Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration

Liu, Yue; Chesnut, Megan; Guitreau, Amy; Beckham, Jacob; Melvin, Adam T.; Eades, Jason; Tiersch, Terrence R.; Monroe, W. Todd

doi:10.1071/rd19154

Cited by 10 publications

(9 citation statements)

References 8 publications

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“…Before cryopreservation, adjustment of sperm concentration is critical for success for aquatic species [ 14 , 22 ]. Our results showed a positive relationship to sperm concentration, with fertilization increasing from 5% to 98% by increasing the sperm concentration.…”

Section: Discussionmentioning

confidence: 99%

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Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources

Huene

Koch

Arregui

et al. 2022

Animals

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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. One limitation of this model system is the lack of long-term storage for genetic resources. The goal of this study was to establish a generalizable cryopreservation approach for Hydractinia that would support future repository development for other cnidarian species. Specific objectives were to: (1) characterize basic parameters related to sperm quality; (2) develop a generalizable approach for sperm collection; (3) assess the feasibility of in vitro fertilization (IVF) with sperm after refrigerated storage; (4) assess the feasibility of IVF with sperm cryopreserved with various sperm concentrations; (5) evaluate feasibility of cryopreservation with various freezing conditions, and (6) explore the feasibility of cryopreservation by use of a 3-D printed open-hardware (CryoKit) device. Animal husbandry and sperm collection were facilitated by use of 3-D printed open hardware. Hydractinia sperm at a concentration of 2 × 107 cells/mL stored at 4 °C for 6 d were able to achieve 50% fertilization rate. It appeared that relatively higher sperm concentration (>5 × 107 cells/mL) for cryopreservation could promote fertilization. A fertilization rate of 41–69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 108/mL, in 0.25 mL French straws. Samples cryopreserved with the CryoKit produced a fertilization rate of 72–82%. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species.

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Section: Discussionmentioning

confidence: 99%

“…Adjustment of sperm concentration is critical in cryopreservation for aquatic animals [ 14 ]. Sperm were collected into 50-mL conical tubes and centrifuged for 20 min at 1500× g , which resulted in a visible white pellet.…”

Section: Methodsmentioning

confidence: 99%

Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources

Huene

Koch

Arregui

et al. 2022

Animals

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“…However, the lack of reliable hardware for quality management has substantially impeded repository development for aquatic species for decades [6]. For example, standard hemocytometers are commonly used in sperm counting due to their low cost; however, the chamber height of 100 µm was not designed for counting of fish sperm (<10 µm for most aquaculture species) and can lead to erroneous counts [7]. Commercial solutions for sperm counting are costly, uncustomizable, and fail to address the diversity of aquatic species (e.g., sperm size differences between fish and amphibians can be 10-fold).…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, microfabrication ("Lab-on-a-chip") technologies have been applied for the creation of novel devices (microdevices) to enable quality assessment of samples to facilitate routine quality control programs. For example, microdevices have been developed for evaluation of sperm motility [8,9] and concentration [7] of aquatic species. These devices can be standardized with community-level efforts and customized to accommodate certain species.…”

Section: Introductionmentioning

confidence: 99%

Low-Cost Resin 3-D Printing for Rapid Prototyping of Microdevices: Opportunities for Supporting Aquatic Germplasm Repositories

Zuchowicz

Belgodere

Liu

et al. 2022

Fishes

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Germplasm repositories can benefit sustainable aquaculture by supporting genetic improvement, assisted reproduction, and management of valuable genetic resources. Lack of reliable quality management tools has impeded repository development in the past several decades. Microfabricated open-hardware devices have emerged as a new approach to assist repository development by providing standardized quality assessment capabilities to enable routine quality control. However, prototyping of microfabricated devices (microdevices) traditionally relies on photolithography techniques that are costly, time intensive, and accessible only through specialized engineering laboratories. Although resin 3-D printing has been introduced into the microfabrication domain, existing publications focus on customized or high-cost (>thousands of USD) printers. The goal of this report was to identify and call attention to the emerging opportunities to support innovation in microfabrication by use of low-cost (<USD 350) resin 3-D printing for rapid prototyping. We demonstrate that low-cost mask-based stereolithography (MSLA) 3-D printers with straightforward modifications can provide fabrication quality that approaches traditional photolithography techniques. For example, reliable feature sizes of 20 µm with dimensional discrepancy of <4% for lateral dimensions and <5% for vertical dimensions were fabricated with a consumer-level MSLA printers. In addition, alterations made to pre-processing, post-processing, and printer configuration steps improved print quality as demonstrated in objects with sharper edges and smoother surfaces. The prototyping time and cost of resin 3-D printing (3 h with USD 0.5/prototype) were considerably lower than those of traditional photolithography (5 d with USD 80/prototype). With the rapid advance of consumer-grade printers, resin 3-D printing can revolutionize rapid prototyping approaches for microdevices in the near future, facilitating participation in interdisciplinary development of innovative hardware to support germplasm repository development for aquatic species.

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“…Automated instrumentation has been used to reduce human error, such as flow cytometry [14], microvolume spectrophotometry [15], and computerassisted sperm analysis (CASA) [16]. However, these types of equipment were designed for sophisticated research and thus costs tens of thousands of dollars [17]. The use of automated equipment solely for evaluation of sperm concentration is cost prohibitive to most facilities that do not specialize in cryopreservation or reproductive biology [18].…”

Section: Introductionmentioning

confidence: 99%

Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species

Belgodere

Liu

Reich

et al. 2022

Fishes

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Accurate determination of sperm concentration in aquatic species is important for assisted reproduction and cryopreservation, yet is challenging as current counting methods are costly or not suitable for many species. The goal of this work was to develop a simple (single-piece and single-layer photolithography) sperm counting chamber (SSCC) for aquatic species. Goldfish (Carassius auratus) and zebrafish (Danio rerio) sperm were used for evaluation in the device, which was created with soft lithography. Four designs with different geometries were evaluated for counting accuracy. Open-corner and open-midpoint designs were the most accurate with no significant differences (P > 0.05) for most of the target sperm concentrations (0.5–1.0 × 108 cells/mL). The open-corner design was not significantly different from the Makler® counting chamber intended for human sperm cells (P = 0.6) but was significantly different from a hemocytometer (P < 0.001) intended for other cell sizes. Material cost of device production was USD 16 per unit, including photolithography supplies, glass slide and coverslip, and polydimethylsiloxane. The cost can be reduced to USD 2 per unit with repeated wafer casts. This device could be further refined for resin 3-D printing and sharing via open-hardware approaches and modified to best suit species specific applications.

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Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration

Cited by 10 publications

References 8 publications

Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources

Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources

Low-Cost Resin 3-D Printing for Rapid Prototyping of Microdevices: Opportunities for Supporting Aquatic Germplasm Repositories

Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species

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