A new, simple and efficient liquid nitrogen free method to cryopreserve mouse spermatozoa at -80 °C

Abstract

The mouse is widely used for biomedical research and an increasing number of genetically altered models are currently generated, therefore centralized repositories are essentials to secure the important mouse strains that have been developed. We have previously reported that spermatozoa of wild type and mutant strains frozen using standard laboratory protocols can be transported in dry ice (-79 °C) for 7 days and safely stored in a -80 °C freezer for up to two years. The objective of this new study was to compare the effects of the freezing techniques using LN₂ or -80 °C freezer on fertility of frozen-thawed mouse spermatozoa. After thawing, sperm fertility was comparable (P > 0,05) between the LN₂ and the -80 °C samples for at least 1 year. Furthermore, we showed that it is possible to freeze and store mouse semen directly at -80 °C and eventually transfer it to LN₂ irrespective of storage time. This study is relevant because it shows for the first time that mouse spermatozoa can be efficiently frozen and stored at -80 °C with no use of liquid nitrogen for a long period of time. A new, simple, efficient and flexible, liquid nitrogen free, method was developed for freezing and maintaining spermatozoa of wild type and mutant C57BL/6N lines. Lines on this genetic background are used in collaborative research infrastructures for systematic phenotyping, e.g. the International Mouse Phenotyping Consortium (IMPC) and therefore largely cryopreserved in repositories like EMMA/Infrafrontier. The importance of this finding will be especially useful for small laboratories with no or limited access to liquid nitrogen and for laboratories generating many mouse mutant lines by CRISPR/Cas9 who do not want to saturate the limited space of a LN₂ tank, using a more accessible -80 °C freezer. This study underlines, once more, that mouse spermatozoa are very resistant and can be frozen, transported, shared and stored at -80 °C for a long time without a significant loss of viability. This new approach simplifies the freezing process and facilitates the long term storage of mouse spermatozoa at -80 °C, always allowing the transfer to LN₂ for indefinite storage without noticeable detrimental effects.

Keywords: -80 °C freezer; Biorepository; Cryopreservation; In vitro fertilization (IVF); Mouse spermatozoa.