Cryopreservation of late embryos and early larvae in the oyster and hard clam

Abstract

Cryopreservation of shellfish embryos and larvae may facilitate aquaculture management and stock enhancement programs. Late embryos and early larvae of oysters (Crassostrea gigas) and hard clams (Meretrix lusoria) were selected to establish the Cryopreservation protocols. Survival rates ranging from 62.3 to 75.1% were obtained in oysters using a stepwise freezing protocol. Late embryos or early larvae of oysters (4 h at 28 °C after artificial fertilization) were equilibrated in 2 M dimethyl sulfoxide (DMSO) + 0.06 M trehalose plus sea water for 10 min at 27 °C and were then cooled at −1 °C/min from 0 °C to −12 °C. Straws containing more than 1000 embryos were held at −12 °C for 10–15 min allowing equilibration after seeding. Embryos/larvae were then slowly cooled at −2 °C/min to −35 °C and allowed 10–20 min for equilibration before quenching in liquid nitrogen. After rapid thawing in a water bath at 28 °C, they were placed in sea water to remove DMSO. Besides an increase in survival rate, embryos/larvae that survived exhibited rotary motion immediately following thawing. For hard clam embryos/larvae with the cryoprotectants 2 M DMSO + 0.06 M glucose, survival rates ranging from 73.3 to 84.2% were achieved using a similar freezing protocol. In a further simplified protocol without seeding, embryos/larvae were brought rapidly from room temperature to 0 °C and then to −7 °C. After holding at −7 °C for 3 minutes, a slow freezing rate of −0.3 °C/min was chosen until −35 °C was reached. Five minutes later, they were quenched in liquid nitrogen. Vitrification freezing of 8 h oyster larvae along a modified Drosophila protocol resulted in an average survival rate of 14.7%.