The number of genetically engineered strains of mice is increasing at an exponential rate, creating a critical need for improved, cost-effective methods to store and archive these mouse strains. Use of cryopreserved spermatozoa would provide such a method. However, although mouse spermatozoa were """"""""successfully"""""""" frozen in 1990, the fundamental cryobiological factors that affect the ability of spermatozoa to participate in fertilization after freezing-thawing remain largely unknown. Current methods used to cryopreserve murine spermatozoa result in less than about 5% to 10% cryosurvival. This causes a reduction in efficiency (more sperm used per insemination) and/or efficacy (suboptimal pregnancy rates). The thesis of this application is that an understanding of the fundamental data with murine sperm have provided basic information regarding permeability and osmotic and mechanical properties of these cells. Knowledge of the water and cryoprotectant permeability is needed to predict the optimal values for major steps involved in cryopreservation. From the cryoprotectant permeability coefficient, the optimum procedure for adding and removing the cryoprotectant can be determined. Knowledge of cell water permeability and its temperature coefficient allows determination of the cooling rate that is low enough to preclude lethal intracellular freezing. Knowledge of mechanical sensitivity is required to take steps to circumvent it. The experimental approach proposed is to: a) perform experiments to determine effects of extender components on osmotic tolerance limits of murine sperm; b) use the permeability values of mouse sperm to water and various cryoprotectant solutes, to predict optimum conditions for adding and removing cryoprotectants and to optimize the cooling and thawing rate; c) develop an optimal cryopreservation procedure based on these fundamental biophysical properties; and d) experimentally test these predictions through in vitro fertilization and embryo transfer. Information gained from these experiments should aid in defining and minimizing loss of sperm function after cryopreservation, and provide a foundation for improvement in breeding efficiency when using frozen semen.
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