Establishment of a Practical Sperm Cryopreservation Pathway for the Axolotl (Ambystoma mexicanum): A Community-Level Approach to Germplasm Repository Development.

The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25-800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks' Balanced Salt Solution (HBSS) at osmolalities of 100-600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7-8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications.

Exploring pathways toward open-hardware ecosystems to safeguard genetic resources for biomedical research communities using aquatic model species.

Development of reliable germplasm repositories is critical for preservation of genetic resources of aquatic species, which are widely utilized to support biomedical innovation by providing a foundational source for naturally occurring variation and development of new variants through genetic manipulations. A significant barrier in repository development is the lack of cryopreservation capability and reproducibility across the research community, posing great risks of losing advances developed from billions of dollars of research investment. The emergence of open scientific hardware has fueled a new movement across biomedical research communities. With the increasing accessibility of consumer-level fabrication technologies, such as three-dimensional printers, open hardware devices can be custom designed, and design files distributed to community members for enhancing rigor, reproducibility, and standardization. The overall goal of this review is to explore pathways to create open-hardware ecosystems among the communities using aquatic model resources for biomedical research. To gain feedback and insights from community members, an interactive workshop focusing on open-hardware applications in germplasm repository development was held at the 2022 Aquatic Models for Human Disease Conference, Woods Hole, Massachusetts. This work integrates conceptual strategies with practical insights derived from workshop interactions using examples of germplasm repository development. These insights can be generalized for establishment of open-hardware ecosystems for a broad biomedical research community. The specific objectives were to: (1) introduce an open-hardware ecosystem concept to support biomedical research; (2) explore pathways toward open-hardware ecosystems through four major areas, and (3) identify opportunities and future directions.

Modeling of cryopreservation pathway operation at an aquatic biomedical stock center for zebrafish.

Aquatic biomedical model organisms play a substantial role in advancing our understanding of human health, however, comparably little work has been directed towards developing dependable, high-throughput storage programs for valuable genetic resources. The Zebrafish International Resource Center (ZIRC) has developed a standardized cryopreservation pathway and stored thousands of genetic lines in their repository for use by the biomedical research community. This has yet to be replicated in other facilities, and an overall repository-level pathway has never been analyzed for aquatic species. To encourage repository development for other biomedical models and to improve the ZIRC storage process and system, this study used discrete-event simulation modeling to systematically analyze the cryopreservation pathway for efficiency, and to identify improvements. The models reflected "real-world" working conditions and were used to simulate key outputs, such as production capacity over time (throughput) and steps in the process that limit production (bottlenecks). With these models, recommendations were identified to eliminate waiting times and increase efficiency. These included following proper husbandry protocols because male quality significantly affected production time, and the use of part-time operators to assist with steps that had longer Waiting Times (i.e., time samples spent in a queue) to increase production capacity. Simulation process modeling is a powerful tool that can improve the operations of existing repositories. It can also support repository development at other biomedical stock centers, and at other facilities devoted to aquatic species such as research, conservation, and aquaculture production hatcheries.

Transitioning from a research protocol to a scalable applied pathway for Xenopus laevis sperm cryopreservation at a national stock center: The effect of cryoprotectants.

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center-level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 106 sperm/mL) on post-thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10% DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%-13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species.

Untangling the Gordian Knot of Aplysia sea hare egg masses: An integrated open-hardware system for standardized egg strand sizing and packaging for cryopreservation research and application.

The California sea hare (Aplysia californica) provides a powerful biomedical model system for studying aspects of neurological development and damage, behavior, aging, and hypoxia. Aplysia encapsulate their zygotes within strands that result in tangled egg masses that greatly complicate culture and experimentation. The historical and current importance of Aplysia for biomedical research and the mounting climate crisis necessitates protection of Aplysia genetic resources. The goal of this work was to prototype open-hardware sizing, processing, and packaging devices for A. californica early life stages suitable for integration into a cryopreservation pathway. The Strand Centi-Sizer was a low-cost, fused filament fabrication 3-D printable device that increased experiment preparation efficiency and standardized the cutting of egg strands customizable to user needs. A downstream system of 3-D printed devices was also prototyped to address inefficiencies in handling of egg strand sections for processing and packaging into existing cryopreservation straw platforms. Time studies were conducted comparing manual methods (i.e., no specialized equipment) with open hardware to demonstrate utility of the devices and to encourage community members to design and prototype new devices to address recurrent and novel problems in other aquatic animals that produce egg strands. Improvements in design could further increase efficiency, standardization, and reproducibility, and extend the application of these devices to other research communities, such as shrimp or salamander spermatophores, sea anemone body part (e.g., pedal lacerate) cryopreservation, or study areas such as vitrification.

