From gamete collection to database development: Development of a model cryopreserved germplasm repository for aquatic species with emphasis on sturgeon

Development of a cryopreserved sperm repository could greatly benefit the study and recovery of endangered species. Currently, a national repository for endangered aquatic species does not exist. This dissertation addressed the development of methods for the creation of a model germplasm repository, which included methods to reduce bacterial contamination within sperm samples, verify sample identification, store sperm (refrigerated or cryopreserved), evaluate sperm quality before and after thawing, and maintain sample and broodstock information. Although these methods were developed for sturgeon, they could be adapted for any aquatic species.; Accurate species identification of sperm samples is essential for an archival repository. A polymerase chain reaction and restriction digest method was developed to identify three sturgeon species, and separate the remaining species into distinct groups. Bacterial contamination of sperm samples can lead to sample deterioration and transmission of pathogens. Twenty-seven species of bacteria from 15 genera were isolated from sperm samples of sturgeon in this study. Addition of antibiotics did not lengthen refrigerated storage time. Cryopreservation procedures developed for sturgeon sperm did not reduce bacterial numbers in thawed samples compared to pre-freeze numbers. Seminal plasma osmolality (100 mOsmol/kg) and sperm activation data (reduction in potassium ion concentration) were used to develop extenders. Hanks' balanced salt solution or modified Hanks' balanced salt solution (mHBSS) at 100 mOsmol/kg allowed refrigerated storage of sperm for as long as 21 d. Sperm were cryopreserved using mHBSS (100 mOsmol/kg), methanol (5% or 10%), a cooling rate of 22–24°C/min, and 0.5-ml straws. A dual-staining flow cytometry technique was used to determine gamete quality in sperm samples before and after thawing. The procedure was validated by evaluating membrane integrity in mixed populations of untreated cells and heat-treated membrane damaged cells. When used with thawed samples, whenever membrane integrity was correlated (r2 > 0.8068) to percent fertilization and hatch, motility was also correlated (r2 > 0.7650) to percent fertilization and hatch. A computer database was developed using Microsoft® Access to maintain inventory and quality control of sample information. The database is currently being used at the Warm Springs Fish Technology Center of the United States Fish and Wildlife Service.