Finfish Breeding and Hatchery Management (2+1)

Cryopreservation  of  fish spermatozoa:

Handling of spermatozoa prior to freezing
● Collect  spermatozoa from young and mature male, avoiding contamination with urine, mucus, water, faeces, etc.
● One may inject male with a spawning agent to ensure higher milt volume
● Carry out motility test to ensure milt quality
● Select spermatozoa showing 70% or more motility for cryopreservation
                        
Extender    
● Select a suitable extender containing inorganic and organic chemicals, resembling that of blood or seminal plasma.
● Add a small drop of milt to a drop of extender taken on a pre-focused slide and mix and observe
● Ensure that  the diluted spermatozoa  are non-motile

Cryoprotectant

● Add a suitable cryoprotectant (e.g.  DMSO) to extender – milt mixture
● The optimum concentration of cryoprotectant is 5-15% of the total volume of the diluent (extender + milt + cryoprotectant).
● Allowed the cryoprotectant to penetrate into the spermatozoa, which may vary from a few minutes to several minutes.

Storage containers

● Fill diluted spermatozoa in polypropylene vials (1-2 ml) or as pellets (40-200 µl) or  in 0.25 or 0.50 ml plastic straws
● If plastic straws are used, seal it with PVC gel
                   
Dilution ratio

● Maintain  spermatozoa : diluent ratio of 1:1to 1:10, depending upon species.
● The dilution ratio should be such that the spermatozoa need not be diluted further at the time of fertilization.
 
Cooling/freezing rate

● Cool/freeze the straws in an igloo box or a programmable cooling  chamber; the optimum rate is between 10 and 450C per minute.
● Use liquid nitrogen (-196⁰C) as a cryogen for freezing and storing spermatozoa
● Store frozen spermatozoa samples in vapor phase or immerse under liquid nitrogen.

 Warming/thawing rate

● Thaw the preserved spermatozoa samples by agitating  them in a hot-water bath at 37⁰C for 10-15 seconds.
● Maintain rapid thawing rates of 50-70⁰C per minute

Viability of cryopreserved spermatozoa
 
● Thaw the cryopreserved spermatozoa only when required for checking motility.
● Record motility, fertilization and hatching rates, fry survival, etc. for judging the post-thaw viability/fertility and compare these values with those of freshly collected spermatozoa