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Major steps used for slow cryopreservation
1. Addingcryoprotectiveagents(CPA)beforefreezing
 Non-penetrating cryoprotectants (sucrose, trehalose etc)
• Dehydration and protection of membranes
 Penetrating cryoprotectants (DMSO, PrOH, ethylene glycol, glycerol etc) • Permeation and protection against IIF
2. Relativelyslowcoolingrateinaregionofcriticaltemperatures(-10°Cto-60°C) 3. Thawingofthecells
4. RemovingCPAsafterthawing
Cryodamage in spermatozoa
Spermatozoa less sensitive to cryopreservation damage ~ other cells  high fluidity of the membrane
 Low water content (<50%)
However, specific cryophysical behaviour and cryodamage affects different levels and functions :
1. Decreaseinmotility:post-thawsamplesshowalmost50%reductioninmotility
2. Decreaseinvitality
3. Morphology
 vacuolation in the cytoplasm  amorphous sperm head
 midpiece anomalies
4. Increaseinacrosomaldefects
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