Embryomax ksom medium 1x

GOs were harvested by sequential digestion of P12 ovaries in 1 mg/ml collagenase (034–10533, Wako Pure Chemical), 0.25% trypsin-EDTA (35555–54, Nacalai Tesque), and 0.5% protease type XIV (P5147, Sigma-Aldrich). FGOs were harvested from adult ovaries (10 to 12 weeks old) by needle puncture and pooled in M2 medium (Sigma). To obtain MII oocytes, superovulation was performed by injecting females at 4 to 5 weeks old sequentially with inhibin antiserum (CARD HyperOva, Kyudo) and human chorionic gonadotropin. Cumulus-oocyte complexes were collected from the ampulla of oviducts and fertilized with C57BL/6J sperm in modified human tubal fluid (CARD mHTF, Kyudo). Cumulus cells were removed by capillary washing, and fertilized eggs were cultured in EmbryoMax KSOM Medium (1X) w/ 1/2 Amino Acids (Merck Millipore) at 37°C and 5% CO₂. After overnight culture, two-cell embryos were transferred into the oviducts of pseudo-pregnant ICR females.

Before obtaining MII oocytes, inhibin antiserum (CARD HyperOva, Kyudo) and human chorionic gonadotropin were sequentially injected into 11–18-week-old females. Cumulus-oocyte complexes were collected from the ampulla of oviducts and fertilized with JF1 spermatozoa in modified human tubal fluid (CARD mHTF, Kyudo). Cumulus cells were removed by capillary washing, and fertilized eggs were cultured in EmbryoMax KSOM Medium (1X) w/ 1/2 Amino Acids (Merck Millipore) at 37 °C and 5% CO₂. After overnight culture, two-cell embryos were transferred into the oviducts of pseudo-pregnant ICR females. Embryos and placentas were dissected from the uteri of the pregnant females on embryonic day 9.5.