CRYOPRESERVATION OF SPERM

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Storage of spermatozoa is a logical approach for preserving transgenic mouse genes as insurance against disaster to animal colonies, prevention of genetic drift and maintenance of unused lines until needed. Current cryopreservation techniques for mouse sperm are inefficient and result in poor sperm survival and low fertility. The objectives of this research are to: 1) develop laboratory assays which evaluate multiple attributes of individual mouse sperm in a population to provide an estimate of fertilizing potential; (necessary to improve the efficiency of sperm cryopreservation), 2) develop effective methodologies to cryopreserve mouse sperm, and 3) develop simple techniques, such as artificial insemination, using cryopreserved mouse sperm to produce viable offspring. To accomplish these goals, experiments are designed to develop analyses for mouse sperm based on flow cytometry, fluorometry, computerized motion analysis, and in vitro fertilization; data will be compared to determine fertilizing potential of sperm from three strains of mice. Subsequent experiments are designed to improve cryopreservation techniques by investigating additives for freezing media, using additives selected because of anticipated protection of sperm plasma or acrosomal membranes. In addition, liposomes and lipophilic compounds will be used to modify composition of sperm membranes before freezing, thereby facilitating cell cryo-survival. Finally, we will test feasibility of obtaining mouse offspring without killing female mice or using sophisticated laboratory equipment. These experiments will test the feasibility of collecting, freezing and using frozen-thawed sperm in artificial insemination programs rather than costly and technically demanding in vitro fertilization programs to propagate mice. By incorporating use of several mouse strains into the experiments of this project, techniques developed should not be strain dependent, and results should yield efficient protocol(s) for the collection, cryopreservation and utilization of frozen-thawed mouse sperm, permitting practical storage of sperm from diverse transgenic lines of mice.