In Vitro Fertilization in Abhd2 Mutant Mice

Sperm from the cauda epididymides of 2-month-old C57Bl/6N male mice were allowed to swim out in Embryomax Human Tubal Fluid medium (HTF, Millipore, MR-070-D) and incubated for capacitation, 1 h at 37°C, 5% CO₂. To collect eggs from seven Abhd2^+/+, eight Abhd2^+/–, and seven Abhd2^–/– females, superovulation of 3.5- to 4-week-old mice was performed using i.p. injection of PMSG and hCG as described above. Thirteen hours after hCG injection, the females were euthanized, and the cumulus–oocyte complexes were collected from the oviduct ampulla in HTF medium and counted. IVF was performed as previously described (Navarrete et al., 2015 (link)) using 300,000 sperm/ml HTF medium. After fertilization, the zygotes were washed in KSOM + AA media, supplemented with 4 μl of BSA and divided into ∼15 zygotes/10 μl of KSOM/BSA for culture at 37°C, 5% CO₂. Three and a half days following IVF, the number of fertilized eggs was determined as the percentage of embryos that had reached morula or blastula stage.