Use of frozen human semen is now essential for artificial insemination by donor programs; as this permits the storage of semen until each donor is retested for the human immunosuppressive virus after sufficient time has elapsed for seroconversion. """"""""Self- preservation"""""""" programs for men who undergo vasectomy or who may be expected to experience iatrogenic sterility secondarily (e.g. , as a result of chemotherapy) may also benefit from sperm banking. Current methods used to cryopreserve human semen result in only about 50% cryosurvival which causes a reduction in efficiency and/or efficacy. In addition, sperm from a number of species with agricultural and/or research importance, such as the boar and the mouse, have very low survival using current methods of cryopreservation. The thesis of this proposal is that an understanding of the fundamental cryobiology of mammalian sperm will permit the development of optimal procedures for cryopreservation. Knowledge of the permeability of a cell to water and to cryoprotectants can be powerful tools in predicting the likely optimum values for the major steps involved in freezing. From the value of the permeability coefficient for the cryoprotectant one can compute the optimum procedure for adding and removing the cryoprotectant without osmotic shock. Knowledge of the permeability of the cell to water and its temperature coefficient allows one to predict the cooling rate likely to be low enough to preclude lethal intracellular freezing. The experimental approach proposed is to (a) perform experiments necessary to determine physical parameters of sperm cells (permeability to water and to cryoprotectants, nucleation temperature at which ice forms, critical tonicity) (b) use these parameters to theoretically determine optimum conditions for adding and removing cryoprotectant and to optimize cooling rate and thawing rate (c) experimentally test these predictions (d) evaluate the interactions between cooling rates and warming rates (e) evaluate the role of unfrozen fractions and of cell shrinkage (f) develop an optimum dilution procedure and (g) develop an optimum cryopreservation procedure based on a thorough understanding of the interactions among cryoprotectant concentration, cooling rate, warming rate, and dilution procedure. Information derived from these experiments should aid in defining and minimizing the nature of cryopreservation injury, characterize and minimize the loss of sperm function after cryopreservation and provide a foundation for improvement in clinical outcome when using frozen semen.
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