Long term survival of cryopreserved mussel larvae (Mytilus galloprovinciallis)

Due to the economic importance of the mussel Mytilus galloprovincialis in the Spanish aquaculture, there is a growing interest in developing alternative methods to ensure the supply of good-quality biological material and to minimize the uncertainty and variability that might be a source of economic risk for this sector. The aim of this work was to develop a successful cryopreservation protocol for Mytilus galloprovincialis larvae and study long-term effects by assessing survival and development post cryobanking. We evaluated the effect of previously studied cryoprotecting agent, Ethylene glycol (EG) for cryopreservation of trochophore larvae and D-veliger larvae 48 h and 72 h old, following an existing preliminary protocol for M. galloprovincialis mussel trochophores. The protocol selected for cryopreservation consisted on holding at 4 °C for 2 min, then cooling at 1 °C/min to −12 °C, holding for 5 min, then cooling at −35 °C with a rate of 1 °C/min, then plunging into liquid nitrogen for storage. Thawing took place by immersion in 35 °C water bath. With this protocol the initial percentage of short-term developed a D-larvae (48 h incubation post-thaw) was 48.9 ± 7.6% with 10% EG + 0.2 M Trehalose (TRE). This initial test only studied the trochophore larvae until they reached the D-stage (48 h old); in the present experiment experiments, we replicated this study but also carried out a long-term larval rearing with cryopreserved trochophore larvae in comparison to fresh larvae. This experiment allowed a comparative post-freezing analysis of both larval development and survival. Larval settlement was also characterized after 13 days. The data revealed that until settling, the survival of the cryopreserved larvae was slightly lower than the 2.8% reported in the larval rearing for Greenshell™ mussel. Over time, there was an initial difference in size of cryopreserved larvae when compared to controls, but from day 17 onwards the size between both types of larvae began to stabilize. Attending to settlement, we obtained a 64% of success of cryopreserved larvae respect to the control