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.

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.

Evaluation of industrial and consumer 3-D resin printer fabrication of microdevices for quality management of genetic resources in aquatic species.

Aquatic germplasm repositories can play a pivotal role in securing the genetic diversity of natural populations and agriculturally important aquatic species. However, existing technologies for repository development and operation face challenges in terms of accuracy, precision, efficiency, and cost-effectiveness, especially for microdevices used in gamete quality evaluation. Quality management is critical throughout genetic resource protection processes from sample collection to final usage. In this study, we examined the potential of using three-dimensional (3-D) stereolithography resin printing to address these challenges and evaluated the overall capabilities and limitations of a representative industrial 3-D resin printer with a price of US$18,000, a consumer-level printer with a price

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.

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 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.