamate (NCG) supplementation during gestation improves reproductive performance in sows after conventional artificial insemination. However, whether NCG can improve reproductive performance and change fecal microbiota and serum metabolite levels during pregnancy in sows after fixed-time artificial insemination (FTAI) remains unclear. Two hundred multiparous sows were assigned a diet from mating until farrowing: control (corn–soybean meal) or NCG supplementation (0.05% NCG). At days 30, 70, and 110 of gestation and after farrowing, maternal microbial diversity and serum metabolites were studied. Supplementation of NCG increased the number of piglets born alive and the litter weight (all p < 0.05) and altered the fetal microbial community during gestation. Some genera were particularly abundant at different time points during gestation and after farrowing, but none were commonly abundant across all four time points. Metabolic analysis revealed that NCG supplementation significantly increased the serum concentrations of NCG, ferulic acid, cinnamoylglycine, 3-phenyllactic acid, and gamma-glutamylglutamic acid in the NCG group compared with levels in the control group. Our results reveal that NCG supplementation during gestation improves reproductive performance in sows after FTAI, exerting both direct (increased serum NCG levels) and indirect effects (altered intestinal microbiome and serum metabolites) on sow reproduction and, ultimately, improving placental and fetal development.

Placentation and placental steroidogenesis are important for pregnancy and maternal–fetal health. As pregnancy progresses, the main site of progesterone (P4) synthesis changes from the corpus luteum to the placenta, in which placental trophoblasts are the main cell type for P4 synthesis. Therefore, this study investigated the effects of P4 on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Porcine placental trophoblasts were treated with different concentrations of P4 for 48 h in a serum-free medium in vitro. Cell number, steroidogenesis, and relevant gene and protein expression levels were detected. A high dose of P4 (10.0 μM) significantly increased P4 (p < 0.01), androstenedione (p < 0.05), testosterone (p < 0.05), and estradiol (p < 0.05) production in porcine placental trophoblasts compared with that in control cells, while a low dose of P4 (1 × 10⁻³ μΜ) had no marked impact on steroid production. The mRNA expression of apoptosis-related genes (CASP3, CASP8, and Bax) (p < 0.05) and steroidogenesis-related genes (CYP11A1, CYP19A1, and StAR) (p < 0.01) was upregulated, and the expression of HSD3B and HSD17B4 was inhibited (p < 0.05) in the porcine placental trophoblasts treated with high doses of P4. Low doses of P4 had a lighter effect on gene expression than high doses. The expression of apoptosis-related proteins CASP3 (p < 0.05), and Bax (p < 0.01) and steroidogenesis-related proteins CYP19A1 (p < 0.05) and StAR (p < 0.01) was raised, but the proliferation-related protein CCND2 (p < 0.01) was downregulated in the pTr cells treated with high dose of P4. In comparison, a low dose of P4 inhibited the expression of Bax, CYP11A1 (all p < 0.01), and CCND2 (p < 0.05), but the expression of CASP3 (p < 0.05) and StAR (p < 0.01) was upregulated. In summary, excessive P4 can induce the apoptosis of porcine placental trophoblasts and lead to abnormal steroidogenesis in the placenta and hormone imbalance.