Background: Cold storage of equine semen at 4°C is routinely used in artificial insemination programs. However, oxidative stress induced during storage impairs sperm quality and fertilizing ability. Moringa oleifera leaf extract (MOLE) is a rich natural source of polyphenols, vitamins, and minerals, which have been documented to possess antioxidant properties in semen preservation of several livestock species, but its effects on equine semen are yet to be explored.
Objectives: The present work aimed to evaluate the dose-dependent effects of MOLE supplementation on oxidative stress biomarkers in equine semen stored at 4°C for 48 h and to determine the optimal concentration that provides the maximum antioxidant protection without over-supplementation.
Methods: Six ejaculates collected from three clinically healthy, fertility-proven stallions (two ejaculates per stallion; n = 6 ejaculates) were divided into four equal aliquots and supplemented with MOLE at 0 (Control), 2.6, 5.3 or 10.7 µL/mL of extender. Oxidative stress Ebiomarkers, such as total antioxidant capacity (TAC), catalase activity (CAT), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyls (PC), were measured after 48 h of cold storage at 4°C.
Results: The optimal antioxidant effects were observed at 5.3 µL/mL MOLE supplementation, which significantly increased TAC, CAT activity, and GSH concentrations, and simultaneously reduced MDA and protein carbonyls content compared to the control (P < 0.01). The 2.6 µL/mL dose had intermediate improvements. At 10.7 µL/mL, antioxidant parameters were decreased, whereas markers of oxidative damage were increased. Values reached the control group indicate the reductive stress in a dose dependent manner. At 5.3 µL/mL, the scene was different. This is the best redox balance for cold-preserved equine semen which was reached by MOLE. Antioxidant defenses, both enzymatic and non-enzymatic, were strengthened. Meanwhile, lipid peroxidation and protein oxidation were reduced. The present study revealed a clear biphasic dose-response pattern. Protective effects were lost when supplementation exceeded 10.7 µL/mL. This pattern fits well with the hormetic and reductive stress mechanisms.
Conclusions: These results support MOLE as a natural antioxidant for equine semen extenders and provide evidence-based recommendations for best practices in semen preservation.