n improved vitrification protocol for the fast and safe storage of mouse oocytes

Abstract Cryopreservation methods for archiving and distributing mouse strains mostly focus on freezing embryos or sperm. Although protocols for the cryopreservation of wild-type mouse oocytes are available, these methods have not been widely adopted in large biomedical research facilities. This is partly due to a lack of validation of the available methods on a large scale using a range of genetically modified oocytes. Furthermore, some of the existing methods report a relatively low rate of fertilization, requiring either the zona pellucida to be physically breached or the inclusion of cumulus cells to enable efficient fertilization, interventions which might be incompatible with maintaining hygiene barriers. Existing methods also often use cryovials rather than straws or slimline vitrification devices, which are more practical for storage and handling. Here, we present a robust vitrification protocol for large-scale oocyte cryopreservation, achieving high viability and fertilization rates comparable to fresh oocytes. We have extensively tested the protocol for in vitro fertilization of many genetically altered strains, using both genetically altered and wild-type C57BL/6J oocytes and sperm. Providing an archive of cryopreserved oocytes harboring genetically modified alleles separately from archives of cryopreserved sperm allows multiple allele combinations to be generated during the rederivation process. This reduces subsequent breeding steps and the need to maintain live stocks of mice. Furthermore, cryopreserving oocytes for later use enables them to be obtained from females at the optimal age, thereby reducing the number of mice required and providing greater scheduling flexibility for subsequent genetic modification and rederivation work.