A Modified-Herringbone Micromixer for Assessing Zebrafish Sperm (MAGS).

Sperm motility analysis of aquatic model species is important yet challenging due to the small sample volume, the necessity to activate with water, and the short duration of motility. To achieve standardization of sperm activation, microfluidic mixers have shown improved reproducibility over activation by hand, but challenges remain in optimizing and simplifying the use of these microdevices for greater adoption. The device described herein incorporates a novel micromixer geometry that aligns two sperm inlet streams with modified herringbone structures that split and recombine the sample at a 1:6 dilution with water to achieve rapid and consistent initiation of motility. The polydimethylsiloxane (PDMS) chip can be operated in a positive or negative pressure configuration, allowing a simple micropipettor to draw samples into the chip and rapidly stop the flow. The device was optimized to not only activate zebrafish sperm but also enables practical use with standard computer-assisted sperm analysis (CASA) systems. The micromixer geometry could be modified for other aquatic species with differing cell sizes and adopted for an open hardware approach using 3D resin printing where users could revise, fabricate, and share designs to improve standardization and reproducibility across laboratories and repositories.

Design, alpha testing, and beta testing of a 3-D printed open-hardware portable cryopreservation device for aquatic species.

Efforts in development of germplasm repositories to preserve genetic resources of aquatic species are impeded globally by a lack of standardized, inexpensive, reproducible, and portable cryopreservation technologies. The present work demonstrates a 3-D printed standardizable freezing device that can be used with nitrogen vapor shipping dewars for on-site sperm cryopreservation for aquatic species and be distributed as open-source The SDPCD could hold 22 French straws (0.25-mL or 0.5-mL) and a quick-release ring design could eject straws directly into a canister inside a dewar by pressing a button after freezing. The final prototypes produced cooling rates of 1 to 64 °C/min for 0.25-mL straws, and 3 to 37 °C/min for 0.5-mL straws with material cost of US$3.5 for a single device and US$1,820-2,562 for batch production of 20 replicates. Progressing through design, prototyping, and testing was delineated to help guide development of other open-source devices within cryopreservation user communities.

Survival and growth of triploid eastern oysters, Crassostrea virginica, produced from wild diploids collected from low-salinity areas.

Triploid Eastern oysters have been reported to suffer greater mortalities than diploids when exposed to low-salinity (<5) conditions in the U.S. Gulf of Mexico and Atlantic estuaries. As such, the effect of broodstock parentage was investigated on the low-salinity tolerance of triploid progeny produced by mating diploid females (collected from three Louisiana estuaries differing in salinity regimes) with male tetraploids at two hatcheries. Diploid crosses were also produced using the wild broodstocks to verify expected differences in low-salinity tolerance among diploid progeny and between ploidy levels. All progeny were deployed at low and moderate-salinity (averages of 9.3 and 19.4) field sites to monitor monthly growth and mortality. Sex ratio, gametogenic stage, gonad-to-body ratio, condition index, and Perkinsus marinus infection were also measured periodically at both field sites Although high triploid mortality at the low-salinity site prevented complete analysis, results indicated that diploid parentage had little effect on triploid survival at low salinity. Broodstock parentage affected diploid mortality and growth, although results did not match with predictions made based on historical salinity at broodstock collection sites. Ploidy level had the largest effect on triploid survival and growth followed by the hatchery site where the oysters were produced.

Initial assessment of the toxicologic effects of leachates from 3-dimensional (3-D) printed objects on sperm quality in two model fish species.

The use of 3-dimensional (3-D) printing is gaining popularity in life sciences and driving innovation in fields including aquatic sperm cryopreservation. Yet, little is known about the effects leachates from these objects may have on biological systems. In this study, we investigated if exposure to leachates from 3-D printed objects fabricated from different photo-curable resins could affect sperm quality in two model fish species, zebrafish (Danio rerio) and goldfish (Carassius auratus). Leachates were collected following contact periods of 10 min and 22 h with objects manufactured using a mask LCD resin printer and three different commercially available resins (i.e., standard, eco-friendly, and impact-resistant). Sperm cells were exposed to the leachates for 18 min, and parameters related to sperm motility, cell count, and membrane integrity were evaluated. All experiments were blinded. Leachate originating from contact with impact-resistant resin for 10 min significantly reduced the cell count of zebrafish sperm, while leachate originating from contact with standard resin for 22 h significantly increased the beat cross frequency of goldfish sperm. The changes were not observed across species and no adverse effects were recorded in percent motility, velocity, amplitude of lateral head movement, or membrane integrity of sperm. Our findings demonstrate that exposure to leachates from certain 3-D printed resins can affect sperm quality, while other resins may support sperm quality evaluation. Further investigations are warranted to assess other parameters, effects, and their biological relevance for a variety of aquatic species.

