Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing.

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen. To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3°C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3°C with dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), 1-2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30min. A first evaluation of freezing rates was made by testing four freezing curves: -1, -3, -6 and -10°C/min. Then, an optimization was made by testing four freezing curves: -2.5, -3.0, -3.5 and -4°C/min. The selected temperature for short term conservation has been 3°C, because only this temperature has allowed good sperm motility conservation after 3h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me(2)SO. EG and MetOH to all concentrations and Me(2)SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate -3°C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa.

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20+/-1 degrees C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20+/-1, -2, -1, -0.5 and -0.25 degrees C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted. Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.