This dissertation addressed comparative studies of sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas, with an emphasis on the development of standardized and optimized protocols. This includes comparative ultrastructural differences between sperm from diploid and tetraploid oysters, methods for the rapid estimation of sperm concentration, optimization of cryopreservation, and evaluation of the mechanisms for sperm agglutination (formation of clumps or elongated "noodles") in thawed samples. Currently, cryopreserved sperm has not been commercialized in any aquatic species, and standardization and optimization could greatly benefit the potential commercialization of its use. In oysters specifically, cryopreserved sperm from tetraploids would facilitate the production of all-triploid seedstocks.
In this study, sperm from tetraploid oysters were 25% larger in linear dimensions (lengths and widths), and 53% had 5 mitochondria compared to 4 in diploids. Spectrophotometric methods for rapid estimation of sperm concentration were developed and validated. The effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization. Combination of the cryoprotectants polyethylene glycol (PEG; formula weight of 200) and methanol (for sperm from diploids) or PEG and propylene glycol (for sperm from tetraploids) were effective in retaining post-thaw motility only when PEG was at low concentrations (2-6%). Such effectiveness was especially manifested with sperm from tetraploids, for example, post-thaw motility as high as 50% was obtained with combined cryoprotectant. Sperm of tetraploid Pacific oysters were more susceptible to damage from cryopreservation procedures than were those of diploids, and male-to-male variation was significant for sperm from diploid and tetraploid oysters. Sperm agglutination was mainly due to the lack of sufficient cryoprotectant for specific sperm concentrations. These findings demonstrated the importance of standardization in sperm concentration and other procedures during cryopreservation. In addition, the systematic optimization of cryopreservation protocols involving interactions of multiple factors, recognition of male-to-male variation, and development of assays for sperm tolerance prior to freezing are all approaches important for the future potential commercialization of cryopreserved sperm in Pacific oysters and for other aquatic species as well.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-04152005-100148 |
| Date | 18 April 2005 |
| Creators | Dong, Qiaoxiang |
| Contributors | Standish K. Allen, Terrence R. Tiersch, Stanley P. Leibo, Robert P. Romaire, John E. Supan, Jill A. Jenkins, Fred Sheldon |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-04152005-100148/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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