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.

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.

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.

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.

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.

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.

An open hardware 3-D printed device for measuring tensile properties of thermoplastic filament polymers at cryogenic temperatures.

With the emerging recognition of open scientific hardware, rapid prototyping technology such as three-dimensional (3-D) printing is becoming widely available for fields such as cryobiology, and cryopreservation, where material selection for instruments and hardware has traditionally been problematic due to extreme low temperatures. A better understanding of the mechanical properties of 3-D printing thermoplastics at cryogenic temperatures is essential to material selection, part design, and printing optimization. The goal of the present study was to explore the feasibility of development for a 3-D printed device ('CryoTensileDevice') to hold a test specimen in liquid nitrogen and be mounted in standard mechanical testing systems to evaluate 3-D printing material behaviors at cryogenic temperatures. The CryoTensileDevice was prototyped with flexible filaments with a per-unit material cost of < US$5 and a printing time of < 5 h. The commonly used printing filament polylactic acid (PLA) was selected to evaluate the utility of the CryoTensileDevice. At room temperature, the CryoTensileDevice did not significantly (P > 0.05) affect PLA tensile measurements such as Young's modulus, yield stress, yield strain, stress at break, or strain at break. With the CryoTensileDevice, specimens 3-D printed with PLA at 50%, 75%, and 100% infill rates had comparable tensile properties when tested at room and liquid nitrogen temperatures. The PLA showed superior performance in tensile properties in comparison to acrylonitrile butadiene styrene (ABS). This device can assist characterization of 3-D printing approaches for cryogenic work, and opens a pathway for future innovations to create a variety of 3-D printed devices to study a wide range of material properties for cryogenic applications.

SIMULATION MODELING OF A HIGH-THROUGHPUT OYSTER CRYOPRESERVATION PATHWAY.

The genetic resources of oysters in Louisiana and the Gulf of Mexico are threatened due to high unexplained oyster mortality. Germplasm repositories are collections of cryopreserved genetic material stored alongside associated information that are used to protect genetics resources and facilitate breeding programs in agricultural industries. Therefore, there is great need for oyster repositories. Development of repositories has been slow despite research on high-throughput cryopreservation protocols because of logistical complexities. The goal of this study was to begin to address the gap between cryopreservation research and repository development in oyster aquaculture by modeling a cryopreservation protocol to understand and improve the process. The steps of a high-throughput cryopreservation protocol were defined and mapped in a process flow diagram. A simulation model was created using time study data, and key bottlenecks in the process were identified. Finally, model variations using alternate types of devices (tools or equipment) were created to address the identified bottlenecks. The model was found to accurately simulate the cryopreservation process. Parameters such as number of straws frozen per oyster, batch size, and number of operators significantly affected how the model performed and device choices produced substantial improvements. Simulation modeling has the potential to inform how cryopreservation pathways and repository systems in aquatic species should be structured and operated. There is ample opportunity for future work such as analyzing the impacts of production scale on cryopreservation processes.

An Impedimetric Sensing Probe Based on Printed Circuit Board Technology for Monitoring in Cryobiology Applications.

Cryopreservation of living cells is an effective tool for protection, maintenance, and distribution of genetic resources, which involves exposure to cryogenic temperatures and requires precise control over various parameters to avoid potential cell damages. Hundreds of protocols have been reported for cryopreservation of aquatic species, but replicating them is challenging without a reliable monitoring technique during a cryopreservation process. In this work, we aim to use electrical impedance as a monitoring parameter to assist standardization of cryopreservation processes and reporting. Specifically, this paper reports an impedance sensing probe compatible with cryogenic temperatures and conventional containers in cryopreservation of aquatic species based on printed circuit board technology its characterization in cryopreservation conditions including different sperm extenders (buffer) compositions and concentrations, presence of cryoprotectant, and multiple cooling rates. The developed probe based on printed circuit board (PCB) technology shows a capability of measuring conditions during cryopreservation differentiating among samples with different buffer contents and cryoprotectants. The probe also demonstrates the capability to distinguish different cooling regimes and detect phase change phenomena. This PCB-based sensing platform provides quantitative impedance measurement data during the cryopreservation process at sample preparation, cooling, and while frozen. Technology such as this offers opportunities for improving the reproducibility of protocols generated by the aquatic species community and can be made widely available as open hardware.

Development of an open hardware 3-D printed conveyor device for continuous cryopreservation of non-batched samples.

