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.