The aim of this proposal is to develop an algorithm using assisted reproductive techniques to reconstitute valuable genetic strains of mice from stored spermatozoa. The ability to store spermatozoa is urgently needed because of the exponentially increasing numbers of mutant and transgenic strains of mice being produced for the study of human diseases. The cost of preserving these strains using conventional breeding programs is so astronomical that the storage of strains will soon be curtailed. The algorithm is also needed to facilitate the rescue of strains of mice with defective sperm or whose males are incapable of breeding. A working algorithm can be identified using current assisted reproductive techniques: artificial insemination, in vitro fertilization, intracellular sperm injection, embryo culture and embryo transfer. The overall efficiency of any particular path in this algorithm is defined as the number of pups born x 100/number of oocytes exposed to sperm. The overall efficiencies of the paths using IVF and ICSI, employing cryopreserved sperm and freeze-dried sperm spermatozoa from five strains of mice commonly used in bioengineering, will be determined. The results will reveal the extent that the overall efficiencies are genetically determined. The results will also allow a determination of whether the newly described freeze-drying route of preserving sperm is a workable option. Research is also proposed that will systematically examine the dehydration of spermatozoa from a biophysical point of view. Attempts will be made to optimize the freeze-drying techniques. Studies will also investigate whether the alternative evaporative route of dehydration is more effective. Other studies will be concerned with improving the current methods of in vitro fertilization, intracytoplasmic sperm injection and embryo transfer in mice, the results of which will be incorporated in a final algorithm.