Post-euthanasia cryopreservation of epididymal sperm is a common method to preserve and distribute valuable mouse models worldwide. However, the euthanasia method used prior to sperm collection must not adversely affect sperm quality. The most common method of euthanasia in mice is CO₂ asphyxiation, but its effect on sperm quality is largely unknown. The objective of this study was to determine the effects of CO₂ euthanasia on C57BL/6 mouse sperm post-recoveries and following freezing and thawing. First, post-mortem sperm concentration, progressive motility, curvilineal velocity (VCL), average path velocity (VAP), and progressive velocity (VSL) were analyzed for mice euthanized via either CD, high flow CO₂ (100%), or low flow CO₂ (30%) displacement/minute, respectively. Then, in-vitro fertilization and embryonic development rates were determined using frozen-thawed sperm from each euthanasia method. There were no differences in sperm concentrations, progressive motility, VAP or VCL for fresh or frozen-thawed sperm. Frozen-thawed sperm collected from the CD group had higher VCL values compared to sperm collected from the low flow group (P=0.039). There were no differences in fresh sperm, or between sperm collected from the CD vs high flow CO₂ or high flow vs low flow CO₂. There were no differences between groups for fertilization (P=0.452) and blastocyst development rates (P=0.298) from using frozen-thawed sperm collected between the groups. The results suggest that CO₂ euthanasia method can serve as an alternative to CD in the case of technical or personal reasons to obtain optimal quality mouse sperm for cryopreservation and comply with the animal and human welfare.

Post-euthanasia cryopreservation of epididymal sperm is a common method to preserve and distribute valuable mouse models worldwide. However, the euthanasia method used prior to sperm collection must not adversely affect sperm quality. The most common method of euthanasia in mice is CO₂ asphyxiation, but its effect on sperm quality is largely unknown. The objective of this study was to determine the effects of CO₂ euthanasia on C57BL/6 mouse sperm post-recoveries and following freezing and thawing. First, post-mortem sperm concentration, progressive motility, curvilineal velocity (VCL), average path velocity (VAP), and progressive velocity (VSL) were analyzed for mice euthanized via either CD, high flow CO₂ (100%), or low flow CO₂ (30%) displacement/minute, respectively. Then, in-vitro fertilization and embryonic development rates were determined using frozen-thawed sperm from each euthanasia method. There were no differences in sperm concentrations, progressive motility, VAP or VCL for fresh or frozen-thawed sperm. Frozen-thawed sperm collected from the CD group had higher VCL values compared to sperm collected from the low flow group (P=0.039). There were no differences in fresh sperm, or between sperm collected from the CD vs high flow CO₂ or high flow vs low flow CO₂. There were no differences between groups for fertilization (P=0.452) and blastocyst development rates (P=0.298) from using frozen-thawed sperm collected between the groups. The results suggest that CO₂ euthanasia method can serve as an alternative to CD in the case of technical or personal reasons to obtain optimal quality mouse sperm for cryopreservation and comply with the animal and human welfare.