Doctor of Philosophy (PhD)
Renewable Natural Resources
The goal of this project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of germplasm for all aquatic species. Vitrification (freezing by formation of “glass” rather than crystalline ice) is simple, fast, inexpensive, can be potentially used to preserve samples in the field, and offers new options for germplasm management especially appropriate for small fishes. Sperm were studied from freshwater fish (channel catfish Ictalurus punctatus), viviparous freshwater fish (green swordtail Xiphophorus hellerii), and marine fishes (spotted seatrout Cynoscion nebulosus, red snapper Lutjanus campechanus, red drum Sciaenops ocellatus, and southern flounder Paralichthys lethostigma). To reduce toxicity, combinations of cryoprotectants at reduced concentrations with incorporation of trehalose and polymers were used to enhance glass formation. For freezing, samples were suspended on 10-µL polystyrene loops and plunged into liquid nitrogen. Thawing was done at 24ºC using Hanks’ balanced salt solution at 300 mOsmol/kg for freshwater species, and seawater at 1,020 mOsmol/kg for marine species. Quality after vitrification was evaluated by sperm motility, membrane integrity and when possible fertility. Post-thaw motility of sperm in marine fishes was higher (as high as 70%) than in freshwater fishes (as high as 20%). The percentage of membrane-intact sperm for marine fishes was ~20% except for southern flounder (11%). For freshwater fishes, the percentage of membrane-intact sperm for swordtail was low (<12%) compared to channel catfish (~50%). Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations (40 – 60%) required for vitrification. This research yielded the first successful vitrification of sperm in these fishes and production of offspring from vitrified sperm in channel catfish, green swordtail, and southern flounder. Sperm vitrification offers an alternative approach to conventional cryopreservation for conservation of valuable genetic lineages, such as endangered species, model strains used in research, and improved farmed strains. Furthermore, sperm vitrification could be used to transport cryopreserved sperm from the field to the laboratory to expand genetic resources available for germplasm repositories. This technique could be utilized to reconstitute genetic lines, and as a new option for conservation biology in imperiled aquatic species.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Cuevas Uribe, Rafael, "A general approach for vitrification of fish sperm" (2011). LSU Doctoral Dissertations. 3597.