Abstract
Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 × 105 °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7-6.0 μL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 107 sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.
Highlights
The initial progress in the present study provides a foundation and guidance for further community-driven efforts in the development of vitrification devices with efficient storage systems
Altsmall volumes can limit the usage of vitrification in animals that produce large numbers of hough the small volumes can limit the usage of vitrification in animals that produce large oocytes, it has great potential for small-bodied species with miniscule sperm volumes [24]
Vitrification devices developed in the present ofstudy sample for distributed applied germplasm repositories
Summary
There are two major sperm cryopreservation approaches. Conventional cryopreservation (‘equilibrium freezing’ or ‘slow cooling’) requires identifying and achieving the ideal cooling rates (e.g., 5–40 ◦ C/min from 4 to −80 ◦ C) that can be fast enough to minimize the toxic solution effects and slow enough to minimize intracellular ice formation [1]. This approach is ideal for processing large numbers of samples in batches, but specialized equipment can cost tens of thousands of dollars (e.g., >US$20,000 for a computer-programmed freezer). An alternative and relatively new method for sperm cryopreservation is vitrification, by which the liquid is cooled at
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