An integrated in vitro antioxidant/ in vivo metabolomics approach unravels the synergistic effects between oregano essential oil and vitamin C as a nutritional strategy for alleviating heat stress in rabbits

Abstract This study investigated the synergistic antioxidant effects of Oregano essential oil (OEO) and vitamin C (Vit-C) in rabbits exposed to heat stress using both in vitro and in vivo approaches. A preliminary in vitro DPPH assay demonstrated that combining half-concentrations of OEO and Vit-C yielded superior antioxidant activity compared to the full concentration of each individual compound. To test these findings in vivo, 24 adult male rabbits were allocated to four groups for 35 days under naturally occurring heat stress conditions (air temperature = 30.58 ± 1.04, relative humidity = 71.29 ± 3.83). The control group received no treatment, while the second (Vit-C) and third (OEO) groups received full recommended doses of vitamin C (0.025% of drinking water) and oregano essential oil (0.02% of drinking water), respectively. The fourth group (Syn) received a mixture of half doses of each of Vit-C (0.0125%) and OEO (0.01%) offered to the second and the third groups. Results showed that the Syn group exhibited greater feed intake (P = 0.003), superoxide dismutase activity (P = 0.004) and higher total antioxidant capacity and IgM levels (P 0.001 for both) compared to other groups. Moreover, the treated groups demonstrated higher crude fiber (P = 0.021), and neutral detergent fiber (P = 0.034) digestion coefficients than the control group. Untargeted plasma metabolomics identified 459 metabolites, and multivariate analysis confirmed treatment effects on metabolic profiles (Adonis: P = 0.034; ANOSIM: P 0.001). Pairwise Adonis comparison revealed a distinction between the Vit-C and OEO group (P = 0.024), and between the Vit-C and Syn group (P = 0.029). The Syn group showed elevated levels of L-Phenylalanine, Oleoethanolamide glucuronidase, and 4-Methyl-5-thiazoleethanol, and reduced Pyridoxine levels. These findings demonstrate a synergistic interaction between OEO and Vit-C, suggesting that the strategy of combining half of the recommended doses of both substances is more effective than their full individual doses in mitigating heat stress effects. This approach offers a promising nutritional intervention for enhancing resilience and productivity in heat-stressed rabbits, with potential applications in other animal production systems.