Vitrification
For vitrification aliquots of 30µl of prepared sperm mixed with a sucrose solution were directly dropped into liquid nitrogen using micropipettes. A stainless strainer was present in the liquid nitrogen to capture the formed spheres. This spheres were then transferred into pre-cooled cryovials and stored in liquid nitrogen for further storage.
The vitrification protocol followed the procedure described in “A simple method of human sperm vitrification” . Swim up sperm (30 microliters) combined with 0.5 M sucrose solution in a 1:1 ratio were dropped into liquid nitrogen using a micropipette, causing them to harden into spheres that sank into a stainless steel tea strainer. These spheres were quickly transferred into cryovials previously cooled in liquid nitrogen for future storage.
After vitrification, prompt transfer into cryovials in liquid nitrogen was crucial to maintain their integrity. Larger volumes necessitated the transfer of more spheres, increasing the risk of spontaneous thawing during the process. This transfer procedure was vital for achieving high post-warming survival and motility.
For devitrification/warming, the spheres were removed from storage after a week and placed in 5 ml of prewarmed gamete handling fluid supplemented with human serum albumin at 42 degrees Celsius for 10 seconds.