Production of progeny mice by intracytoplasmic sperm injection of repeatedly frozen and thawed spermatozoa experimental technique.

Methods for generating genetically engineered mice have progressed, and the number of valuable mouse strains has increased rapidly, requiring methods for managing and maintaining these strains. Sperm cryopreservation and assisted-reproduction techniques, such as intracytoplasmic sperm injection (ICSI), can contribute greatly; however, the number of possible progeny is limited due to the finite number of cryopreserved preparations. The ability to refreeze and reuse sperm preparations would extend the utility of each cryopreserved sperm preparation. The purpose of this study was to develop a reproduction protocol involving ICSI that yielded live progeny after repeated freezing and thawing of a cryopreserved sperm preparation. We used mouse sperm subjected to repeat freezing and thawing in TYH medium for in vitro fertilization. Three inbred strains of laboratory mice--C57BL/6J, BALB/cA, and C3H/HeN--were reproduced by ICSI after reuse of a sperm preparation that had been frozen and thawed repeatedly. In particular, C57BL/6J progeny could be reproduced from spermatozoa frozen and thawed 10 times. From these results, we conclude that the reuse of cryopreserved spermatozoa can extend the opportunities for reproduction of progeny from cryopreserved sperm and can increase the utility of cryopreserved preparations as bioresources. Our results broaden management options regarding bioresource banking, particularly for mice.