Establishment of a Practical Sperm Cryopreservation Pathway for the Axolotl (Ambystoma mexicanum): A Community-Level Approach to Germplasm Repository Development

Simple Summary Amphibians are vitally important in biomedical research and conservation. Preserving germplasm materials (e.g., sperm, egg, embryos) in cryopreserved form can reduce the costs and risks associated with maintenance of live organisms. The goal of this work was to establish an initial practical sperm cryopreservation pathway for axolotls (Ambystoma mexicanum) to provide a foundation for development of germplasm repositories. The specific objectives were to (1) establish an efficient approach for sperm collection and quality evaluation; (2) evaluate the effects of osmotic pressure of extender solutions on sperm quality during refrigerated storage; (3) evaluate the effects of cryoprotectants on post-thaw sperm quality; (4) evaluate the feasibility of fertilization with thawed sperm; and (5) establish a cryopreservation pathway by integrating the findings into a comprehensive process flow map. With this approach, axolotl offspring were produced with cryopreserved sperm for the first time. Future research can build from this work with the goal of developing a germplasm repository based on community-level integration at the Ambystoma Genetic Stock Center. 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 mu L of sperm fluid was collected at concentrations of 1.6 x 10(6) to 8.9 x 10(7) 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 degrees C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 degrees 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.