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Below is a method for zebrafish sperm cryopreservation that is an
adaptation of the Harvey method (Harvey et al., 1982). We have introduced two
changes to the original protocol that both streamline the procedure and increase
sample uniformity. First, we normalize all sperm volumes using freezing media
that does not contain the cryoprotectant (i.e. methanol). Second, cryopreserved
sperm are stored in cryovials instead of capillary tubes. The rates of sperm
freezing and thawing (ΔºC/time) are probably the two most critical variable to
control in this procedure. For this reason, do not substitute different tubes for
those specified. Working in teams of 2 it is possible to freeze the sperm of 100
males/team in ~2 hrs.