069 Advances in shellfish germplasm cryopreservation: Current status and future directions

• Published in: Cryobiology Vol. 67; no. 3; p. 417
• Main Authors: Adams, Serean L., Gale, Samantha L., Hilton, Zoë, Smith, John F., Robin Tervit, H., Burritt, David, Rusk, Adam, McGowan, Lindsay T., Alfaro, Andrea C.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.12.2013
• Subjects: Crassostrea gigas; Haliotis; Haliotis iris; Marine; Ostrea chilensis; Panopea zelandica; Perna canaliculus; PSE, New Zealand
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2013.09.075

Cryopreservation can be a powerful tool in shellfish aquaculture for selective breeding programmes and hatchery production. Most species that are commercially farmed are broadcast spawners, releasing sperm and eggs into the environment where they fertilize and develop. However, some species are brooders (e.g. flat oysters (Ostrea chilensis)), which retain and rear embryos until they reach later developmental stages. Sperm cryopreservation has been successfully achieved for several farmed species including the Pacific oyster (Crassostrea gigas), greenshell™ mussel (Perna canaliculus) and abalone (Haliotis iris). In general, dimethyl sulphoxide and trehalose are the preferred cryoprotectants (CPAs) and simple methods to control cooling rate (e.g. floating rack on liquid nitrogen; methanol/dry ice bath) have worked well for our main species of interest (Pacific oyster, greenshell™ mussel and abalone). Interestingly, sperm perform better in CPAs that are prepared in Milli-Q water as opposed to seawater, indicating that a lower salt concentration during freezing is beneficial. Recent studies with flat oysters and geoducks (Panopea zelandica) have also resulted in successful sperm cryopreservation. Shellfish oocyte and larvae cryopreservation has proven more challenging, however, successful cryopreservation of Pacific oyster oocytes has been achieved. Pacific oyster oocytes are able to be fertilized and develop to normal spat post-thawing. However, for other species (e.g. greenshell™ mussel) and for larvae, cryo-injuries are not always immediately apparent post-thawing but manifest during development and are often lethal. greenshell™ mussel oocytes are irretrievably damaged when they reach -6 to -8°C and attempts to overcome this sub-zero chilling injury by vitrification have been unsuccessful. Towards understanding these injuries and their effects, we investigated the effect of cryopreservation on the meiotic spindle of greenshell™ mussel oocytes. Results show that the meiotic spindle undergoes major changes during both CPA addition and cooling. However, the spindle is able to re-form normally in most instances given sufficient time post thawing. For a proportion of oocytes, cryopreservation appears to trigger oocyte activation. The effect of cryopreservation on oxidative stress in greenshell™ mussel oocytes has also been investigated. Although, there are changes in oxidative stress parameters as a result of cryopreservation and strong correlations between fertilization, development and oxidative stress parameters (lipid hydroperoxides, superoxide dismutase, protein carbonyls, total glutathione and glutathione state), preliminary attempts to mitigate oxidative stress using antioxidants have so far failed to enable normal development following cooling, suggesting that oxidative stress may be a secondary rather than primary stress event. Research on shellfish larval cryopreservation utilizing SEM and confocal microscopy has also been carried out for greenshell™ mussel. Later larval stages (D-larvae) appear more resilient to cryopreservation than trochophore larvae, but abnormalities are still apparent (e.g.shell abnormalities and delayed organogenesis). Future studies are aimed toward elucidating mechanisms of injury using new tools such as proteomics and then targeting susceptible cellular processes for protection during cryopreservation. Source of funding: This research was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802). Conflict of interest: None declared. serean.adams@cawthron.org.nz