The major goal of this application is to develop a simple and efficient method for preservation of valuable mouse genomes by using ICSI with preserved ejaculated spermatozoa. The hypothesis of this application is that ejaculated spermatozoa can be repeatedly retrieved from a male, preserved, and then used for ICSI yielding live offspring with high efficiency. The collection of spermatozoa from mice is limited to techniques that involve euthanasia and result in a death of the male, precluding him from further breeding. A method for repeatedly obtaining ejaculates allows for keeping the male alive and able to breed. In the Specific Aim 1 we will test if ejaculated preserved spermatozoa injected into the oocytes by ICSI allow obtaining live offspring with high efficiency. We will repeatedly obtain spermatozoa from several C57BL/6 males and preserve them by conventional eryopreservation and by simple freezing without cryoprotection. We will use ICSI to inject these spermatozoa into the oocytes from C57BL/6 females to produce embryos. We will evaluate the effects of these methods on sperm DNA integrity by analysis of patemal chromosomes in the zygotes and perform embryo transfer and produce live offspring. Finally, we will apply the proposed technology to one mutant mouse strain (azh - abnormal spermatozoon head-shape) with fertility problems to demonstrate its usefulness in preservation of a medically important mouse strain. In the Specific Aim 2 we will evaluate the mechanism of chromosome degradation in ejaculated preserved mouse spermatozoa. We will explore the biological significance of observed phenomenon that spermatozoa exposed to uterine content are more susceptible to DNA damage. We will establish if the unknown factor present in the uterus after mating affects live spermatozoa or rather interferes with the conditions of unprotected sperm freezing. We will also analyze independently the effects of two compounds of the uterine content: uterine fluid and seminal vesicle fluid. The significance of this proposal is that it will vigorously test the novel approach of using ICSI with preserved ejaculated spermatozoa to maintain valuable mouse genomes. This method will allow increasing the overall efficiency of reconstituting mouse strains and will accelerate sharing of given genetic material (i.e. a novel mutation) within the scientific community. Our expectations are that the results of this application will allow us to recommend ICSI with preserved ejaculated spermatozoa as a simple and efficient method for preservation of valuable mouse genomes.