Idebenone enhances cryosurvival of ram sperm after conventional slow freezing but impairs quality following kinetic vitrification

Idebenone (IDB) is a synthetic analog of coenzyme Q10 with potent antioxidant properties and mitochondrial protective activity. In sperm cryopreservation, IDB has been proposed as a cryoadditive to mitigate oxidative stress and preserve functional parameters during and after freezing procedures. In this sense, this study evaluated the effect of IDB supplementation in conventional slow freezing (CS) and kinetic vitrification (VIT) media on the cryosurvival of ram spermatozoa. Eighteen ejaculates were collected from three adult rams (six per male) using an artificial vagina during the mating season (October–December). Each ejaculate was divided into four aliquots to form four treatments, respectively, based on IDB supplementation (10 µM vs 0 µM [control]) in TTG-EY extender (tris, TES, glucose, and 6% egg yolk) and the cryopreservation method (CS vs VIT): CS+IDB, CS-Control, VIT+IDB, and VIT-Control. All samples were adjusted to a concentration of 50 × 106 sperm/mL. Sperm samples from the CS+IDB and CS-Control treatments were added 5% glycerol, loaded into 0.25-mL straws, and then frozen by exposing them to static liquid nitrogen (LN2) vapors. Samples from VIT+IDB and VIT-Control treatments were prepared by adding 100 mM sucrose and directly submerging 30-µL droplets into LN2. Straws (60 per treatment) and cryotubes (36 per treatment) were stored in a cryogenic tank for 3 months. A factorial ANOVA and Fisher’s post hoc multiple comparison tests were used to evaluate the interaction effects between IDB supplementation and cryopreservation method on sperm kinematics, membrane integrity (plasma and acrosome), DNA fragmentation, and oxidative stress. The results showed that CS + IDB significantly improved (P < 0.05) sperm kinematics (i.e. motility, curvilinear and straight-line velocities, straightness, linearity, amplitude of lateral head displacement, and beat-cross frequency), viability, and acrosomal integrity, while significantly reducing DNA fragmentation compared with CS-Control. Likewise, sperm from the VIT + IDB treatment showed significantly lower oxidative stress and DNA fragmentation than the VIT-Control (P < 0.05). However, VIT+IDB samples also exhibited a significant and undesirable reduction (P < 0.05) in several kinematic parameters (e.g. velocities, linearity, and oscillation) after warming compared with VIT-Control. In conclusion, idebenone supplementation in the freezing medium improved post-thaw sperm kinematics, viability, and acrosomal integrity and reduced DNA fragmentation. In vitrification, however, although idebenone reduced oxidative stress and DNA damage, it negatively affected postwarming sperm kinematics.

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