Effects of developmental stage, seawater concentration and rearing temperature on cryopreservation of Pacific oyster Crassostrea gigas larvae

ABSTRACT: For the development of a stepwise cryopreservation technique for larvae of the Pacific oyster Crassostrea gigas , various conditions were examined. Larvae at 9, 12, 15, 18 and 21 h after insemination were cooled at a rate of −1°C/min (seeding at −8°C for 15 min) and then plunged into liquid nitrogen at −35 or −40°C using 1.5 M dimethyl sulfoxide (DMSO) and 250 mM trehalose as cryoprotectants. Among these larvae, 15 h after insemination (the trochophore stage before formation of the shell gland) showed the highest motility and the best external appearance after thawing. Trochophore larvae were cryopreserved in preservation media containing different dilutions (1/4, 1/6, 1/8, 1/10 and 1/30) of seawater. Larvae preserved in the 1/4 seawater medium showed the highest appearance of shelled larvae 4 days after thawing. Trochophore larvae reared in seawater at 21, 25 or 29°C were cryopreserved for 8 months and then reared at 26°C after thawing. Larvae reared at 25°C showed the highest survival rate and normal larval ratio at day 6 after thawing, although larvae reared at 21°C showed the highest rates until day 4. One larva developed at 25°C succeeded to settle.