M Çeşmeci, R Arici, S Yağcioğlu, H Şenlikci, A Eser, A Baran
Cryopreservation of epididymal spermatozoa is an important approach for preserving canine genetic material when ejaculated semen cannot be collected due to sudden death, injury or castration. However, epididymal spermatozoa are particularly susceptible to cryodamage because they lack the protective proteins and antioxidants present in seminal plasma. Trehalose, a non-permeating disaccharide with membrane-stabilising and osmoprotective properties, may mitigate these effects, although its efficacy in canine epididymal sperm cryopreservation remains insufficiently characterised. This study aimed to determine the optimal trehalose concentration for preserving the post-thaw functional and structural quality of canine epididymal spermatozoa cryopreserved in a Tris-egg yolk extender. Epididymal sperm samples were collected from the testes of four dogs per replicate (n = 28) and cryopreserved with 0, 50, 75 or 100 mM trehalose across seven experimental replicates. Post-thaw sperm motility and kinematic parameters were evaluated using computer-assisted sperm analysis. Plasma membrane and acrosome integrity, viability, mitochondrial membrane potential and DNA fragmentation were assessed by flow cytometry, while membrane functionality was evaluated using the hypo-osmotic swelling test. Motility parameters were also assessed following thermal stress incubation. Trehalose supplementation significantly improved plasma membrane integrity at all concentrations and enhanced acrosome integrity at 100 mM (P < 0.05). However, trehalose had no significant effects on sperm motility or kinematic parameters, viability, mitochondrial membrane potential, DNA integrity or hypo-osmotic swelling responses (P > 0.05). Overall, trehalose supplementation, particularly at 100 mM, improved the structural preservation of canine epididymal spermatozoa during cryopreservation, although these benefits did not translate into improvements in post-thaw functional parameters.