Principles of Genetics and Breeding

Cryopreservation of fish sperm

Short-term preservation or storage of gametes ranging from a few hours or a few weeks, usually at low temperatures, can also be used in hatcheries to overcome temporary shortage of gametes, asynchrony in artificial and natural spawning, transportation of gametes and selective breeding. First successful cryopreservation of fish sperm was reported in the 1950s. The success of the cryopreservation of sperm depends on the seasonality, varying sperm quality, collection techniques, diluents used, precise freezing and thawing regimes, storage conditions, and post-thaw fertilization techniques.

The development of successful protocols for long-term freezing will allow the storage of disease-free gametes of desirable strains and species for future use and also facilitate the comparison and evaluation of new strains of fish with original parental stock at minimum cost. In addition, frozen gene banks may be used in conjunction with live gene banks. A major advantage of this approach is that the effective population size can be dramatically increased at relatively low cost by storing spermatozoa from a large number of individual males. Unlike sperm of higher vertebrates fish spermatozoa remain quiescent and become activated the moment they come in contact with water. In most of the fishes spawning in freshwater, spermatozoa remain motile for 2-3 min, and in carps energetic movement of sperm is only for a short duration of 30-60 secs during which they are capable to fertilize the eggs.

Cryopreservation methodology includes proper collection of fish milt; addition of extenders to prevent depletion of sperm energy reserve and to maintain sperm in quiescent condition but alive; using of cryoprotectants reduce thermal shock;, and freezing and thawing techniques to minimize sperm damage.

Materials

Hank's Balanced Salt solution

Cryoprotectants - Methanol

- Glycerol

- DMSO (10%)

Straws, Cryocan and its accessories

Procedure

1. Collect the milt directly by pressing the abdomen of the matured male fish (in carps) or cut the testes and homogenize (in the case of catfish) in 10 ml HBSS (refrigerated at 4°C)

2. Dissolve the cryoprotectants to HBSS or 0.9%NaCl.

3. Dilute the sperm suspension at 1:4 in the various cryoprotectant solution.

4. Draw the suspension into 0.5 ml french straws.

5. Equilibrate for 15 min at 10°C prior to freezing.

6. Place the straw in a strainer tray suspended above liquid nitrogen in a circular insulated tank.

(visits on cryopreservation centers, demonstration of pituitory gland for harmone extraction

Visit on selective breeding centers)