Title page for ETD etd-04152005-100148


Type of Document Dissertation
Author Dong, Qiaoxiang
Author's Email Address qdong@agctr.lsu.edu
URN etd-04152005-100148
Title Comparative Studies of Sperm Cryopreservation of Diploid and Tetraploid Pacific Oysters
Degree Doctor of Philosophy (Ph.D.)
Department Renewable Natural Resources
Advisory Committee
Advisor Name Title
Terrence R. Tiersch Committee Chair
Jill A. Jenkins Committee Member
John E. Supan Committee Member
Robert P. Romaire Committee Member
Standish K. Allen Committee Member
Stanley P. Leibo Committee Member
Fred Sheldon Dean's Representative
Keywords
  • oyster sperm cryopreservation
Date of Defense 2005-03-17
Availability unrestricted
Abstract
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.

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