Conventional Freezing
The conventional freezing method was performed following the guidlines of SpermFreeze Solution: Quinn’s Advantage Sperm Freezing Medium (Cooper Surgical Trumbull CT). SpermFreeze solution was added to the seminal sample in cryotube vials at a 1:1 ratio. The cryovials were kept at room temperature for10 minutes, after which they were cooled in a vapor state for 20 minutes. They were placed horizontally, approximately 3 cm above the liquid nitrogen surface, reaching a temperature of approximately -70 to -90°C. Finally, the cryovials were submerged in liquid nitrogen and stored at -196 ⁰C.