The party is over, all the fun has been had, everyone is toweled off and has had a smoke. 

The eggs are in buckets and the milt is in bags; both are over ice. Now what – after all that trouble of raising prime broodfish and all the hassles of spawning, it would be an ultimate waste if the crew did not get the best fertilization rates biologically possible. There are some basics to ensure good frothy fun with sex in a bucket. These deal with milt quality, activation and water chemistry.  Here are the details.

When the mature sperm is in the collecting ducts of the testis, it is immotile. This is achieved by high osmotic pressure, high ion concentrations (mainly potassium – K⁺) and pH of around 7. Upon release of the sperm into fertilization media, it is the dilution of the K⁺ ions and quick change in osmotic pressure that cause the sperm to become motile. A dilution of at least 1:1,000 is necessary for sperm activation; the rates of fertilization for Rainbow Trout are optimal around the 1:1,000 mark with sharp declines after 1:10,000.

At lower dilution rates, not all the sperm are activated, so a sample of the milt may appear to be long-lived as more of the sperm are activated over time. This can give a false reading of motility and fertilization capacity because some of the sperm die while others are waiting to be activated. Dilution rates that are high, such as when more milt is added than necessary can result in low fertilization rates. In this case, a zealous worker will pour all the remaining milt from the baggy onto the eggs thinking more is better. In doing so, the milt is not diluted properly, all the sperm are not activated and fertilization sucks.

A good rule of thumb is about 5 ml per hen (8,000 – 13,000 Atlantic eggs) and activate with a litre of diluent. The books say 1 – 3 ml of milt per litre of eggs.

There are several methods to determining sperm motility. Most involve the use of a binocular or monocular dissecting scope. The most common is to active a small sample on a microscope slide (recall dilution influences). Use ovarian fluid to activate the sample. It contains Na+ ions, dilutes the ample and has chemical attractants that really get sperm going. Sort of like the sound of opening a cold beer to a farm worker.

The most common measure of motility is a global movement based on an arbitrary scale of 1-3 or 3-5 for activity – or sometimes hot, warm or cold milt. Another relative measure is the duration of wiggling or survival time for 50% of the population. Sometimes in these assessments the intensity of the movement is included. The results are often characterized as a percentage of activation, with 80% being the putative cut-off for use. Really keen researchers use tracking programmes that are computer driven. Here, a computer tracks the path of individual sperm and measures both duration and distance. It can also measure tail beat frequency and number of sperm per unit area. Don’t try this at home.

Sperm concentration in a sample can be determined by using a haemocytometer that permits counting of blood cells, or in this case sperm by using a microscope. The cells are viewed under high power and are counted against a grid that is etched in a glass chamber of the slide. The volume of the chamber is known, so once the cells are enumerated a volume extrapolation gives the sperm per ml. An AA Spectrophotometer is another way, but that costs time and money. A third way is to use a hematocrit centrifuge. The milt is collected in capillary blood collection tubes and centrifuged at 10K rpm for 3 – 5 min. The proportion of clear liquid to packed cells is the spermatocrit. The spermatocrit will depend on SW or FW spawning and if the males have been induced or not (induced males give much more production). This is the most common method.

Fertilization is the most conclusive measure of gamete quality. Most commonly, it is measured by a 24 hr fertilization check for cell divisions or by eyed egg rate. This measure relates to a series of conditions at time of fertilization such as optimal pH, motility, concentration/dilution of sperm, temperature and inherent egg quality.

The optimal pH for egg fertilization in salmonids is about 8. As well, a little Na⁺ is welcomed in the mix as it changes the ionic composition of the fertilization medium. Some researchers purport that 5% seawater or 30‰ X 5% equals 1.5 grams per litre sodium increases fertilization rate by as much as 20% when combined with 1 mM Ca⁺⁺. This does not take into account the inherent salinity of ovarian fluid and this result is not published in the literature. However, there is a general consensus that a sodium bicarbonate buffer in the activation buffer will aid in the stabilization of a higher pH and donate Na⁺ to the cause.

Temperature at time of fertilization is important. Sperm burn out at rapidly at temperatures of 10°C or higher. The beat frequency of their little tails is inversely proportional to the temperature, which accounts for the crash and burn. It is important that the eggs, milt and activation fluid are all at temperatures between 5° and 10°C to optimize fertilization.

There are two common methods of activation: wet and dry. The wet method has the buffered diluent added to the eggs prior to the addition of milt. The dry method has the milt mixed with the eggs and then add the diluent for activation. The method of choice is personal and should be based on experience and past performance. Water quality (e.g. hardness) of the activation fluid may be the difference.

One last note: sperm eat up oxygen. The little dudes can eat up 20-40m l of O₂ per 10¹⁰ cells per hour. This might not seem like much, but given that there are 6X10¹⁰ sperm per gram of testis produced, a fair bit of O² will be consumed (sperm from a large fish may need 5+ litres of oxygen in 24 hours). Therefore, it’s not only important to oxygenate milt with an overlay and place the baggy on ice, but it’s also important to recheck milt before using.

Optimization of fertilization rates at a given facility is a learned practice. The recipes and information here is not provided as a "be all to end all" solution, rather as an invitation to explore these ideas. The goal is to improve performance and maximize broodstock potentials.