title: Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment.
abstract: Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo-larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo-larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5M plus trehalose 0.04M. The optimised protocol developed begins with an initial hold at 4°C for 2min, followed by cooling at -1°Cmin(-1) to -12°C. At this point a seeding step was incorporated with a 2min hold, followed by a second cooling at -1°Cmin(-1) to -80°C. After a final hold of 2min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71-98% of controls) was obtained when cryopreserved blastulae were further incubated for 72-96h in artificial seawater. We conclude that embryo-larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.
authors.affiliation: Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Cabo Estai - Canido, 36200 Vigo, Galicia, Spain. juan.bellas@vi.ieo.es
authors.forename: Juan
authors.initials: J
authors.lastname: Bellas
authors.forename: Estefanía
authors.initials: E
authors.lastname: Paredes
mesh_terms: D000818:Animals; D001681:Biological Assay; D036703:Blastula / Q000187:drug effects / Q000254:growth & development*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000196:embryology*; D004784:Environmental Monitoring / Q000379:methods*; D005260:Female; D007814:Larva / Q000187:drug effects / Q000254:growth & development*; D008297:Male; D047328:Paracentrotus; D012617:Sea Urchins / Q000196:embryology*; D012623:Seawater / Q000032:analysis; D013696:Temperature; D013997:Time Factors; D014199:Trehalose / Q000494:pharmacology; D014876:Water Pollution / Q000032:analysis*
chemical_list: D003451:Cryoprotective Agents; D014199:Trehalose; D004121:Dimethyl Sulfoxide