Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages

Abstract

Global aquaculture production of blue mussel has increased over last years. This work reaffirms the great potential of cryopreservation technique on mussel industry and overcome economic barriers a cause of a traditional and rudimentary management and continue growing. The aim of this work is to set some preliminary basis attending to toxicity of cryoprotecting agents (CPAs) on different development stages of Mytilus galloprovincialis as a start point to develop a stable cryopreservation protocol. Toxicity tests were carried out by using common CPAs (dimethyl-sulfoxide (Me 2 SO), glycerol, (GLY), propylene glycol (PG) and ethylene glycol (EG)) in a range from 0.5 to 3 M on fertilized egg, trochophore larva, and D-larva of Mytilus galloprovincialis. Results evidenced more resistance of older development stages to toxicity. Of all CPAs tested, toxicity testing highlights PG or EG as suitable CPAs for cryopreservation of early development stages; whereas D-larva was unaffected by any of the CPAs tested. Preliminary cryopreservation trials were developed to obtain information into cell cryoprotection. Further research should be focused on membrane permeability and other parameters, such as the balance between toxicity and cryoprotective effect of CPAs.

Development of a method to cryopreserve Greenshell mussel™ (Perna canaliculus) veliger larvae

“…Research on other mollusc species like Pacific oyster (Crassostrea gigas) or blue mussel (Mytilus galloprovinciallis) indicate that there is not strong inter-species variability in the response to cryopreservation. In general, EG is the most used CPA for larvae cryopreservation in combination with other non-permeating CPAs like TRE or polyvinylpyrollidone (PVP) [3,6,8,9,16,19,24,25,26,28]. In this study, it was found that higher concentrations of EG, in combination with TRE at a higher concentration and PVP were generally better for survival than lower EG concentrations.…”

“…The final part of this study investigated whether larval performance post-thawing could be improved by freezing larvae at a later stage. It is well known from many previous studies that D-larvae are more resilient to freezing when compared with earlier developmental stages such as trochophore [6,19,20,21,25]. Although, [8] reported that Pacific oysters cryopreserved from a late D-larvae stage (i.e.…”

“…However, further optimisation of the methodology is required to improve pediveliger yields in order to implement larval cryopreservation in a selective breeding programme. The development of a robust and reproducible freezing procedure requires the study of several factors including selection of a suitable cryoprotecting agent (CPA) according to its balance between toxic effect (yielding cell injuries) and cryoprotection efficiency which is cell-type dependent [4,6,10]. Prior work, focusing on the cryopreservation of P. canaliculus gametes [1,22,23], recommended the use of ethylene glycol (EG) and trehalose (TRE) for oocyte cryopreservation and dimethyl sulfoxide (Me 2 SO) in combination with TRE for sperm cryopreservation.…”

Long term survival of cryopreserved mussel larvae (Mytilus galloprovinciallis)

“…Perna canaliculus D-larvae showed a20% delay in size when compared to controls after 48h incubation post-thaw (Paredes et al 2012, Rusk 2012, Paredes et al 2013, Heres et al 2019.…”

“…Crassostrea gigas, followed by C. virginica are the most popular (Paredes 2015). Regarding mussels, sperm cryopreservation is the most reported followed by larvae due to the difficulty of cryopreserving oocytes successfully, described protocols and cryopreservation information for M. galloprovincialis can be found in few reports like Di Mateo et al 2009, Wang et al 2011, Paredes et al 2013, Wang et al 2014or Heres et al 2019.…”