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

A successful cryopreservation procedure must guarantee on thawing the recovery of the morpho-functional characteristics of the cells to allow it to be used comparably with non-preserved semen. The aim of this work was to optimise all the stages in the cryopreservation procedure, so as to identify a species-specific freezing protocol for sea bass D.labrax spermatozoa. In the first stage of the experiment, on the basis of the toxicity tests at room temperature, the cryoprotectants and the relative concentrations that had the least toxic effect on motility were selected. The capacity of the previously selected cryoprotectant substances was then assessed in the freezing tests: Me2SO 5 and 7%, EG 7 and 10%, PG 7 and 10%. The cryoprotectant that had given the best results in the previous series of experiments, namely EG 10%, was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent + Na-pyruvate + EG10%) and the adaptation procedure (6h at 0¸2 ° C) that had given the best results in the previous stages of the experiment, four freezing gradients were tested: -10, -12, -15, -24 °C/min. In conclusion, the semen that was diluted immediately after collection in the extender already containing the cryoprotectant (EG 10%), equilibrated for 6h at 0¸2 °C and then frozen at a gradient of –15 °C/min showed motility on thawing comparable to that of fresh semen (P = 0.045).