Doctor of Philosophy (PhD)


School of Renewable Natural Resources

Document Type



Fishes of the family Goodeidae are considered to be among the most imperiled taxa in the world. The goal of this project was to provide a foundation for development of sperm repositories for goodeids and to use them as a model to assist conservation programs for imperiled freshwater live-bearing fishes. Development of such repositories is challenging because of unusual reproductive characteristics of live-bearing fishes, such as sperm bundles, internal fertilization, and the bearing of live young. Standardized methods were established to evaluate quality-related attributes of sperm bundles. The different features between activation of free and bundled sperm were investigated and the positive effects of isotonic solutions, extracellular Ca2+, and alkalinization on activation were identified. To investigate molecular mechanisms, a novel method was established for integration of sperm bundles into cell imaging techniques. Based on this, signaling mechanisms regulating sperm activation of goodeids were proposed: upon spawning the alkaline environment within the female reproductive tract activates Ca2+ channels in the sperm plasma membrane, leading to a rapid increase in intracellular Ca2+ that activates flagella movement. A generalized cryopreservation protocol was developed for live-bearing goodeids using sperm bundles from three species (Xenotoca eiseni, Goodea atripinni, and Ataeniobius toweri): sperm bundles suspended in Ca2+-free Hanks’ balanced salt solution at 300 mOsmol/kg were mixed with 10% DMSO as cryoprotectant for 20 min of equilibration, loaded into 0.25-ml French straws, and cooled at a rate of 10 ºC/min at 4.0 × 106 bundles/ml. Based on this protocol, live young X. eiseni were produced with artificial insemination with cryopreserved sperm. A total of 29 live young were produced from 117 females inseminated with thawed sperm, and 12 were produced from 81 females inseminated with fresh sperm, demonstrating the feasibility of cryopreservation. This dissertation project enables future establishment of sperm repositories to assist conservation and recovery for imperiled live-bearing goodeids. A strategy with a comprehensive conservation and recovery approach combined with germplasm repository methodologies was developed. This would involve efforts including habitat restoration and captive breeding, and strong collaborations among individuals, agencies, and facilities with different specialized expertise and capabilities.



Committee Chair

Tiersch, Terrence