Cryopreservation at -80 °C impacts sperm integrity and fertility in a mouse strain-dependent manner

We previously developed a simple method to cryopreserve and maintain B6N spermatozoa at -80 °C, instead of using liquid nitrogen (LN₂), for up to one year without detrimental effects on fertility. The goal of the present study was to test the efficacy of this method in six different mouse strains (129/SvImJCnrm; 129, C57BL/6JCnrm; B6J, C57BL/6NTacCnrm; B6N, BALB/cByJCnrm; BALB/c, Crl:CD1(ICR); CD-1, and FVBN/JCnrm; FVB). At different time points up to one year, we evaluated the integrity and the fertility of the spermatozoa cryopreserved and stored at -80 °C in comparison to the classical LN₂ method. After 1 year, no differences in the in vitro fertilisation (IVF) rate and in the number of dead spermatozoa were observed in 129, B6N, CD-1 and FVB strains. In contrast, spermatozoa from B6J and BALB/c cryopreserved at -80 °C showed a significant reduction in the IVF rate, an increased number of dead spermatozoa and increased morphological abnormalities after 3 months (BALB/c) and 6 months (B6J). In all strains, the total sperm motility decreased after 1 month at -80 °C and increased ultrastructural damage was found in the -80 °C group. The present results indicate that spermatozoa from 129, B6N, CD-1 and FVB can be cryopreserved and stored at -80 °C for at least one year without impacting their fertility. However, spermatozoa from B6J and BALB/c are the most sensitive to temperature. Therefore, cryopreservation and storage of 129, B6N, CD-1 and FVB strains at -80 °C for up to one year is feasible, thereby circumventing the use of LN₂.