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.