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.

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.

UVA-induced photo recovery during early zebrafish embryogenesis.

DNA photorepair has been widely studied in simple aquatic organisms that live in the marine environment, but is less understood in more complex species that live in freshwater. In the present study, we evaluated UVA-induced DNA photo recovery in embryonic stages of zebrafish, Danio rerio, a freshwater model species. Evaluation of UVB exposure and UVA photo recovery of zebrafish embryos revealed different UVB tolerances and capacities for UVA photo recovery at different stages of development. Effective UVA photo recovery was observed at 3h post-fertilization (hpf), 6-7 hpf, and 12 hpf, but not in the early cleavage stage (2-32 cells). UVA photo recovery was most effective during the gastrula stage (6-7 hpf) of development, and less effective at earlier stages (e.g., 3 hpf) or later stages (e.g., 12 hpf). Embryos at the cleavage stage of development were found to be tolerant to extreme levels of UVB exposure, and possible mechanisms were discussed. For embryos at 6-7 hpf, examination of time window (or delay of UVA exposure) that would still permit recovery from UVB exposure suggested a short time period of 2h. The transgenic fli-1 zebrafish with fluorescent vascular structure was used to show that embryos with normal morphological appearance could exhibit a disrupted vascular patterning, suggesting that this endpoint could provide a sensitive tool for detection of UV damage.