Principles of Genetics and Breeding

9.1.4 Preservation

Extenders

To keep the selected sample alive for a certain duration needs preservation. Preservation may be noncryogenic (short term) or cryogenic (long term). For both the preservation milt samples are to be diluted in a slightly hypertonic electrolyte medium termed as extender.

• Extenders have been defined as “a solution of salts, sometimes including organic compounds, which helps maintain viability of cells during refrigeration”.
• Extender keeps the spermatozoa alive but inactive condition, isotonic and also act as buffer. Extenders have been developed for many species. Hanks’ balanced salt solution (HBSS) has been successfully used for catfishes.
• Extenders are formulated at an osmolality that will maintain sperm cells in a non-activated state.
• Use of extenders provides increased storage time and dilutes the sperm to a greater volume, making the sperm easier to work with. Specific dilution ratios should be optimized for each species.
• Extenders should be sterilized or sterilized water should be used for preparation.

Some of the workable extender for carps are as follows .

A. NaCl – 6.5g; CaCl_{2 –} 30g; KCl– 3.0g; NaHCO₃ – 0.2g; distilled water – 1000ml .

B. NaCl -750mg; CaCl₂ -23mg; KCl -38mg, NaHCO₃ – 100mg; NaHPO₄ – 41mg MgSO₄ – 23mg in 100ml distilled water.

C.NaCl – 750mg; CaCl₂ – 20mg; KCl – 20 mg; NaHCO₃ – 20mg in100ml distilled water.

Dissolve NaHCO₃ separately and mix.

Addition of cryoprotectant

• Cryoprotectants are chemicals that allow cells to survive freezing protocols.
• Cryo-injuries, the degeneration of the sperm membrane during cooling and thawing is regarded as the principal cause of reduced post-thaw viability. They are grouped into two broad categories: those that are able to permeate cells and those that are not.
• Examples of permeating cryoprotectants include DMSO (Dimethylsulphoxide), methanol and glycerol.
• Examples of non-permeating cryoprotectants include sugars such as glucose or sucrose, polymers such as dextran, milk and egg proteins and antifreeze proteins such as those found in polar fishes.
• The cryoprotectants most widely used and studied are DMSO and a combination of DMSO and glycerol.
• Cryoprotectants are added to extenders to minimize the stress on cells during cooling and freezing. Methanol is highly toxic for carps but used in tilapia. For carps, DMSO is considered to be the best.
• The cryoprotectant should not be mixed directly in the milt sample. It is mixed first with extender in the required ratio and kept in refrigerator. It is very important to maintain isothermal condition while mixing the milt with diluent.