Diogo Patrícia, Martins Gil, Quinzico Isa, Nogueira Rita, Gavaia Paulo J, Cabrita Elsa
Faculty of Sciences and Technology, University of Algarve, 8005-139, Faro, Portugal.
Centre of Marine Sciences, University of Algarve, 8005-139, Faro, Portugal.
Fish Physiol Biochem. 2018 Dec;44(6):1443-1455. doi: 10.1007/s10695-018-0500-6. Epub 2018 Apr 13.
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.
斑马鱼精子冷冻保存是管理和备份转基因和突变品系等宝贵遗传资源的基本方法。冷冻保存通常需要液氮进行储存，这既昂贵又危险。我们的目标是评估电动超低温冰箱（-150°C）是否是斑马鱼精子储存的可行替代方案。斑马鱼精子在相同条件下（-20°C/分钟）进行冷冻保存，分别储存在液氮或超低温冰箱中，并在1周、1个月和3个月后解冻。评估精子活力、膜完整性和受精能力。在整个储存期间，活力和孵化率没有显著差异。此外，我们旨在了解直接在超低温冰箱中（-66°C/分钟）进行冷冻保存是否可以提高解冻后精子质量。像之前一样以-20°C/分钟的速度进行冷冻，并与通过直接放入超低温冰箱（-66°C/分钟）以快速冷却速率冷冻保存的样品进行比较。根据活力、生存力、DNA片段化和凋亡（膜联蛋白V）评估精子质量。与其他处理相比，-66°C/分钟的冷却速率显示出显著更高的膜和DNA完整性，以及晚期凋亡细胞数量更少。这项研究表明，在超低温冰箱系统中进行斑马鱼精子冷冻保存和储存是可行的，并且直接在超低温冰箱中快速冷却速率可提高解冻后精子质量。
