Semen is routinely frozen in special cryo-vials or cryo-straws.
Freezing of semen is most commonly curried out manually by gradual time exposure of the specimen to nitrogen vapor prior to plunging it into nitrogen liquid at -196 °C for storage. Alternatively, programmable controlled rate freezers can also be used to perform this task. Either of the two approaches necessitates the use of cryoprotectants. The latter serve as means to decrease the freezing point at which the semen solidifies and essentially effect their action by reducing the amount of salts and solutes present in the liquid phase hence avoiding ice formation within the spermatozoa. There are two types of cryoprotectants; penetrating and non-penetrating. Glycerol is the most commonly used penetrating cryoprotectant (intracellular and extracellular action) while egg yolk, sugars (such as sucrose and fructose) and amides supplement the medium as non-penetrating cryoprotectants (extracellular action only). Semen is diluted at equal volumes with cryoprotectant medium, added in a drop wise fashion and thoroughly mixed at room temperature. Sufficient time (10-15min) must be allowed for the cryoprotectant substances to interact with the sperm cells for proper equilibration prior to the freezing procedure taking place.
The technique used to thawing semen specimens is simpler and less time consuming than the freezing procedure, albeit is equally important pertaining to the healthy recovery of spermatozoa and maintenance of their normal biological activities. The commonest method entails exposure of semen cryo-vials to warm running water (~ 36 to 37° C) until it liquefies while if in straws these are thawed in a thermostat set at similar temperatures.
Following thawing the semen specimen must be processed by centrifugation in culture media so the cryoprotectant is removed.
Freezing and subsequent thawing may incur both functional and structural damage to spermatozoa as a consequence of thermal and/or osmotic shock, cellular dehydration and crystallization (intracellular and extracellular). An either too rapid or too slow freezing rate may be detrimental due to deranged cell volume alterations associated with the influx or efflux of water in and out of the cells. Similarly, a higher temperature at thawing accounts for and increased the rate of intracellular and extracellular recrystallization. The latter affirms the importance of rigorous freezing and thawing protocols. Currently, there are no reports in literature documenting evidence of increased miscarriage rates or chromosomal abnormalities in offspring conceived from cryopreserved spermatozoa when compared to natural conceptions.