A great challenge among communities participating in germplasm repository development is to obtain suitable cryopreservation equipment and devices. Commercial programmable freezers are costly and thus unaffordable to many users. Self-made devices have substantial variability among users, resulting in few opportunities for standardization across communities. The development of open hardware with the increasing accessibility of three-dimensional (3-D) printing offers rapid prototyping and easy fabrication of devices by users around the world at low cost. The present study explored the feasibility of developing operational prototypes of 3-D printed motorized cryopreservation devices for continuous freezing of non-batched samples. A controlled cooling conveyor device (CCCD) was designed and fabricated to cryopreserve sperm samples in straws that were loaded onto chain links suspended over liquid nitrogen held in a Styrofoam box. Cooling rates of 5 to 34 °C/min for 0.5-ml French straws were produced by adjusting the height of conveyor chains, slopes, and liquid nitrogen mass. The plunge temperature (-47 °C to -61 °C) was controlled by adjustment of conveyor speed. The cooling curves from the CCCD were comparable to a commercial programmable freezer. There were no significant differences in post-thaw motility of sperm from ornamental (Koi) common carp (Cyprinus carpio) among samples frozen with the CCCD and those frozen with a commercial programmable freezer. The post-thaw sperm motility was consistent among samples frozen in the CCCD across a 15-min time span. The CCCD prototypes in the present study proved to be feasible and functional as low-cost, customizable, portable, and yet standardizable options for freezing of individual (non-batched) samples. Additional design alternatives are proposed to facilitate further adaptation and development by diverse user communities.

A generalized approach for sperm cryopreservation in the genus Pomoxis: Sperm cryopreservation and fertilization efficiency of black-stripe black crappie, Pomoxis nigromaculatus.

Approaches for white crappie, Pomoxis annularis sperm cryopreservation have led to interest in applying similar methods to black-stripe black crappie, Pomoxis nigromaculatus. Their rarity in wild populations makes them a preferred phenotype for hatchery use. Sperm cryopreservation procedures were compared between black-stripe black crappie and white crappie for sperm motility and egg fertilization rate. There was no difference in black-stripe black crappie sperm motility after thawing between 5% dimethyl sulfoxide (DMSO, 45% motility) and 10% methanol (50% motility). However, fertilization rates were higher (p < .001) for sperm cryoprotected with 5% DMSO (38 ± 8%) than 10% methanol (22 ± 7%). Hatchery use requires sperm-to-egg ratios and fertilizing potential of single doses (i.e., 0.5 ml straw). Using black-stripe black crappie sperm (2.5 × 108 sperm/ml; 5% DMSO), the highest fertilization (27%) was found using single straws with 785 eggs (0.25 ml); total sperm:egg ratio: 159,000:1; motile sperm:egg ratio: 71,700:1. Therefore, sperm of two Pomoxis species could be cryopreserved using 350 mOsmol/kg Hanks' balanced salt solution as an extender, 5% DMSO as a cryoprotectant, cooling at 40°C/min, and thawing for 8 s at 40°C to maintain sperm motility and fertility. Basic protocols can be generalized within a genus if variables such as sperm concentration, process timing, and sample volumes are controlled.

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

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 µm×100 µm) and a PDMS base platform to create a known volume with a 10-µm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations.

3-D Printed Customizable Vitrification Devices for Preservation of Genetic Resources of Aquatic Species.

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.

Three-dimensional printing can provide customizable probes for sensing and monitoring in cryobiology applications.

Cryopreservation has been recognized as a powerful tool for long-term preservation of genetic resources. However, the outcomes of cryopreservation by different user groups often vary due to inconsistency in procedures and freezing equipment. Herein, we report on the feasibility of providing customizable sensing probes with three-dimensional (3-D) printing to monitor cryopreservation phenomena. The objectives were to: 1) introduce 3-D printing as a fabrication method for developing customizable probes to be used in cryogenic applications; 2) design and fabricate an example of a 3-D printed sensing probe and multiplexer capable of detecting phase-change phenomena based on quantitative data regarding sample electrical resistance and temperature, and 3) demonstrate the sensing platform in cryopreservation conditions and in combination with a custom-made 3-D printed freezing device. The sensing probe developed was designed to fit within standard 0.5-ml French straws. Phase-transition phenomena were detected by analyzing electrical resistance changes. The quantitative data from this device in conjugation with a 3-D printed freezer rack provided cryopreservation capability with high reproducibility and offered an alternative to expensive programmable freezers. The use of 3-D printing provided flexibility to develop new sensing probes or modify existing designs based on specific needs. After initial prototyping, fabrication, and testing of 3-D printed sensing probes, particularly useful designs can lead to the reduction of variation in performing standardized cryopreservation protocols.

