Cryopreservation of sperm and larvae of the European flat oyster (Ostrea edulis)

Cryopreservation of sperm and larvae of the European flat oyster (Ostrea edulis) was carried out to assess their performance characteristics using methods commonly employed in fish spermatozoa and other oyster species. Sperm was cryopreserved in Calcium-free Hanks balanced salt solution in the presence of cryoprotectants methanol and dimethyl-sulfoxide (DMSO) at concentrations of 5 or 10%. The highest post-thaw motility (progressive: 3 +/- 6%, static: 8 +/- 10%) was observed with 10% DMSO with a noted decrease in comparison to fresh sperm (progressive: 13 +/- 6%, static: 63 +/- 23%). Membrane integrity of spermatozoa was measured by live-dead fluorescent staining. The highest percentage of membrane-intact cells (60 +/- 6%) was also observed when 10% DMSO was used. A 30-min exposure of trochophorae and veliger larvae to 5, 10, 15 or 20% DMSO was carried out to test the toxicity of this cryoprotectant on early life stages, resulting in the highest survival of veligers (99 +/- 2%) exposed to 10% DMSO. Cryopreservation of veligers was carried out in two steps [(i) at 13 cm above the level of liquid nitrogen for 10 min, (ii) 10 cm for 12 or 23 min] in the presence of 5 or 10% DMSO. The highest post-thaw survival of larvae (59 +/- 31%) was observed at 12 cm of the second step and 10% DMSO. However, none of the larvae survived for 24 h after thawing probably due to the inaccuracies of the freezing protocol, thus, further studies are needed to standardize cooling conditions.