64. Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and blue mussel (Mytilus galloprovinciallis)

Abstract

The Pacific oyster Crassostrea gigas is among the most farmed aquaculture species worldwide. The most farmed bivalve species in Spain is the mussel, Mytilus galloprovinciallis (FAO). Due to the variability of spat recruitment from year to year, and in response to the economic importance of these two bivalve species, there is an increasing interest in developing hatchery spat production. Cryopreservation of shellfish embryos and larvae can improve aquaculture management and enhance breeding design flexibility. The aim of this study was to develop a cryopreservation for trochophore larvae of Pacific oysters and blue mussels larvae for future aquaculture purposes. Gametes from mature Pacific oysters were obtained during the natural breeding season by gentle stripping of the gonad (Marlborough Sounds, New Zealand). Blue mussel gametes were obtained by thermal cycling mature broodstock ( Ria de Vigo Spain). Oocytes were fertilized and after 16–20 h of incubation, trochophores were collected by gently siphoning onto a mesh screen held in seawater. For each experiment, at least three pools of trochophores were produced. The ability of trochophores to develop to the D-larval stage following treatment was assessed against a control. Experiment 1: Five ethylene glycol (EG) concentrations in combination with trehalose (TRE) and/or polyvinylpyrrolidone (PVP) were tested for toxicity for both species. Experiment 2: Based on experiment 1, selected cryoprotectant (CPA) concentrations were then used to evaluate two cooling regimes for both species. Trochophores diluted with CPAs were aspirated into 0.25 mL straws and loaded into temperature controlled freezers that were programmed to hold at 0 °C for 5 min, then cool to −10 °C at 1 °C min−1, hold for 10 min, then cool at either 0.5 or 1 °C min−1 to −35 °C, after which, straws were plunged into liquid nitrogen. For thawing, straws were thawed at 35 °C and 0.1% Bovine Serum Albumin (BSA) included in the thawing medium. Experiment 3: A final extra experiment was performed to compare the % of normal D-larvae resulting from cooling mussel trochophores at 0.5 or 1 °C min−1, and also the variation of larvae size after 48, 72 and 96 h incubation. 10% EG + 0.2 M TRE was used. For trochophores of both species, the no observed effect concentration (NOEC) was 15% EG and the lowest observed effect concentration (LOEC) was 20% EG. Development of thawed oyster trochophores through to D-larval stage was highest (60 ± 6.7%) when trochophores were frozen in 10% EG at a rate of 0.5 °C min−1. For mussels, there was large variation between replicate pools with several treatments producing ∼50% normal D-larvae from thawed trochophores and one pool producing 80% normal D-larvae when frozen in 10% EG + 0.2 M TRE at a cooling rate of 0.5 °C min−1. For both species measurements of D-larvae size were performed along the experiments. Extended research must be done to study the long-term viability of resulting D-larvae by examining their ability to develop to the eyed-larval stage and scale-up issues for application in selective breeding and hatcheries.