Effect of Synthetic Cholesterol (Synthechol®) Supplementation in an Egg Yolk-free Extender on Dog Sperm Cryopreservation

BACKGROUND: SyntheChol^® is a new synthetic, non-animal-derived cholesterol that is easily dissolved in ethanol, ready to use, and behaves in a similar way as natural cholesterol. Therefore, it could be used as a substitute of natural cholesterol in dog sperm freezing extender. OBJECTIVE: To evaluate the effect of supplementing an egg yolk-free (EY-free) extender with synthetic cholesterol (SyntheChol^®) on cryopreserved dog sperm. MATERIALS AND METHODS: Spermatozoa (1 × 10⁸ sperm/mL) were suspended in EY-free extender supplemented with 0% (control), 0.25, 0.5, 1, 2, 4, or 6% SyntheChol^® (Extender 1), cooled at 4 °C for 1 h, and diluted (1:1, v/v) with Extender 1 containing 1 M glycerol. The spermatozoa were then cooled to 4 °C for 30 min. Sperm-containing straws were frozen using LN₂ vapor. Sperm motility (computer-assisted sperm analysis, CASA), sperm membrane integrity (SYBR-14 and PI staining), and acrosome integrity (FITC-PSA) were evaluated after thawing. Thereafter, optimal concentrations were determined (0.25, 0.5, 1, or 2%) and used to evaluate reactive oxygen species (ROS) generation, apoptosis, and the gene expression of motility-related sperm mitochondria-associated cysteine-rich protein, apoptosis-related B-cell lymphoma 2 (BCL2), and BCL2-associated X protein ( BAX) in cryopreserved sperm. RESULTS: Sperm progressive motility, membrane integrity, and acrosome integrity were markedly greater in the SyntheChol^®-supplemented groups (0.25, 0.5, 1, or 2%) than in the control group. Only BAX expression was significantly reduced in the SyntheChol^® groups (0.25, 1, or 2%) compared with the control group. However, there were no significant effects on the ROS generation or apoptosis index. CONCLUSION: SyntheChol^® (0.25, 1, or 2%) proved to be effective in reducing the BAX gene expression level and improving sperm progressive motility, and membrane and acrosome integrity.