Going for a cold one will never be the same. There are a number of differences between beer and milt, first among them consumption, but the idea of refrigeration to preserve quality links the two.

Reasons for collecting milt at one time and using it later are manifold. Primary is breeding programmes where a ‘Northern Dancer’ family has superior breeding potential for use in a selective breeding programme. Other needs include: endangered species preservation, increasing genetic diversity, gene banks and construction of hybrids.

Getting there is half the fun

Before thinking about milt preservation, the quality of the milt has to be established. This means the fish has to be ripe and in season. Induction of maturation and spermiation with spawning agents is probably a good idea as it lessens the threat of a no-show male. The choice of good males is further backed up by examining milt quality. While motility is the standard test, fertility is the best measure. In practical methods, a quick motility check done with a good activating fluid such as ovarian fluid, is indicated.

Getting good uncontaminated milt from a prime male is also important. In SW species, salt can spoil the day’s fun. In FW, milt can become diluted or activate the sperm so all the fun is over quickly. One of the big spoilers is urine in the sample.

In the case of obtaining pure milt, catheters are indicated. This means plumbing the depths of the male’s naughty bits with a tube and drawing out milt using a syringe.  his prevents contaminants from getting to the sample and storage in a watertight, sterile container. Dog and cat catheters from a vet are good, or simple tubing can be adapted.

Chilling

Most milt can be kept for weeks at 4^oC under special conditions notably those prevent activation. The use of a diluent or extender (See NA August 2002) will help preserve milt integrity. As well, gas exchange is important. If storage of milt exceeds the biological limits of sperm, then long-term storage is necessary.

Principles of cryopreservation

There are three main components of cryopreserving milt: 1) cryoprotectants, 2) freezing rate and storage and 3) thawing rate.

Cryoprotectants

Brian Harvey and Joachim Carolsfeld of the World Fisheries Trust (www.worldfish.org) have written the book¹ (chapter, really) on cryopreservation of milt. They describe the need for cryoprotectants to preserve cellular integrity by preventing the rapid formation of water crystals (ice) inside and outside the cell. The theory is that if ice forms rapidly in the sperm, it explodes, it if forms rapidly outside the cell, the sperm dehydrates due to increased salt concentrations.

Ideal cryoprotectants enter the cell to ‘regulate’ ice formation and also be outside the cell to control salt concentrations. There are three components to the cryoprotectants: intracellular, extracellular and a stabilizer. The most common intracellular component is DMSO (dimethylsulfoxide). This can be substituted, but the literature suggests that DMSO is pretty much universal². Next, to maintain salt/water balance, sugar is added. This acts as antifreeze, much as in a car radiator. The third component is a protein, usually powdered milk or egg yolk. These preserve membrane integrity and preventing salt damage.

There are a number of recipes for cryoprotectants. If it is used once and works, that usually becomes the Golden Rule for that species. Unfortunately, optimization of formulas is seldom done which leads to dogma concerning species-specific recipes.

Protectants and diluents are usually added to milt at 35-50%. Again, different recipes abound for different fish and optimization is important. Of course, it is noteworthy that recipes should not contain restricted chemicals or those toxic to eggs or sperm.

Freezing

Freezing rate for the milt is critical. The idea here is to move water out of the sperm cells before it forms crystals and not too fast that it forms ice when it gets outside the cell. A suggested rate of cooling is10 - 40^oC with a final temp of -120^oC. The optimal freezing rate will be fast enough to minimize exposure of the sperm to the diluent or cryoprotectant and slow enough to allow water to leave the cell.

It is important to keep the storage temperature low. Deep freezes that hit – 80^oC are not cold enough. It has to be liquid nitrogen (LN2) cold: -196^oC. This ensures that biochemical reactions don’t occur. Even so, degradation is still likely.

The thawing of the sample is also crucial. The thaw should be rapid to 25^oC, but only until the sample thaws. Then add the milt to the eggs in a normal proportion.

Mechanics

Once the milt sample is collected, the extenders and protectants are added and the fluid equilibrated to be homogeneous. The milt is drawn into cryo straws of 0.5 – 5 mls volume. Use a machine, don’t suck it up (Duh!). The small straws are plugged with cotton, the larger horse straws are plugged with a little steel ball. Use something else. On thawing, the pressure mounts and the little ball flies. It’s all fun until someone puts an eye out.

Chill the straw in dry ice noting that the final temp here is –80^oC. Then plunge the straw into a dewar containing LN2. Wait for the females to ripen.

Pros and cons

Cryopreserved milt can be a solution to asynchronous spawning between males and females, preserve genetic material and lend flexibility to breeding programmes. It is not for the uninitiated and requires some level of skill and wit. Fertilization is never that of fresh milt, either. The cost has to be weighed against the benefit, but that is up to the programme directors.

From a sociological perspective, the construction of hybrids can be controversial. Hybrids are made for their perceived or measured performance values as cultured or sport fish. However, they can escape and may be viable in the wild. As with cost, the risk has to be assessed.