Cryopreservation of European flat oyster ( Ostrea edulis ) larvae using a controlled-rate freezer

Larvae of the European flat oyster (Ostrea edulis) were cryopreserved using two protocols originally developed for other bivalvian species with several modifications. Culture of the European flat oyster went through several setbacks due to parasitic diseases, thus, assisted reproductive technologies such as larval cryopreservation can aid in prompt restoration of lost populations and thus facilitate farming of the species. Veliger larvae were diluted in two basic extenders (0.4 M trehalose or 0.4 M sucrose) supplemented with ethylene glycol (EG), dimethyl-sulfoxide (DMSO) or propylene glycol (PG) in concentrations of 10-20% with or without the addition of polyvinylpyrrolidone (average MW-40,000; PVP-40). Larvae were loaded into 0.25-ml or 0.5-ml straws and cryopreserved in a controlled-rate freezer using two slow cooling profiles. Assessment of larval survival 20 min or 24 h post-thaw revealed that the cooling profiles and the type of extender used did not have a significant effect on survival rates. On the other hand, the type of cryoprotectant, straw volume and the time of assessment had a significant main effect on larval survival percentages (p < 0.001 in all three cases). Survival percentages of 70-80% were observed 20 min post-thaw which decreased to 40-60% at 24 h. Generally, EG and DMSO resulted in higher post-thaw larval survival than did PG when larvae were cryopreserved in 0.25-ml straws, whereas, these differences were not detected when 0.5-ml straws were employed. Results of these experiments will allow later studies on long-term survival of cryopreserved European flat oyster larvae.