The effect of lactose, sucrose, and trehalose supplementation on dog sperm cryopreservation: cooling times, and adjustment of extender pH

BACKGROUND: Glycerol toxicity in semen extenders necessitates the development of glycerol-free alternatives for canine sperm cryopreservation. OBJECTIVE: We evaluated the effects of disaccharides at different concentrations, cooling time, and extender pH adjustment before freezing and post-thaw incubation on sperm parameters and gene expression. MATERIALS AND METHODS: Spermatozoa were processed using glycerol-free Tris (GFT) supplemented with 50–200 mM disaccharides in Experiment 1 (E1). In Experiments 2 (E2) and 3 (E3), 100 mM lactose (L), trehalose (T), or sucrose (S) was used. Additionally, pH-adjusted forms of lactose (LP), trehalose (TP), and sucrose (SP) were used in E3. Samples were equilibrated at 4°C for varying times, frozen in LN2 vapour, and stored in LN2. Post-thaw progressive motility and viability were assessed. ROS, mitochondrial membrane potential MMP (JC−1/PI), and sperm mitochondrial cysteine-rich protein (SMCP) gene expression were analyzed in E3. RESULTS: In E1, 100 mM enhanced progressive motility across all disaccharides ( P <0.05). In E2, optimal cooling times improved motility (50–70 min for trehalose, 50 min for sucrose) and viability (30–50 min for sucrose). In E3, pH adjustment (6.85) of the extender improved motility in S, LP, and SP after 30 min post-thaw incubation at 24°C ( P <0.05). Additionally, S increased live sperm with high MMP, while LP and SP increased LLMMP, with no effect on SMCP gene expression. CONCLUSION: Cryopreservation using GFT supplemented with 100 mM sucrose, 50 min cooling at 4°C, and 30 min post-thaw incubation at 24°C improved sperm motility, viability, and mitochondrial potential.