Date of Award

2014

Document Type

Thesis

Degree Name

Master of Science (MS) in Biology

Department

Biology

Abstract

Cryopreserervation is a powerful tool for the long term storage of genetic material from endangered species. While mammalian gametes have long been freezable, eggs and embryos from fish have yet to be successfully frozen. Furthermore, protocols for freezing fish sperm have low success rates. In an effort to overcome the limitations in gamete manipulation currently impeding successful fish gamete cryopreservation, I outline two experiment using zebrafish (Danio rerio) as a model for freshwater fish species. First, I attempted to extend working time in both eggs and sperm by incubating the gametes in rainbow trout oviductal fluid (RTOF), RTOF + HEPES, or RTOF under a blood-gas atmosphere. Incubation in RTOF under a blood gas atmosphere significantly extended viability in both gametes to 24 hours. Second, I attempted to minimize the number of sperm necessary to fertilize a single egg by reducing the volume in which the eggs were fertilized and by comparing the traditional fertilization system to a novel inverted drop technique. There were no differences between fertilization rates with sperm concentrations ranging from 1575 to 4 sperm/μL . Furthermore, the inverted drop technique improved overall fertilization. Both increased gamete manipulation time and reduced sperm waste have the potential to improve cryopreservation success in fish gametes.

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