Scabiosa atropurpurea mitigates oxidative stress and maintains the viability of cryopreserved ram sperm: evidence from oral and extender supplementation

• AES improves biochemical stability of frozen–thawed ram semen • Extender supplementation enhances membrane integrity • Oral AES strengthens systemic antioxidant defenses • Combined strategy reduces cryo-induced sperm damage • Natural antioxidant approach for semen preservation This study evaluated the effects of oral supplementation with Scabiosa atropurpurea aqueous extract (AES) and/or its inclusion in the semen extender on the post-thaw semen quality and oxidative status of ram semen. Ten “ Queue Fine de l’Ouest” rams were assigned to either a control (n = 5) or a treated group (n = 5) receiving AES orally (50 mL/day, 2 mg/kg). Semenwas collected, pooled, and divided into five aliquots, then diluted with extenders containing 0–1 g/L AES, resulting in 10 experimental conditions. After thawing, several parameters were assessed: sperm motility, kinetics, viability, morphology, membrane integrity, oxidative stress markers (TAS, TOS, OSI, MDA), ATP and Ca²⁺ concentrations, DNA fragmentation, and HSP70levels. Oral AES supplementation did not significantly affect total motility (P > 0.05); however, itimproved antioxidant status by increasing TAS (22.0 ± 0.5 vs. 20.3 ± 0.3 mmol/L; P< 0.05) and reducing MDA (5.2 ± 0.3 vs 3.5 ± 0.3 µmol/L; p < 0.05) and OSI (1.1 ± 0.0 vs 0.7 ± 0.0; P < 0.05). The inclusion of AES in the extender reduced the proportion of damaged spermatozoa (24.0 ± 1.1% vs. 37.7 ± 1.2%; P < 0.05) and improved both the ATP ratio (2.1 ± 0.0 vs. 0.9 ± 0.0mg/L per g protein) and Ca²⁺ concentration (32.9 ± 1.0 vs. 22.6 ± 1.1 mg/L per g protein; P< 0.05). The combined supplementation protocol (2 mg/kg oral AES + 1 g/L extender AES) provided the most effective protection against oxidative stress and significantly enhanced membrane integrity, ATP production, and Ca²⁺ levels in post-thaw sperm. In conclusion, AES supplementation improves the biochemical stability and cellular integrity of cryopreserved ram semen.