With the aim of establishing cryopreservation protocols for oyster (Crassostrea gigas) embryos, toxicity of single or combined cryoprotectants to oyster embryos was investigated. In experiments on the toxicity tolerance of oyster embryos, four conventional cryoprotectants, namely, acetamide (A), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG), with concentrations from 1 to 5 M were used to test the toxicity tolerance of oyster embryos at eight different developemental stages. On a molar-equivalent basis, DMSO appeared to be less toxic to PG, EG and acetamide in general. Oyster embryos were tolerant to low concentrations of all cryoprotectants tested in the range from 1 M to 2 M for all developmental stages. Early stage embryos were more vulnerable to high concentration (4 M and 5 M) cryoprotectants tested than late stage embryos. Experiments were subsequently performed to study the combined effects of DMSO and acetamide adding trehalose or glucose in reducing toxicity to 4 hour oyster embryos. The use of trehalose or glucose reduced toxicity of high cryoprotectant concentrations. Survival was higher when DMSO was combined with acetamide than when using DMSO or acetamide alone at high concentration (5 M). By adding trehalose to combined 5 M DMSO and acetamide (total concentration) an average survival rate of 67 ± 7% was obtained in contrast to 8 ± 5% or 2 ± 1% survival when testing with 5 M DMSO or 5 M acetamide, respectively.
