Nitrogen vapor immersion: An accessible alternative for honey bee (Apis mellifera L.) semen cryopreservation

Semen cryopreservation is a valuable conservation tool and is often used in livestock species to accelerate artificial selection of desirable traits. Recently, semen cryopreservation has been successfully introduced to honey bees, bolstering trait selection for breeders and aiding conservation efforts for threatened bee populations. Current cryopreservation methods use slow-programmable freezing to achieve long-term storage of honey bee germplasm. However, the equipment necessary for this method is costly and time consuming to use, making it less accessible to breeders and researchers. We tested two cost and time efficient alternatives to slow-programmable freezing, vitrification and vapor immersion using two freezing devices, the CryoLock and microdialysis tube. Semen was preserved in either 20, 40, or 60% dimethyl sulfoxide (Me₂SO). The post-thaw sperm viability (% living sperm) and subjective motility (0-5 scale) of these techniques were compared to those of slow-programmable frozen semen and non-frozen controls. Semen frozen in microdialysis tubes produced higher motility and sperm viability than semen frozen with the CryoLock device. The same trend was observed between vapor immersion and vitrification, with vapor immersion proving superior. Vapor immersed semen dialyzed with 20% Me₂SO produced statistically similar sperm motility (4 ± 0.41) and viability (73.51% ± 5.56%) to slow-programmable freezing (4.25 ± 0.25, 80.61% ± 4.20%) and the non-frozen control (4.5 ± 0.28, 93.39% ± 0.90%). Optimization of the dialysis process and freezing rate may further increase the post-thaw sperm quality. Nonetheless, these results show promise for an effective replacement to slow-programmable freezing that maintains high sperm quality while increasing accessibility.