Principles of Genetics and Breeding

9.1.7 Protocol for cryopreservation

• The efficiency of cryopreservation is greatly enhanced if the prefrozen milt is diluted with a suitable extender.
• The chemical constituents are weighed accurately and dissolved in double distilled water.
• The desirable pH of the solution is 7.2 to 7.3. The solution kept under refrigeration can be used safely for a month.

Preparation of diluent

The ratio of extender and cryoprotectant in different diluents are as follows:

A. Glycerol - 10 ml

DMSO - 5 ml

Extender - 90 ml

B. Glycerol -10ml

DMSO -8 ml

Extender -90ml

C. DMSO -15ml

Extender - 65ml

The working ratio of milt and diluent is 1:4 for carps and 1:5 for tilapia.

Cryoprotectant toxicity

• Care must be taken when freezing live material since the water it contains expands during ice crystal formation, potentially rupturing the cell.
• Cryopreservation requires a careful management of cell water content, through the use of cryoprotectants.
• Essentially gametes or embryos are bathed in an ‘anti-freeze’ solution containing cryoprotectants and are then frozen down to the temperature of liquid nitrogen (-196^oC). When required for use, the material is gently thawed.
• As the cells cool below 0^oC, water begins to freeze out of solution and this increases the concentration of the remaining solution. With further reduction in temperature the solution gets more concentrated as more water freezes out.
• Eventually a point is reached – the eutectic point – at which the whole solution freezes.
• The cryoprotectant mixture used must effectively protect the cells against damage during this cooling process. Gametes and other cells have an internal concentration of solutes (an osmolality) that must be balanced against a similar concentration of solutes outside the cell.
• Initially, when cells are placed in a cryoprotectant, the cells shrink as water leaves the cell to dilute the cryoprotectant on the outside and then the cryoprotectant enters the cell until equilibrium is reached.
• To reduce the mechanical dangers of this, cryoprotectant can be added at slowly increasing concentrations. If cells are cooled too rapidly, lethal ice crystals can form, but cooling too slowly allows extended contact with the cryoprotectant solution that can be toxic.
• Once the cells have reached their eutectic point and no more water comes out of solution (-35^oC to -80^oC), they can be rapidly cooled to -196^oC by plunging them into liquid nitrogen.
• Non-penetrative cryoprotectants are often used to offset problems of cell expansion and the formation of ice crystals during thawing and, as with cooling, the rate of temperature change can be critical.
• Depending upon dose, most cryoprotectants are toxic to cells. To minimize toxic effects by cryoprotectants, they can be added slowly, at cool temperatures and should be diluted with extender solutions (e.g.1:1) prior to addition to sperm.
• Some cryoprotectants can cause exothermic reactions when mixed with extenders. Care should be taken to make sure that mixtures are cooled before addition to sperm.
• Always report cryoprotectant concentration clearly in molarity or percent (or both).