An automated modular open-technology device to measure and adjust concentration of aquatic sperm samples for cryopreservation.

Repositories for aquatic germplasm are essential for safeguarding valuable genetic diversity for species relevant to aquaculture, biomedical research, and conservation. Development of aquatic germplasm repositories is impeded by a lack of standardization within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as cell concentration, that strongly affect the success and reproducibility of cryopreservation. The wide dissemination and use of specialized tools and devices as open hardware can improve processing reliability and save costs. The goal of the present work was to develop and prototype a modular and open-technology approach to help to standardize the cell concentration of germplasm samples prior to cryopreservation. The specific objectives were to: 1) design and fabricate prototypes of the automated concentration measurement and adjustment system (CMAS), incorporating custom peristaltic pumps and optical evaluation modules, and 2) evaluate the performance of the CMAS with biological samples. Linear regression models were obtained for estimation of aquatic sperm concentration >108 cells/mL and for algae concentration > (3 × 105) cells/mL. Algae were diluted with extender medium by an automated process, resulting in a dilution precision of ±12.6% and ±6.7% in two trials, attaining means of 89% and 71% of the target cell concentration. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm and can invite new users, makers, and developers into the open-technology community. This will increase the reach and capabilities of much-needed aquatic germplasm repositories.

Simulation analysis of high-throughput oyster cryopreservation at three scales of production.

Cryopreservation and germplasm repositories offer a variety of potential benefits to aquaculture industries. Despite this, no comprehensive repository systems exist for any prominent aquaculture species. A species that could greatly benefit from the use of cryopreserved sperm and repository storage is the eastern oyster, Crassostrea virginica. High-throughput cryopreservation protocols already exist for this species, and the easy transport of frozen sperm could facilitate selective breeding programs that address pressing challenges currently faced in the industry, such as mortality due to low-salinity conditions. This study addressed the gap between cryopreservation protocols and repository development in the oyster industry by creating simulation models to evaluate cryopreservation needs at three different scales of production. The effects of high-throughput device options and three key parameters (straws per oyster, batch size, and number of operators) on production capacity, time, and cost were evaluated. Recommendations for decisions concerning cryopreservation pathways and repository creation were given at each scale of production. Relative values of broodstock, juvenile oysters, and oyster sperm sold at hatcheries were also discussed. In general, repositories operating at higher production levels benefited from the economy of scale, could use automated high-throughput equipment options, and could hire more labor without drastically increasing production costs.

Energetic budget of diploid and triploid eastern oysters during a summer die-off.

Triploid oysters are widely used in off-bottom aquaculture of eastern oysters, Crassostrea virginica. However, farmers of the Gulf of Mexico (GoM) and Atlantic coast estuaries have observed unresolved, late-spring die-offs of triploid oysters, threatening the sustainability of triploid aquaculture. To investigate this, the physiological processes underlying oyster growth (e.g., feeding, respiration) and mortality of one-year-old diploid and triploid oysters were compared in early summer following an uptick in mortality. It was predicted that higher triploid mortality was the result of energetic imbalances (increased metabolic demands and decreased feeding behavior). Oyster clearance rates, percentage of time valves were open, absorption efficiency, oxygen consumption rates (basal and routine), ammonia excretion rate were measured in the laboratory and scope for growth was calculated. In addition, their condition index, gametogenic stage, Perkinsus marinus infection level, and mortality were measured. Mortality of triploids in the laboratory was greater than for diploids, mirroring mortality observed in a related field study. The physiological parameters measured, however, could not explain triploid mortality. Scope for growth, condition index, and clearance rates of triploids were greater than for diploids, suggesting sufficient energy reserves, while all other measurements where similar between the ploidies. It remains to be determined whether mortality could be caused from disruption of energy homeostasis at the cellular level.

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

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.

A 3-D printed vitrification device integrated with French straws.

The goal of this work was to develop prototypes of open-hardware vitrification devices for sperm cryopreservation that can be integrated with existing straw platforms. The open-hardware Vitrification Device for French Straws (VD-FS) is low-cost, customizable, 3-D printable, standardized, and allows long-term sample storage and identification. The feasibility was shown for vitrifying and storing samples with multiple configurations. The results can be improved by design alternation and evaluation of various vitrification solutions. This is the first complete open-hardware vitrification device that can be integrated with existing French-straw storage systems, providing a foundation for future community-level modifications and improvements.

