Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. However, one limitation of this model system is the lack of long-term storage for genetic resources. Our goal in this study was to establish a generalizable approach to sperm cryopreservation that would support future repository development and could be applied to many species according to available resources. Our approach was to: 1) Assess sperm characteristics and standardize collection and processing; 2) Assess acute toxicity to cryoprotectants, and 3) Evaluate and refine freezing conditions to permit post-thaw fertilization and produce viable offspring. By following this approach, we found that Hydractinia sperm incubated in 5% DMSO, equilibrated at 4°C for 20 min, and cooled at a rate of 20°C/min to -80°C at a cell concentration of 10 8 -10 9 /mL in 0.25-mL aliquots were able to fertilize 150-300 eggs which yielded offspring that could metamorphose into juvenile polyps. In addition, improvements were made for processing sperm using a customized 3-D printed collection system. Other opportunities for improvement include optimizing the volumetric sperm-to-egg ratio for fertilization. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, this application-based approach highlights the long-term value of establishing repository-level resources that can be expanded to fit community needs.

中文翻译：

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低温保存Hydractinia symbiolongicarpus精子，以支持基于社区的知识库开发，以保存遗传资源

Hydractinia symbiolongicarpus是一种新兴的模式生物，正在开发尖端的基因组工具和资源以用于越来越多的研究领域。但是，该模型系统的局限性在于缺乏对遗传资源的长期存储。我们在这项研究中的目标是建立精子冷冻保存的通用方法，以支持将来的储存库开发，并根据可用资源将其应用于许多物种。我们的方法是：1）评估精子的特征并标准化收集和加工；2）评估对防冻剂的急性毒性，并且3）评估和改善冷冻条件，以允许解冻后受精并产生可行的后代。通过这种方法，我们发现，Hydractinia将精子在5％DMSO中孵育，在4°C下平衡20分钟，然后以20 mL / min的速度冷却至-80°C，以10 mL -10 9 / mL的细胞浓度以0.25 mL的等分试样进行冷却能够使150-300个卵受精，产生后代，这些后代可能会变成幼虫息肉。此外，使用定制的3D打印采集系统对精子的处理进行了改进。其他改进机会包括优化受精的精子与卵子的体积比。建立Hydractinia的存储库功能研究社区对于遗传资源的未来开发，维护，保护和分配至关重要。更广泛地讲，这种基于应用程序的方法强调了建立存储库级资源的长期价值，该资源可以扩展以满足社区需求。