Cryopreservation Of Salmonid Sperm

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.

Understanding the reproductive physiology and endocrinology of fishes is essential for captive maturation and seed production in the field of aquaculture. Studying the spermatology of fishes is a comparatively new focus in aquaculture, which has emerged as an important area of fish research over the past two decades. In this regard, the cryopreservation of fish gametes is a crucial aspect. Moreover, energetics studies of gametes have become essential, considering the loss of vigour in the spermatozoa after cryopreservation.The latest development in this context is the cryopreservation of spermatogonial stem cell, which is also covered in the book, along with detailed information on embryo cryopreservation in fishes and crustaceans. The role of cryopreservation in conservation programmes is another important aspect, one that will especially interest biologists.This book addresses central issues in fish gamete cryopreservation and breeding, while also reviewing the history of cryopreservation. Its most unique feature is the breadth of its coverage, from basic information on reproduction in fishes, to such advanced topics as embryo cryopreservation. Chiefly intended as a handy troubleshooting guide, the book represents a valuable resource for research students in related fields.

Numerous fishery researchers have attempted to preserve viable fish sperm, but only three investigations were successful in the fertilization of fresh eggs with frozen and thawed sperm: Blaxter (1953); Hoyle and Idler (1968); and Graybill and Horton (1969). Significant advances reported in this thesis were: the development of suitable life extenders for semen of steelhead trout (Salmo gairdneri), and coho (Oncorhynchus kisutch) and Chinook (0. tshawytscha) salmon; the selection of a life protector composed of 8.0% dimethyl. sulfoxide; the verification that the best rate of freezing semen was -30 C/mm, arid that the best method of thawing frozen sperm was in a 4 C water bath; and the determination that no equilibration time was necessary when fresh semen was diluted with extenders and protectors. Application of the above techniques led to the successful cryo-preservation of male gametes of steelhead trout and Chinook and coho salmon in liquid nitrogen ( -196 C). Frozen and thawed sperm was mixed with fresh eggs and resulted in maximum fertilizations of 59.1% for steelhead trout, 38.4% for spring Chinook salmon, and 79.2% for coho salmon. Details of the chemical constituents of the extenders and protectors tested are appended.

The large amount of information on fish reproduction available is not always readily accessible to all interested parties. Written to appeal to aquaculturalists, conservation managers, and scientific researchers, Methods in Reproductive Aquaculture provides an overview of available techniques and addresses ways to improve depleted stocks of endange