CRYOPRESERVATION OF LATE EMBRYOS AND EARLY LARVAE IN THE OYSTER AND HARD CLAM

Cryopreservation of shellfish embryos and larvae may facilitate aquacu lture management and stock enhancement programs. Late embryos and earl y larvae of oysters (Crassostrea gigas) and hard clams (Meretrix lusor ia) were selected to establish the cryopreservation protocols, Surviva l rates ranging from 62.3 to 75.1% were obtained in oysters using a st epwise freezing protocol. Late embryos or early larvae of oysters (4 h at 28 degrees C after artificial fertilization) were equilibrated in 2 M dimethyl sulfoxide (DMSO) + 0.06 M trehalose plus sea water for 10 min at 27 degrees C and were then cooled at -1 degrees C/min from 0 d egrees C to -12 degrees C. Straws containing more than 1000 embryos we re held at -12 degrees C for 10-15 min allowing equilibration after se eding. Embryos/larvae were then slowly cooled at -2 degrees C/min to - 35 degrees C and allowed 10-20 min for equilibration before quenching in liquid nitrogen. After rapid thawing in a water bath at 28 degrees C, they were placed in sea water to remove DMSO. Besides an increase i n survival rate, embryos/larvae that survived exhibited rotary motion immediately following thawing. For hard clam embryos/larvae with the c ryoprotectants 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 fu rther simplified protocol without seeding, embryos/larvae were brought rapidly from room temperature to 0 degrees C and then to -7 degrees C . After holding at -7 degrees C for 3 minutes, a slow freezing rate of -0.3 degrees C/min was chosen until -35 degrees C was reached. Five m inutes later, they were quenched in liquid nitrogen. Vitrification fre ezing of 8 h oyster larvae along a modified Drosophila protocol result ed in an average survival rate of 14.7%. (C) 1997 Elsevier Science B.V .