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.

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.

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.

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.

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.

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.