RESUMO
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.
RESUMO
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.