Workshop report: Cryopreservation of aquatic biomedical models.

The genetic resources of aquatic biomedical model organisms are the products of millions of years of evolution, decades of scientific development, and hundreds of millions of dollars of research funding investment. Genetic resources (e.g., specific alleles, transgenes, or combinations) of each model organism can be considered a form of scientific wealth that can be accumulated and exchanged, typically in the form of live animals or germplasm. Large-scale maintenance of live aquatic organisms that carry these genetic resources is inefficient, costly, and risky. In situ maintenance may be substantially enhanced and backed up by combining cryopreserved germplasm repositories and genetic information systems with live animal culture. Unfortunately, cryopreservation has not advanced much beyond the status of an exploratory research for most aquatic species, lacks widespread application, and methods for successful cryopreservation remain poorly defined. For most aquatic species biological materials other than sperm or somatic cells are not comprehensively banked to represent and preserve a broad range of genetic diversity for each species. Therefore, new approaches and standardization are needed for repository-level application to ensure reproducible recovery of cryopreserved materials. Additionally, development of new technologies is needed to address preservation of novel biological materials, such as eggs and embryos of aquatic species. To address these goals, the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) hosted the Cryopreservation of Aquatic Biomedical Models Workshop on January 7 to 8, 2017, in conjunction with the 8th Aquatic Animal Models of Human Disease Conference in Birmingham, Alabama. The goals of the workshop were to assess the status of germplasm cryopreservation in various biomedical aquatic models and allow representatives of the scientific community to develop and prioritize a consensus of specific actionable recommendations that will move the field of cryopreservation of aquatic resources forward. This workshop included sessions devoted to new approaches for cryopreservation of aquatic species, discussion of current efforts and approaches in preservation of aquatic model germplasm, consideration of needs for standardization of methods to support reproducibility, and enhancement of repository development by establishment of scalable high-throughput technologies. The following three broad recommendations were forwarded from workshop attendees: 1: Establish a comprehensive, centralized unit ("hub") to programmatically develop training for and documentation of cryopreservation methods for aquatic model systems. This would include development of species-specific protocols and approaches, outreach programs, community development and standardization, freezing services and training of the next generation of experts in aquatic cryopreservation. 2: Provide mechanisms to support innovative technical advancements that will increase the reliability, reproducibility, simplicity, throughput, and efficiency of the cryopreservation process, including vitrification and pipelines for sperm, oocytes, eggs, embryos, larvae, stem cells, and somatic cells of all aquatic species. This recommendation encompasses basic cryopreservation knowledge and engineering technology, such as microfluidics and automated processing technologies. 3: Implement mechanisms that allow the various aquatic model stock centers to increase their planning, personnel, ability to secure genetic resources and to promote interaction within an integrated, comprehensive repository network for aquatic model species repositories.

On-Site Capabilities of a Mobile Laboratory for Aquatic Germplasm Cryopreservation.

Cryopreservation of genetic material can become an important tool for user groups in imperiled fishes, wild fisheries, aquaculture, and biomedical research. Persistent challenges within aquatic species cryopreservation are standardization and reliable collection of diverse, high quality samples. The overall goal of this study was to work with different user groups and cryopreserve sperm on-site at their facilities to evaluate the uses and challenges of a mobile laboratory with high-throughput and quality control capabilities comparable to those of a specialized centralized facility. The objectives were to demonstrate collection and cryopreservation of sperm of: 1) large-bodied freshwater Blue Catfish (Ictalurus furcatus) for aquaculture; 2) small-bodied freshwater Xiphophorus for biomedical and imperiled repository development, and 3) saltwater Red Snapper (Lutjanus campechanus) for wild fisheries research. Over the course of this project, the mobile laboratory traveled more than 4,000 km collecting germplasm from more than 650 male fishes. A total of 136 Blue Catfish were processed in 2015 and 2016 resulting in a total of 6,146 0.5-mL French straws. A total of 521 males from 11 different species in the genus Xiphophorus were processed over 4 d in 2015 resulting in a total of 488 0.25-mL French straws. And, a total of 17 Red Snapper males were processed during 2015 resulting in a total of 316 0.5-mL French straws. This is the first development of a mobile laboratory with high-throughput capability for aquatic species. User groups would no longer be limited to germplasm resources that can only be shipped as samples or transported as live animals to a central cryopreservation facility. Mobile laboratories create opportunities to collect higher quality germplasm, provide access to new species, and enable direct cooperation, including training, with a wide variety of user groups and applications.

