Combined hCG and ketoprofen treatment improves dairy cow pregnancy rates through proposed complementary mechanisms: Luteal metabolic reprogramming and endometrial PGF2α suppression.

We investigated whether combining human chorionic gonadotropin (hCG) with ketoprofen after timed artificial insemination (TAI) improves pregnancy rates and explored the underlying mechanisms through serum metabolomics and in vitro endometrial cell analysis. Dairy cows (n = 710) were allocated to three groups: hCG + ketoprofen (1500 IU hCG on day 7 and ketoprofen at 3 mg/kg on days 15-16 of TAI; n = 249), hCG only (1500 IU hCG on day 7 of TAI; n = 246), or control (n = 215). After adjusting for parity, days in milk, body condition score, and milk yield using multivariable logistic regression, the overall treatment effect was significant (χ² = 7.24, df = 2, P = 0.027). The hCG + ketoprofen group had a higher pregnancy rate than the control (adjusted OR = 1.63; 95% CI: 1.12-2.37; P = 0.028, Tukey-adjusted). However, the hCG-only group did not differ significantly from the control group (adjusted OR = 1.15; 95% CI: 0.79-1.68; P = 0.756, Tukey-adjusted). Serum metabolomic profiling by liquid chromatography-mass spectrometry (LC-MS) identified 17 significantly differential metabolites between hCG + ketoprofen and hCG only groups, with KEGG pathway enrichment revealing unsaturated fatty acid biosynthesis as the most prominently altered pathway. In lipopolysaccharide (LPS) challenged bovine endometrial epithelial cells (BEECs), ketoprofen significantly enhanced cell viability (0.5998 ± 0.0043 vs. 0.4498 ± 0.0037; p < 0.0001), reduced reactive oxygen species (ROS) accumulation, and suppressed PGF2α secretion by 36% (29.2 ± 2.77 vs. 45.6 ± 5.36 pg/mL; p < 0.0001). Additionally, ketoprofen downregulated the expression of key prostaglandin synthesis and steroidogenesis genes, including PTGS2, PLA2G4A, PTGFR, VEGFA, STAR, and CYP11A1. These findings suggest that hCG + ketoprofen treatment may enhance pregnancy rates through potentially complementary mechanisms, though these proposed mechanisms remain hypothetical and require in vivo confirmation.