Restricted Scope for Administrative Supplement: Ambystoma Genetic Stock Center

PROJECT SUMMARY The Ambystoma Genetic Stock Center (AGSC) is a P40 Research Resource that distributes laboratory axolotls (Ambystoma mexicanum) to researchers and educators nationally and internationally. The AGSC is currently working on a project to develop a reproducible protocol for cryopreserving axolotl sperm, which is needed to efficiently manage axolotl stocks within the AGSC and the broader axolotl community. Early results from this project found that axolotl spermatids are viable for only a short time after they are packaged into structures called spermatophores. This presents a challenge to develop a sperm cryopreservation protocol because there is a narrow window of time within which spermatophores can be harvested to obtain viable sperm. Additionally, spermatophores were found to be laid by males at various times of the day, more often at night and not during the workday. Temporal parameters for collecting spermatophores with viable sperm have not been defined because the mating process has never been rigorously monitored to know when spermatophores are deposited. Until these parameters are defined, it will not be possible to collect large numbers of viable spermatophores from which to develop and optimize methods for cryopreservation. There is critical need to develop a standard protocol for collecting spermatophores with viable sperm. There is also critical need to optimize and gain capacity for sperm freezing and thawing at the AGSC, and to integrate in vitro fertilization into the workflow. To address these needs, the AGSC will hire a full-time employee with previous experience in cryopreservation and axolotl biology. The new employee will work with undergraduate student workers to monitor axolotl courtship during the day and night for the purpose of recording behavioral data and collecting spermatophores. Spermatophores will be frozen and thawed using reagents and conditions recommended by the Aquatic Germplasm and Genetic Resources Center (AGGRC) at the Louisiana State University Agricultural Center and experiments will be performed to assay sperm viability and in vitro fertilization success. Protocols arising from the proposed work will be made publicly available. This administrative supplement will accelerate progress in developing an axolotl sperm cryopreservation protocol and building AGSC capacity to manage germplasm for the axolotl community. .