Standardization of a protocol for purple sea urchin (Paracentrotus lividus, Lamark 1816) sperm cryopreservation

• Main Author: Gonçalves, Carlota Francisca Cerqueira Fernandes Martins
• Publication Date: 2023
• Format: Master thesis
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10400.1/20552
• topic: Paracentrotus lividus; Conservação; Esperma; Criopreservação; Crioprotetores; Testes de qualidade

Aquaculture, as a result of growing seafood consumption and overexploitation of wild populations, has the potential to provide a sustainable alternative to meet market demand. This study developed reproductive strategies for the production and conservation of the purple sea urchin, Paracentrotus lividus. as this species is highly exploited for their rich content gonads, sea urchin “roe”. In that aim, three experiments were conducted to standardize a protocol for sea urchin sperm cryopreservation: the first experiment tested the optimal freezing rate using two different freezing rates, -5°C/min and -10°C/min, and which storage equipment was more adequate to pack sea urchin sperm, 500 μL straws and 1.2 mL cryovials. Our results showed that while analyzing sperm motility parameters and cell survival, treatments with -10°C/min freezing rates and straws as storage equipment produced significantly higher results, thus they were chosen for the following experiments. As cryoprotectants are one of the most important aspects when selecting a cryopreservation protocol, the second experiment tested three permeable cryoprotectants, DMSO, ethylene glycol, and methanol at different concentrations (5%, 10%, and 20%), and used sperm motility parameters, cell viability, and DNA fragmentation as tools to evaluate post-thaw sperm quality. We discovered through these tests that 20% DMSO had greater results than all the other treatments. Finally, the third experiment involved examining quality tests such as sperm motility, cell viability, DNA fragmentation, and fertilization rates after the addition of a non-permeable cryoprotectant (trehalose or sucrose). The results were clear and adding a nonpermeable cryoprotectant to 20% DMSO had no influence. Further research should be conducted to explore higher cooling rates, more sperm quality testing, and alternative combinations of permeable and nonpermeable cryoprotectants.