Idebenone reduces oxidative stress and differentially improves chromatin integrity in dog and cat epididymal spermatozoa after slow freezing and vitrification

Idebenone (IDB), a synthetic analogue of coenzyme Q10, has been proposed as a promising antioxidant in sperm cryopreservation because of its potential to reduce oxidative damage. This study investigated the effects of IDB on dog and cat epididymal spermatozoa cryopreserved by slow-freezing (SF) or vitrification (VIT) on kinematics parameters, membrane integrity, oxidative stress, and chromatin integrity. Epididymal sperm samples from twenty dogs (n = 40) and eleven cats (n = 22) were recovered by retrograde flushing and slicing-washing, respectively. Four treatments were established according
to cryopreservation method and IDB supplementation (0 [control] or 10 μM): SF-Control, SF+IDB, VIT-Control, and VIT+IDB. Samples subjected to SF were supplemented with 5% glycerol and frozen in static liquid nitrogen (LN₂) vapor, whereas those subjected to VIT were supplemented with 250 mM sucrose and 1% BSA, and directly plunged into LN₂. IDB supplementation did not improve sperm kinematics or membrane integrity in either species (P > 0.05). In dog sperm, both SF+IDB and VIT+IDB showed lower oxidative stress and greater chromatin integrity than SF-Control and VIT-Control, respectively (P < 0.05). In cat sperm, VIT + IDB showed higher post-warming beat-cross frequency than VIT-Control (P < 0.05). In addition, both SF + IDB and VIT + IDB showed lower oxidative stress than their respective controls (P < 0.05). However, improved chromatin integrity in cats was only observed in the SF + IDB treatment compared with SF-Control (P < 0.05). In conclusion, idebenone reduced oxidative stress in dog and cat epididymal spermatozoa, while improved chromatin integrity was observed in dogs after both cryopreservation methods and in cats only after slow freezing.