Paredes E et al. (2022), Handling, Reproducing and Cryopreserving Five E...

ECB-ART-51149

Animals (Basel) 2022 Nov 16;1222:. doi: 10.3390/ani12223161.

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Handling, Reproducing and Cryopreserving Five European Sea Urchins (Echinodermata, Klein, 1778) for Biodiversity Conservation Purposes.

Paredes E , Campos S , Lago A , Bueno T , Constensoux J , Costas D .

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In 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.

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	Figure 1. Sea urchin species, from left to right: Echinus esculentus, Sphaerechinus granularis, Paracentrotus lividus, Echinocardium cordatum and Psamechinus miliaris adults at scale.
	Figure 2. Percentage of fertilization (A) and development to 4-arm pluteus larvae (B) for each species (n = 100) (mean% ± SD). Asterisks mark significant differences among species (p ≤ 0.05).
	Figure 3. Percentage of fertilization after storage of the gametes at room temperature (18 °C) and fridge temperature (4 °C) for four species of sea urchins. The treatment combinations used to observe fertilization after 1, 2, 3, 4 and 72 h of keeping the gametes at 4 °C and 18 °C were the following: (A) 4 °C sperm in the gonad with 18 °C oocyte; (B) 4 °C sperm in gonad with 4 °C oocyte; (C) 4 °C activated sperm with 18°C oocyte; (D) 4°C activated sperm with 4 °C oocyte; (E) 18 °C sperm in the gonad with 18 °C oocyte; (F) 18 °C sperm in the gonad with 4 °C oocyte; (G) 18 °C activated sperm with 18 °C oocyte; (H) 18 °C activated sperm with 4 °C oocyte.
	Figure 4. Relative percentage of fertilization, mean ± SD, obtained with sperm that had been cryopreserved using 1.5 M Me2SO for four species of sea urchins (n = 3 individuals), using a floating rack at 5 cm above liquid nitrogen. Sperm to egg ratio was constant at 10–20:1, and contact time allowed was 15 min before the fertilization assessment. (Controls showed 60 ± 10%, 35 ± 5.5% (unusually low), 95 ± 3% and 98 ± 2.1% fertilization, respectively). Asterisks show significant differences with their specific controls (p ≤ 0.05).
	Figure 5. (A) Cryopreserved blastula embryos of S. granularis larval length after incubation in seawater after different incubation times (from 96 to 192 h at 18 °C) when using Paredes and Bellas’ 2014 cryopreservation protocol and 1.5 M of different CPAs. (B) Picture shows larvae obtained after 192 h of incubation of blastulas and cryopreserved with 1.5 M Gly. Red arrows show normal skeletogenesis, and black arrows point out frequent abnormalities.

References [+] :

Adams, The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus. 2006, Pubmed, Echinobase