Development of cryopreservation procedures for semen of Pacific bluefin tuna Thunnus orientalis

A method for cryopreservation of Pacific bluefin tuna semen, on board ships, was developed in the current study. Semen was collected by squeezing the abdomen of long-line fished tuna. The effect of following variables was tested on post-thaw sperm cell motility: two extender solutions (1% NaCl or 1% glucose), three cryoprotectant compounds (glycerol, DMSO and methanol) at three different concentrations (10, 20 or 30%), the duration of equilibration time (5, 10 or 20 min) in the cryoprotectant solutions and two freezing procedures (immediate immersion in liquid nitrogen or by a two-step freezing procedure). Samples were stored for 7, 30 or 60 days in liquid nitrogen. Post-thaw motility was found only in samples suspended in 1% NaCl (275-290 mOsm/kg) and in those subjected to the two-step freezing procedure. Motility was observed only in 11 of the tested treatment groups and no clear trend was found for the equilibration time in the different cryoprotectant solutions. Sperm cells remained motile for about 480 s after thawing, with those suspended in glycerol showing a distinctive specific pattern. Cells suspended in glycerol showed a gradual increase in motility during the first 20 s after thawing and motility was reduced later after 480 s. The duration of storage in liquid nitrogen did not affect post-thaw motility of sperm cells. The methods suggested for cryopreservation of Pacific bluefin tuna sperm cells based on sperm cell motility, await confirmation by successful fertilization of eggs, whenever they will become available.