Cryopreservation of Pacific salmon and steelhead trout sperm Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/b8515r484

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  • Successful fertilization of fresh steelhead trout, pink salmon, kokanee, chum salmon, fall chinook salmon, and coho salmon eggs with cryopreserved sperm is reported in this thesis. A mean percent fertilization greater than 50 percent was achieved for all species tested except chum salmon. Differences in sperm viability from donor males was observed; sources of variability probably were degree of sexual maturity and inherent physical-chemical properties of semen. Urine contamination of sperm samples was not responsible for male variability. In the cryopreservation of salmonid sperm, dilution ratios of 1:4 and 1:9 were acceptable; and when dimethyl sulfoxide was utilized as the solute moderator, equilibration was unnecessary. Extenders were developed for each species, with the most significant advance being development of a simplified diluent containing only NaCl, NaHCO₃, and lecithin. The preferred life protector was dimethyl sulfoxide, and protection was accomplished at levels between 7.5 and 12.0% (v/v) depending on the species tested. The preferred freezing procedure consisted of placing an ampoule (unsealed) of diluted semen in the vapor of liquid nitrogen (LN₂) for at least 1 mm prior to immersion in LN₂ for storage. Storage times approaching one month did not affect sperm viability as determined by mean percent fertilization of fresh ova; longer holding times appear practicable. The thawing procedure was determined to be critical in terms of sperm survival after freezing. The optimal technique consisted of filling the neck of an unsealed ampoule of frozen sperm with 4-10 C water, and then swirling the ampoule in a 50-60 C bath for 8 sec. When water was added to the ampoule of frozen sperm during thawing, cells equilibrated, thaw rate increased, agglutination decreased, and mean percent fertilization increased. Recommendations for a cryogenic procedure suitable for salmonids were made; potential problems also were identified. Finally, one specific recommendation was proposed which involved production of normally and cryogenically produced embryos which would be monitored throughout their reproductive cycle in an attempt to determine the biological selectivity of the cryogenic procedure.
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