Effects of extender composition, cooling rate, and freezing on the motility of sea bass ( Dicentrarchus labrax, L.) spermatozoa after thawing

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass ( Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me 2SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15 min and 6 h), as well as the effects of adding an energy substrate (1.25 mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent + Na-pyruvate + EG10%) and the adaptation procedure (6 h at 0–2 °C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 °C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6 h at 0–2 °C and then cooled at a rate of 15 °C/min, showed motility on thawing comparable to that of fresh semen ( P=0.045).