Modulation of stress factors for cryopreservation of Paracentrotus lividus (Lamarck 1816) larvae

In the present work we modulated two stress factors salinity and temperature, whose ranges have been previously determined by bioassays using six pre-treatments (18 °C-29.5‰; 18 °C-35‰; 18 °C-39‰ and 20 °C-29.5‰; 20 °C-35‰; 20 °C-39‰), in order to obtain a successful cryopreservation protocol for pluteus larvae of the sea urchin P. lividus (Lamarck 1816). Toxicity tests were performed with different cryoprotectants in a range of 0.5–3 M. Best results pointed out to METH and Me2SO as those more suitable for cryopreservation. First an exploratory cryopreservation experiment with Me2SO supplemented with 0.04 M trehalose (TRE) was tested following the protocol for cryopreservation of embryos (8-h blastula) of Bellas and Paredes, 2011, which did not give satisfactory results. A cryopreservation experiment was performed with both cryoprotectants supplemented with 0.04 M trehalose on 4-arm pluteus larvae (48 h-old) developed in these pre-treatment conditions, followed by a simpler and shorter protocol with a cooling rate of 1 °C/min to −35 °C, achieving for the first time the successful cryopreservation of P. lividus larvae. When larvae were incubated in low salinity or low temperature pre-treatments, they showed delayed larval development and abnormalities. In contrast, pretreatments with high temperature and salinity showed good results. Dimethyl sulfoxide with trehalose proved to be the only effective cryoprotectant for successful cryopreservation of P. lividus larvae. The success of dimethyl sulfoxide is consistent with that described for other cases in previous literature, where dimethyl sulfoxide, although not the least toxic compound, gave the best cryopreservation result.Fundacion BiodiversidadUniversidade de Vigo/CISU

Handling, reproducing and cryopreserving five European sea Urchins (Echinodermata, Klein, 1778) for biodiversity conservation purposes

Financiaciado para publicación en acceso aberto: Universidade de Vigo/CISUGIn this work, five local sea urchin species found in European waters were studied. Four were regular species: Sphaerechinus granularis, Psammechinus miliaris, Echinus esculentus (Linnaeus, 1758) and the edible sea urchin Paracentrotus lividus; and one was an irregular species, Echinocardium cordatum. These five species of sea urchins have been studied regarding their fertility, toxicity of cryoprotecting agents, cryopreservation of different cell types and chilling injury. The baseline fertility is similar in P. lividus, P. miliaris and S. granularis. Nonetheless, the sperm:egg ratio, contact time and development of the fertilization envelope would need to be studied further on a case-by-case basis. Sperm can be maintained inactively in the gonad (4 °C), and oocytes also maintain quality in sea water (4 °C), even after 72 h. Sperm was cryopreserved for four species with some post-thaw intra specific variability, and embryo cryopreservation was only possible for S. granularis. Overall, this study provided a wider vision of the biology and reproduction of these species that will help us develop tools for their biodiversity conservation through cryopreservation.Fundacion Biodiversida