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Electric ultrafreezer (− 150 °C) as an alternative for zebrafish sperm cryopreservation and storage


Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (− 150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (− 20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (− 66 °C/min) could improve post-thaw sperm quality. Freezing at − 20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (− 66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The − 66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.

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Patricia Diogo acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) through the doctoral grant SFRH/BD/97466/2013. This work was partly founded by the FCT and the European Commission (ERDF-COMPETE) through PEst-C/MAR/LA0015/2011 project and by the FCT through UID/Multi/04326/2013 project. The authors acknowledge the support of Ana Marreiros for the statistical analysis and Marco Tarasco for technical support during the samplings.

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Correspondence to Elsa Cabrita.

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Diogo, P., Martins, G., Quinzico, I. et al. Electric ultrafreezer (− 150 °C) as an alternative for zebrafish sperm cryopreservation and storage. Fish Physiol Biochem 44, 1443–1455 (2018).

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  • Storage
  • Ultrafreezer
  • Zebrafish sperm
  • Cryopreservation
  • Cooling rate