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 degrees C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 degrees C with dimethylsulfoxide (Me2SO), 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 30 min. A first evaluation of freezing rates was made by testing four freezing curves: -1, -3, -6 and -10 degrees C/min. Then, an optimization was made by testing four freezing curves: -2.5, -3.0, -3.5 and -4 degrees C/min. The selected temperature for short term conservation has been 3 degrees C, because only this temperature has allowed good sperm motility conservation after 3 h 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% Me2SO. EG and MetOH to all concentrations and Me2SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate -3 degrees 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. (C) 2011 Elsevier Inc. All rights reserved.