2.2. In Vitro Embryo Culture in Microdrops

In this study, commercially available, frozen one-cell murine embryos, in vivo derived from the B6C3F1 × B6D2F1 strain, were purchased from Embryo-Tech (Haverhill, MA, USA). According to the manufacturer’s protocol, straws containing frozen murine presumptive zygotes were exposed to room temperature for 3 min and plunged into a 37 °C water bath for 1 min. The straws were cut at the seal and the plug bisected before pushing the contents into a 40 μL drop of M2 medium (Merck Millipore, Watford, UK) in a 60 mm IVF hydrophobic culture dish. Finally, embryos were rinsed twice in pre-warmed KSOM medium and allowed to rehydrate for 10 min in the final rinse droplet at 37 °C under 5% CO₂, 5% O₂ in a humidified nitrogen atmosphere in a benchtop MINC™ incubator. Finally, 10 embryos were cultured in 40 µL culture microdrops in 35 mm hydrophobic IVF certified dishes (Nunc™, Thermo Fisher Scientific, Warrington, UK), covered with 5 mL of BioUltra mineral oil (Sigma Aldrich). A total of 360 embryos were used for this study.