Introduction
Portuguese oyster suffered a major decline on natural populations in the past decades in Europe and strategies for its restoration and management need to be developed. Cryopreservation is a helpful tool for the management of endangered genetic resources through the preservation of genetic material in ultralow temperatures (Martínez-Páramo et al. 2017). In invertebrates, such as oysters, it is possible to preserve the genetic resources of both parents, through sperm and oocytes cryopreservation. However, it is also possible to preserve the offspring by freezing embryos and larvae. The availability of cryopreserved sperm is useful when there is no synchrony between spawning of males and females. However, the cryopreservation of larvae presents several advantages, since it allows the availability of diploid animals upon thawing, which simplifies its application for aquaculture purposes (Labbé et al. 2018). Cryopreservation methodologies needs to be adapted according to the species and type of biologic material, being necessary to explore the type and toxicity levels of cryoprotectants and use of single or combined freezing solutions. The main goal is to achieve the conditions that produce the lowest levels of cryodamage, improving post-thaw quality. The objective of this work was to develop conservation strategies of Portuguese oyster through sperm and larvae cryopreservation to support this species management.
Materials and methods
Samples from 10 males of Portuguese oyster were collected by gonadal incisions following the protocol described by Riesco et al. (2017). Each sperm sample was cryopreserved with three cryoprotectant medium in a programed biofreezer at -6ºC/min. Cryoprotectant solutions were prepared in filtered seawater containing 20% dimethyl sulfoxide (DMSO) and 20% DMSO added with 0.9 M trehalose or sucrose. Post-thaw sperm motility was analyzed with CASA software. Flow cytometry was applied to determine the cell viability using PI and to detect levels of reactive oxygen species in live post-thaw sperm by combination of DHE and Sytox dyes. MDA quantification was used to assess lipid peroxidation.
Portuguese oyster larvae were produced by broodstock gonadal incisions to collect the gametes followed artificial fertilization and incubation until D stage. Pools of 60,000 D-larvae/mL (n=7-8) were divided in five aliquots, one used as control and the others exposed and cryopreserved to two cryoprotectant solutions. The solutions were composed by 20 % (v/v) ethylene glycol (EG) or DMSO, 2% (w/v) polyvinylpyrrolidone 40000 MW and 0.4 M sucrose in milli-Q water. Larvae samples were exposed for 3 minutes to cryoprotectant solutions in a 1:1 (v/v) proportion to evaluate the toxicity levels of cryoprotectants. For cryopreservation, samples were loaded into 0.5 mL straws and freeze in a programmable biofreezer. Larvae diluted in filtered seawater (FSW) was used as control. D-larvae quality was evaluated before and after cryoprotectant exposure and post-thawing, according to morphology, motility rate and velocity. Moreover, the whole transcriptome profile of fresh larvae, larvae exposed to DMSO solution, and post-thaw larvae were compared.
Results and discussion
Sperm cryopreserved with DMSO supplemented with sugars significantly improved post-thaw sperm viability and reduced levels of lipid peroxidation and ROS in comparison with DMSO treatment. This can be related with the fact that combination of permeable and non-permeable cryoprotectant interact as membrane fluidity regulator and increase the membrane hydrophobicity (Hassan et al. 2017). The supplementation with sugars was successfully applied in other oyster species such as Pacific oyster (Adams et al. 2004) and Australian flat oyster (Hassan et al. 2017).
The toxicity tests showed that larvae exposed to cryoprotectant solutions containing DMSO had no significant differences in morphology and motility when compared to control. Larval movement (VAP) was significantly higher in DMSO treatment than EG treatment, suggesting that DMSO promoted lower toxicity. Cryopreservation reduced larval quality parameters when compared with control. This effect was also described for Pacific oyster by Labbé et al. (2018). Oyster larvae cryopreserved with DMSO solution showed a significant improvement of larval movement when compared to EG treatment. In this way, DMSO as internal cryoprotectant was beneficial in maintaining post-thaw larvae quality. The transcriptomic analysis of Portuguese oyster larvae did not show many differences in gene expression between fresh and cryoprotectant exposed larvae. Contrarily, after cryopreservation there was a significant number of genes in larvae up and down regulated compared with the ones in fresh larvae. This supports the results previously described, which suggested that quality reduction of D-larvae was related to cryopreservation or cold damage.
Conclusion
The current work provided methodological tools to set up a Portuguese oyster gene bank through the establishment of cryopreservation protocols for sperm and larvae to support the aquaculture and restoration programs.
Acknowledgements
Supported by 0139_VENUS_5_E (Interreg POCTEP), ASSEMBLE+ JRA2-H2020-INFRAIA-2016-2017 (No 730984), EBB-EAPA_501/2016 (Interreg Atlantic Area) and received national funds through FCT - Foundation for Science and Technology through project CCMAR/Multi/04326/2021. Catarina Anjos was supported by a FCT fellowship (SFRH/BD/130910/2017).
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