The Need for a Framework Addressing the Temporal Aspects of Fish Sperm Motility Leading to Community-Level Standardization.

Motility is a widely available parameter that can be used to assess sperm quality of aquatic species. Sperm from fishes with external fertilization usually undergo a dynamic and short-lived period of motility after activation. The common practice of assigning a single value at an arbitrary peak of motility presents challenges for reproducibility, community-level standardization, and comparisons across studies. This study aimed to explore statistical approaches to standardize motility reporting, and to develop an initial framework for community-level standards. Sperm samples from 14 zebrafish (Danio rerio) with a total of 21,705 cells were analyzed by use of computer-assisted sperm analysis with data collection starting at 10 s after activation at 5-s intervals for 50 s. Four common motility variables were selected for analyses: curvilinear velocity, straight-line velocity, beat cross frequency, and amplitude of lateral head displacement. Cluster analysis was used to evaluate sperm subpopulations within and among males over time, least-square means was used to explore temporal aspects, and the first derivative of the regression equations was used to calculate the rate of change for the motility parameters. Cluster analysis proved informative, but overlapping ephemeral clusters were not valuable for providing standardization options. Analysis of temporal aspects and rate of change indicated opportunities for standardization by reporting the overall motility-time functions or reporting during stable time windows instead of peak motility or at random times. These approaches could minimize the inconsistencies caused by male-to-male variation and dynamic changes of subpopulations while providing comparable information. An overall temporal framework was identified for motility reporting along the collection-processing-cryopreservation-thawing sequence to provide a basis to support efforts of community-level standardization.

An Open-Hardware Insemination Device for Small-Bodied Live-Bearing Fishes to Support Development and Use of Germplasm Repositories.

Small-bodied live-bearing fishes attract broad attention because of their importance in biomedical research and critical conservation status in natural habitats. Artificial insemination is an essential process to establish hybrid lines and for the operation of sperm repositories. The existing mouth-pipetting technique for artificial insemination of live-bearing fishes has not been substantially upgraded since the first implementation in the 1950s. The goal of this work was to develop a standardized artificial inseminator device (SAID) to address issues routinely encountered in insemination by mouth-pipetting, including lack of reproducibility among different users, difficulty in training, and large unreportable variation in sample volume and pressure during insemination. Prototypes of the SAID were designed as relatively inexpensive ( 0.99) between the piston position and volume. Pressure generation from eight mouth-pipetting operators and SAID prototypes were assessed by pressure sensors. The pressure control by SAID was superior to that produced by mouth-pipetting, yielding lower pressures (31−483 Pa) and smaller variations (standard deviation <11 Pa). These pressures were sufficient to deliver 1−5 µL of fluid into female reproductive tracts yet low enough to avoid physical injury to fish. Community-level enhancements of the SAID prototype could enable standardized insemination with minimal training and facilitate the participation of research communities in the use of cryopreserved genetic resources.

The emerging role of open technologies for community-based improvement of cryopreservation and quality management for repository development in aquatic species.

Genetic resources of aquatic species are of tremendous value, but worldwide these are maintained almost exclusively as live populations. This is extremely expensive and insecure, and largely results from a pervasive lack of production capability, quality management, and reproducibility in cryopreservation that are barriers in development of germplasm repositories. Community-based technology approaches are emerging that can stimulate research previously limited by a lack of affordable, customizable equipment. Open-access technologies can provide for custom design and fabrication not available through traditional manufacturing. This can assist repository development with robust sample production methods and strong quality management, and can greatly improve reproducibility and standardization. Open technologies can support establishment of new communities of users, makers, and developers that collectively strive to develop open hardware in a distributed (i.e., non-centralized) fashion that can yield aggregate throughput. This occurs through use of consumer-level tools, supplies, software, and equipment, free exchange of designs and modifications, and a shared sense of mission. For cryopreservation and repository development, we have identified 14 categories of open hardware for a processing pathway, and six categories for a quality management pathway. Open hardware offers economic incentives to develop repositories for aquatic species, something that has not occurred despite 70 years of research largely focused on protocol development rather than practical applications. Advanced development of custom scientific hardware enhancing open technologies will be facilitated by interdisciplinary collaboration across biological and engineering fields. This manuscript is a contribution to the Special Issue in memory of Dr. Duane Garner, a leader in the sperm biology.

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70-75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

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

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 (

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

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.