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Authors
Advisor(s)
Abstract(s)
Cryopreservation of fish sperm is a key strategy in both biomedical research and biodiversity conservation, enabling the long-term storage of genetic resources while supporting the ethical principles of Replacement, Reduction and Refinement (3Rs). This work integrates two complementary studies to evaluate the biological consequences and cross-species applicability of sperm cryopreservation using the widely adopted ZIRC protocol. In zebrafish (Danio rerio), we compared offspring derived from fresh versus cryopreserved sperm across wild-type, mutant and transgenic reporter lines. Cryopreservation reduced fertilization success, embryo survival and increased larval malformations, with lineage-specific vulnerabilities in transgene expression. Notably, Tg(gata1:GFP) larvae exhibited impaired erythropoiesis and reduced fluorescence, suggesting inherited DNA damage and redox imbalance, while Tg(atoh7:GAP-RFP) showed milder effects and Tg(aldoca:Venus) remained resilient. These findings suggest that cryopreservation can compromise developmental processes and reporter fidelity, raising concerns for the reliability of archived transgenic resources. In parallel, we tested the protocol in giant danio (Devario aequipinnatus), a related cyprinid, to assess its transferability to other species. Fertilization rate was slightly lower, but not significant, however survival rate was lower than in zebrafish, with higher morbidity, largely influenced by parental age rather than the cryopreservation process itself. However, when compared with in vitro fertilizations performed with fresh sperm, these values did not differ significantly, suggesting that cryopreservation itself did not compromise reproductive quality. These results confirm the robustness of the protocol across cyprinids and highlight the importance of gamete quality in reproductive success. Collectively, our findings underscore the triple relevance of sperm cryopreservation: safeguarding genetically modified lines for biomedical applications and reducing the number of animals used in research while providing scalable tools for germplasm biobanking and conservation.
Description
Dissertação de Mestrado apresentada no ISPA – Instituto Universitário para obtenção de grau de Mestre na especialidade de Biologia Marinha e Conservação
Keywords
Conservation Cryopreservation Cryoinjury Giant danio Zebrafish