Effects of CO₂ Euthanasia of C57BL/6 Mice on Sperm Motility, In Vitro Fertilization, and Embryonic Developmental Competence

Cryopreservation of epididymal sperm collected after euthanasia 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 the quality of sperm collected postmortem is largely unknown. The objective of this study was to determine the effects of CO₂ euthanasia of C57BL/6 mice on both freshly recovered sperm and sperm subjected to freezing and thawing. First, sperm concentration, progressive motility, curvilineal velocity (VCL), average path velocity (VAP), and progressive velocity (VSL) were analyzed for mice euthanized by cervical dislocation (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. Neither fresh nor frozen-thawed sperm showed significant differences in sperm concentration, progressive motility, VAP, or VCL when compared to CD and CO₂ groups. However, frozen-thawed sperm collected from CD mice had higher VCL values than did those collected from the low flow mice (P = 0.039). VCL was not different in fresh or frozen-thawed sperm collected after mouse euthanasia by CD as compared with high flow CO₂ or by high flow as compared with low flow CO₂. Frozen-thawed sperm showed no differences among the 3 euthanasia groups for fertilization (P = 0.452) or blastocyst development rates (P = 0.298). The results indicate that CO₂ euthanasia can be used as an alternative to CD to obtain optimal quality mouse sperm for cryopreservation while remaining compliant with welfare requirements.