Trivalent AMH-INH-RFRP DNA Vaccine Enhances Estrus and Ovulation Rates in Buffaloes

Background: Buffaloes have a significant share in the agricultural economies of many developing countries; however, the industry is not well-established because of the low reproductive efficiency of buffaloes. In this study, we designed a novel strategy to enhance buffalo reproductive efficiency and examined the significance of intramuscular AMH-INH-RFRP DNA vaccine immunization on reproductive performance. Methods: Allocated into four groups (n = 30/group), female buffaloes (aged 5–8 years, n = 120) were randomly enrolled in the study. Treatment groups received 10 mL of AMH-INH-RFRP DNA vaccine intramuscularly, once daily for three consecutive days, at the following concentrations: T1—3 × 108 CFU/mL, T2—3 × 109 CFU/mL, and T3—3 × 1010 CFU/mL. All vaccinated groups received a booster vaccination two weeks later, while the control group received 10 mL of phosphate-buffered saline (PBS) intramuscularly on the same schedule. Serum antibody titers against anti-Müllerian hormone (AMH), inhibin (INH), and RF-amide-related peptide (RFRP) were quantified using indirect ELISA on days 14 (post-primary immunization) and 28 (post-booster immunization). Results: Following primary immunization, antibody titers against AMH, INH, and RFRP were significantly higher in the T3 group than in the T1 group (p < 0.05), and booster immunization further increased antibody positivity rates. Serum concentrations of IL-4, IFN-γ, and E2 were significantly elevated in the T2 and T3 groups compared to the control group (p < 0.05), while P4 levels showed no significant differences among groups. Ultrasonography revealed that the ovulatory follicle diameter and dominant follicle growth rate were significantly increased in the T2 and T3 groups compared to the control and T1 groups (p < 0.05). The estrus rate was significantly higher in the T3 group than in the control group (76.67% vs. 40.00%, p < 0.05), and ovulation rates were significantly higher in the T2 (83.33%) and T3 (86.67%) groups compared to the control group (56.67%) (p < 0.05). However, conception rates did not differ significantly between vaccinated and control groups in the primary dose comparison (p > 0.05). In exploratory post hoc analyses, antibody-positive (Ab+) buffaloes exhibited higher E2 concentrations, larger ovulatory follicle diameters, accelerated dominant follicle growth rates, and higher estrus and ovulation rates compared to antibody-negative (Ab−) buffaloes. Furthermore, gestating buffaloes were monitored until calving, revealing no significant effect of the vaccine on newborn calf weight or body size. Conclusions: In total, intramuscular immunization with the AMH-INH-RFRP DNA vaccine, particularly at the highest dose, enhances reproductive performance in buffaloes by stimulating E2 secretion and follicular development.