The role of alkalinization-induced Ca2+ influx in sperm motility activation of a viviparous fish Redtail Splitfin (Xenotoca eiseni).

Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca2+ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca2+ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca2+ and pH levels. Intracellular Ca2+ signals were detected from sperm within bundles, and increased significantly with extracellular Ca2+ and pH levels. Major channel blockers known to inhibit Ca2+ influx (NiCl2, ruthenium red, GdCl3, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55-0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca2+ influx, except for CdCl2, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca2+ channels in the sperm plasma membrane without osmotic shock, and the Ca2+ influx functions as a second messenger to activate motor proteins controlling flagella movement.

A microfluidic device for motility and osmolality analysis of zebrafish sperm.

A microfluidic chip is described that facilitates research and quality control analysis of zebrafish sperm which, due to its miniscule (i.e., 2-5 µl) sample volume and short duration of motility (i.e., <1 min), present a challenge for traditional manual assessment methods. A micromixer molded in polydimethylsiloxane (PDMS) bonded to a glass substrate was used to activate sperm samples by mixing with water, initiated by the user depressing a transfer pipette connected to the chip. Sample flow in the microfluidic viewing chamber was able to be halted within 1 s, allowing for rapid analysis of the sample using established computer-assisted sperm analysis (CASA) methods. Zebrafish sperm cell activation was consistent with manual hand mixing and yielded higher values of motility at earlier time points, as well as more subtle time-dependent trends in motility, than those processed by hand. Sperm activation curves, which indicate sample quality by evaluating percentage and duration of motility at various solution osmolalities, were generated with on-chip microfabricated gold floor electrodes interrogated by impedance spectroscopy. The magnitude of admittance was linearly proportional to osmolality and was not affected by the presence of sperm cells in the vicinity of the electrodes. This device represents a pivotal step in streamlining methods for consistent, rapid assessment of sperm quality for aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the microfluidic device described herein will help improve the reproducibility of studies on sperm and assist development of germplasm repositories.

Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids.

More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5-15% v/v %), equilibration exposure times (1-60 min), cooling rates (5-40 °C/min), concentrations (4 × 104-4 × 106 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200-400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4 × 106 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ±â€¯5% of post-thaw dissociable bundles with 209 ±â€¯10 s of dissociation duration for X. eiseni, 96 ±â€¯9% with 814 ±â€¯14 s for Blackfin Goodea (Goodea atripinni), and 66 ±â€¯2% with 726 ±â€¯25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers.

Activation of free sperm and dissociation of sperm bundles (spermatozeugmata) of an endangered viviparous fish, Xenotoca eiseni.

Knowledge of sperm motility activation for viviparous fishes has been limited to study of several species in Poeciliidae, and the dissociation of sperm bundles is even less understood. The goal of this study was to use the endangered Redtail Splitfin (Xenotoca eiseni) as a model to investigate the activation of sperm from viviparous fishes by study of free sperm and spermatozeugmata (unencapsulated sperm bundles). The specific objectives were to evaluate the effects of: (1) osmotic pressure and refrigerated storage (4 °C) on activation of free sperm, (2) osmotic pressure, ions, and pH on dissociation of spermatozeugmata, and (3) CaCl2 concentration and pH on sperm membrane integrity. Free sperm were activated in Ca2+-free Hanks' balanced salt solution at 81-516 mOsmol/kg. The highest motility (19 ±â€¯6%) was at 305 mOsmol/kg and swim remained for 84 h. Glucose (300-700 mOsmol/kg), NaCl (50-600 mOsmol/kg), and KCl, MgCl2, and MnCl2 at 5-160 mM activated sperm within spermatozeugmata, but did not dissociate spermatozeugmata. CaCl2 at 5-160 mM dissociated spermatozeugmata within 10 min. Solutions of NaCl-NaOH at pH 11.6 to 12.4 dissociated spermatozeugmata within 1 min. The percentage of viable cells had no significant differences (P = 0.2033) among different concentrations of CaCl2, but it was lower (P < 0.0001) at pH 12.5 than at pH between 7.0 and 12.0. Overall, this study provided a foundation for quality evaluation of sperm and spermatozeugmata from livebearing fishes, and for development of germplasm repositories for imperiled goodeids.