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.

Water quality and the CO2-carbonate system during the preconditioning of Pacific oyster (Crassostrea gigas) in a recirculating aquaculture system.

The continued increase of the demand for seed of the Pacific oyster (Crassostrea gigas) has driven the aquaculture industry to produce land-based hatcheries using broodstock conditioning. This has led to the need to create closed systems to control the main factors involved in reproduction (temperature and food). Additionally, reproductive synchronization of broodstocks may be considered to ensure homogeneous maturation and spawning among the organisms. In this work, we synchronized the broodstock reproductive stage of Pacific oysters in a recirculating aquaculture system (RAS) using a "preconditioning" process and evaluated the effect of the water quality and the CO2-carbonate system on preconditioned broodstock. The oysters were kept at 12 °C for 45 days in a RAS containing a calcium reactor (C2) or without a calcium reactor (C1, control). Water quality parameters were measured daily, and the oyster's condition and reproductive development were monitored using condition index, biometrics, and histology, on Days 0, 20, and 45. C1 and C2 systems kept the water quality within the ranges reported as favorable for bivalves. The calcium reactor kept the pH (8.03-8.10), alkalinity (200 mg/L as CaCO3), CO32- (≤ 80 µmol/kg), and Ω aragonite (≤ 1) closer to the ranges reported as optimal for bivalves. However, no significant differences were detected in the total weight and the condition index in C1 and C2. The preconditioning allowed to maintain the organisms in early reproductive development, allowing gametogenesis synchronization to start maturation.

A continuous microwave system for prevention of invasive species during de-ballasting operation--death kinetics.

A continuous microwave heating system was tested for its effectiveness at removing potentially invasive organisms during deballasting operations. Four different organisms, namely Nannochloropsis oculata (microalgae), Artemia nauplii, Artemia adults and Crassosstrea virginica (oyster larvae) normally found in ballast water were investigated in a controlled study to quantify their survival after continuous microwave heating of synthetic ballast water. The experiments were performed in the microwave system using a 2 x 2 factorial design with power (2.5 and 4.5 kW) and flow rate (1.0 and 2.0 lpm) and the organisms subsequently subjected to different holding times. The control treatment was performed in a water bath using the same temperatures and holding times as in the case of the microwave treatment. Overall, the results obtained indicated that the microwave system was more effective in eliminating the organisms when compared with the control treatment. In most cases there were no survivors present after the microwave treatment at holding times above 100 s, and temperatures as low as 50 degrees C particularly for oyster larvae and Artemia adults. The results are promising, indicating that this technology has the potential to be an effective tool in controlling/preventing the introduction of invasive species into native environments.