Finfish Breeding and Hatchery Management (2+1)

Unit 17 - Cryopreservation of fish gametes

17.2. Cryopreservation of fish spermatozoa
The spermatozoa of several species of finfish and shellfish have been cryopreserved and `Sperm banks’ established for some species.
This is because of

• Smaller size (4-6 um)
• Larger number per unit volume (several million spermatozoa / ml milt)
• Repeatability and ease of collection and handling
• Simple membrane (easy to dehydrate or cryoprotect spermatozoa)

Handling of spermatozoa prior to freezing

• Collection of spermatozoa from mature male, avoiding contamination with urine, mucus, water, faeces, etc.
• Males may be injected with spawning agent to ensure higher milt volume
• Motility test to be carried out to ensure milt quality
• Spermatozoa showing 70% or more motility should be selected for cryopreservation

Extender

• Extender is a solution of inorganic and organic chemicals, resembling that of blood or seminal plasma.
• An extender is slightly hypertonic and prevents spermatozoa dehydration/exhaustion.
• Chemical formulations of extenders used for cryopreservation vary widely depending upon the physiological and chemical characteristics of spermatozoa.
• Extenders inhibit motility, but initiate motility when diluted with water or activating solution.

Cryoprotectant

• Cryoprotectant is added to extender – milt mixture to minimize freeze-damage to cells during cooling/ freezing.
• Common cryoprotectnts recommended are – dimethyl sulfoxide (DMSO), methanol, glycerol, DMA, etc.
• The optimum concentration of cryoprotectant is 5-15% of the total volume of the diluent (extender + milt + cryoprotectant).
• Cryoprotectants are of two types- intracellular (penetrating) and extracellular (non-penetrating)

Equilibration period
It is the time allowed for cryoprotectant to penetrate into the sperm cells. It may vary from a few minutes to several minutes.
Storage containers
Diluted spermatozoa are normally stored in polypropylene vials (1-2 ml), as pellets (40-200 µl) and in 0.25 or 0.50 ml plastic and then it forms a seal when comes in contact with a fluid.
Dilution ratio

• The spz : diluent ratio varies between 1:1 and 1:10, depending upon species.
• The dilution ratio should be such that the spz need not be diluted further at the time of fertilization.

Cooling/freezing rate

• Cooling/freezing is considered the most critical variable of cryopreservation.
• The optimum rate is between 10 and 450C per minute.
• Liquid nitrogen (-1960C) is the most commonly used cryogen for freezing and storing spz.
• Frozen spermatozoa samples are stored in vapor phase or immersed under liquid nitrogen.

Warming/thawing rate

• Thawing is also considered an important variable in cryopreservation.
• Very rapid thawing rates are used to avoid recrystallization.
• Slow warming rate may result in recrystallization.
• Thawing of preserved spz is accomplished by agitating them in hot-water bath at 370C for 10-15 seconds.
• Thawing rates of 50-700C are recommended, although higher rates of 100-1500C have also been used.

Viability of cryopreserved spermatozoa

• Spermatozoa stored under LN2 remain fertile indefinite.
• They should be thawed only when required for checking motility.
• Motility, fertilization and hatching rates, fry survival, etc. are the common criteria for judging the post-thaw viability/fertility of cryopreserved spermatozoa.

Minute spermatozoa found within seminiferous lobule