Euthanasia via CO₂ inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development

Abstract

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO2 (H CO2), low flow CO2 (L CO2), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F‐actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two‐cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO2. The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO2 group compared with CD and H CO2 groups (p < 0.001). These data collectively suggest that CO2 associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.

Humanely Ending the Life of Animals: Research Priorities to Identify Alternatives to Carbon Dioxide

“…Conceptually, CAS techniques are attractive because they allow stunning multiple animals at a time without restraint and with minimal direct interaction, which is aversive to mice [129,130]. They also kill animals without gross anatomical damage (unlike some physical methods), which may be important for some scientific purposes; however, physiological disturbances may reduce tissue viability and yields [131]. The majority of CAS alternatives have focused on inert gases which, unlike CO 2 , do not have narcotic properties at normobaric conditions (the notable exception being xenon [132]).…”

Generation of mouse hippocampal brain organoids from primary embryonic neural stem cells

“… CRITICAL: cervical dislocation must be performed by trained and qualified personnel only. Cervical dislocation without prior anesthesia with CO 2 or isofluorane inhalation ensures high yield of healthy, viable embryos 7 , 8 however a fast CO 2 narcosis or isoflurane induction before the cervical dislocation can be performed. Clean the mouse skin with Et-OH 70% and quickly harvest the embryos.…”

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR

“…Briefly, 17-day pregnant BALB/cAJc1 mice (CLEA Japan) were euthanized by cervical dislocation, and the uterus was isolated. Euthanasia by cervical dislocation has been widely used in animal experiment, since it can cause a fast and painless death and avoid damage to the fetus [27][28][29][30][31][32][33][34]. Furthermore, it does not chemically contaminate tissues.…”