Graduate Thesis Or Dissertation
 

Cryo-preservation of viable fish sperm

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  • Fishery researchers have attempted to preserve viable fish sperm for extended periods of time. Only limited success has been achieved with storage of spermatozoa from salmonids. The principal accomplishments of this investigation during the fiscal year 1966-1967 were the development of standard methods for the collection and evaluation of fish sperm, the development of an extender and life protector of spermatozoa, and the development of a freezing and thawing procedure for the live preservation of fish sperm at temperatures of liquid nitrogen (-196 C). Collection of fish sperm was accomplished by means of a suction device with which semen was withdrawn from the genital pore of a fish into a test tube. A modification of the conventional hemocytometric technique was used to determine the concentration of sperm in fish semen. The use of vital stains to differentiate between live and dead sperm cells was investigated and abandoned due to inconsistent results. A subjective method of estimating the viability and relative motility of spermatozoa was established. Experiments were conducted to prolong the viability of sperm cells in the unfrozen state. The deleterious effect of water on prolonging the viability of fish sperm was confirmed. I found that a high percentage of sperm cells remained viable for at least three days if they were refrigerated under aerobic conditions and kept from contact with water. Increased dilution of fish semen with extenders reduced the viability of sperm cells. The freezing point depression of solutions isotonic to fish spermatozoa was found to be -0.51 C. My evidence suggests that the key to maintaining viable fish sperm at 5 C is to provide substances in the extender which can be aerobically metabolized and stored as latent energy by the spermatozoa. Experiments were conducted to prolong the viability of fish spermatozoa in the frozen state. Dimethyl sulfoxide (DMS0) gave the best results of the life protectors studied. Equilibration of the sperm cells in increasing concentrations of a life protector may have increased the viability of the spermatozoa. During several experiments, the optimum concentration of protector increased as the rate of freezing increased. The best survival of viable sperm was obtained in samples that were frozen in Solution 48 in combination with DMSO as a protector. Thawing the ampoules of semen in a 4 C water bath yielded the highest recovery of viable spermatozoa. Alevins were produced from eggs fertilized with cryogenically preserved fish sperm. These alevins appeared to be as normal as young fish produced from unfrozen spermatozoa. The fertility rate was low (0-18%), but to my knowledge this is the first successful attempt to fertilize salmonid eggs with cryo-preserved spermatozoa. The findings in this study warrant further investigation.
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