Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity, cooling rate and thawing temperature

Aquaculture of hard clams Mercenaria mercenaria is a $65 million industry along the east coast and Gulf of Mexico coast in the United States. The goal of this study was to develop a preliminary protocol to cryopreserve trochophore larvae of hard clams. The objectives were to evaluate the: 1) toxicity of cryoprotectants, including dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol and glycerol, at 5, 10, 15 and 20% for exposure time of 1, 15, 30, 45, 60 and 75min; 2) effects of cooling rates (5, 10, 20 and 30 degrees C/min for the first trial; and 1, 3 and 5 degrees C/min for second trial from 4 to -80 degrees C), thawing temperature (30, 40 and 50 degrees C) and their interactions on post-thaw viability. A basic protocol was concluded as: 15-hr trochophore larvae mixed with DMSO or propylene glycol (5, 10%), equilibrated for 15min, cooled in a programmable freezer from 4 to -80 degrees C at a cooling rate of 5 degrees C/min and thawed at 50 degrees C for 6s. With this protocol, the immediate post-thaw trochophore survival was 23 +/- 14%, and survival to D-stage was 27 +/- 14%. This is the first report on larval cryopreservation in the hard clam and would have application for genetic breeding and seed production.