Our experience from other fish species shows that there may be a large variation of sperm concentration between individuals and also between subsequent collections from the same individual. When cryopreserving zebrafish sperm, we therefore recommend pooling sperm from several males for each line. This increases the working volume of each group which in turn allows us to measure sperm concentration and standardize this within each batch prior to freezing.
For the client, this assures that the sample concentration in each straw offers stability and predictability in the fertilization results after thawing. You will know how many sperm cells you have and how many eggs you can expect to fertilize.
We use spectrophotometric measurements to estimate the sperm concentration in each sample with high accuracy. Our calibration curves are based on hundreds of samples tested in-house.
The number of motile sperm in a sperm sample is closely related to the fertilization capacity for eggs that are of good quality. To gain the necessary information as to the quality of the cryopreserved sperm straws, we routinely measure the motility on one sample from each batch that is cryopreserved. Sperm from fish usually have a very short motile period once they are activated by contact with water, as can be seen in the demonstration below.