Plan apochromat 20 na 0

Time-lapse observations in bovine were performed on a Zeiss LSM700 confocal microscope (MIMA2 platform; INRA). The inverted microscope AxioObserver Z1 is coupled with an incubation system including a heating insert P stage, a XL incubator chamber, and an incubator S for delivering of CO₂. All insert and incubators are regulated by specific controllers (TempModule S, Heating Unit XL S, and CO₂ Module S) purchased from Carl Zeiss, Inc. For time-lapse observations, embryos (maximum of six) were cultured in 20 μl drop of SOF medium in IBIDI dish with glass bottom and placed on the inverted microscope with the incubator chamber at 38.5°C and 5% CO₂. Bright field and fluorescence images within different focal planes with 7 μm intervals were recorded, with Zeiss Zen software, every 2 h using a Plan-Apochromat 20× NA 0.8. To improve the signal detection and to decrease toxicity by irradiation, we used a 2.5 Airy unit pinhole size (scaling Z = 7.2 μm) and laser power equal to 0.2% of maximum (laser at 488 nm of 10mW, laser at 555 nm of 1mW). Embryos were followed until the 8- to 16-cell stage and then either returned to culture in the same IBIDI dish up to the blastocyst stage or left under the microscope up to the blastocyst stage with 6 h intervals between each acquisition.