Sperm vitrification as an alternative approach to conventional cryopreservation (equilibrium freezing) allows quick and low-cost sample preservation and is suitable for small-bodied aquatic species with miniscule testis, fieldwork at remote locations, and small-scale freezing for research purposes. The goal of this present study was to develop operational prototypes of 3-dimensional (3-D) printed vitrification devices with innovative components that can provide comprehensive functionalities for practical repository development for aquatic species. The design featured an elongated loop to suspend a thin film of sperm sample in cryoprotectant, a retractable sleeve to protect the vitrified samples and allow permanent labeling, a handle to facilitate processing and storage, and a shaft with annular grooves to guide positioning of the protective retractable sleeve. To span a wide range of sample capacities and configurations, a total of 39 different configurations (3 loop lengths ×13 loop heights) were fabricated by 3-D printing with the thermoplastics polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS). A total of 86 devices were fabricated with ABS filament with a print failure rate of 9%, and 97 devices were fabricated with PLA filament with a failure rate of 20%. Major types of printing failures included disconnected loops, insufficient build surface adhesion, stringing, and inconsistent extrusion. The sample volume capacity ranged from 1-47 μL and had linear relationships to the loop lengths and layer numbers. Vitrified samples were observed in 10-mm and 15-mm loops fabricated with PLA and ABS but not in 20-mm loops. This study demonstrated the feasibility of development of standardized low-cost ($0.05 material cost) devices fabricated by 3-D printing with practical functions including vitrification, volume control, labeling, protection, and storage within conventional systems. These prototypes can be further developed, standardized, and used to assist development of germplasm repositories to protect the genetic resources of aquatic species by user groups such as breeders, hatcheries, aquariums, and researchers.

中文翻译：

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用于保存水生物种遗传资源的 3D 打印可定制玻璃化装置

精子玻璃化作为传统冷冻保存（平衡冷冻）的替代方法，可以快速、低成本保存样品，适用于睾丸微小的小型水生物种、偏远地区的野外工作以及用于研究目的的小规模冷冻。本研究的目标是开发具有创新组件的 3 维 (3-D) 打印玻璃化装置的操作原型，这些组件可以为水生物种的实际存储库开发提供全面的功能。该设计具有一个细长的环，用于将精子样本薄膜悬浮在冷冻保护剂中，一个可伸缩的套筒，用于保护玻璃化样本并允许永久标记，一个手柄，以方便处理和储存，以及一个带有环形凹槽的轴，用于引导保护剂的定位可伸缩套筒。为了涵盖广泛的样品容量和配置，使用热塑性塑料聚乳酸 (PLA) 和丙烯腈丁二烯苯乙烯 (ABS) 通过 3D 打印制造了总共 39 种不同的配置（3 个环长度 × 13 个环高度）。共有 86 个设备使用 ABS 线材制造，打印失败率为 9%，97 个设备使用 PLA 线材制造，打印失败率为 20%。打印故障的主要类型包括环路断开、构建表面附着力不足、拉丝和挤出不一致。样品体积容量范围为1-47 μL，与环长度和层数呈线性关系。在用 PLA 和 ABS 制造的 10 毫米和 15 毫米环中观察到玻璃化样品，但在 20 毫米环中未观察到。这项研究证明了开发通过 3D 打印制造的标准化低成本（0.05 美元材料成本）设备的可行性，该设备具有实用功能，包括玻璃化、体积控制、标签、保护和在传统系统中存储。这些原型可以进一步开发、标准化，并用于协助育种者、孵化场、水族馆和研究人员等用户群体开发种质资源库，以保护水生物种的遗传资源。