Zygote collection

C57BL/6J (JAX Stock # 000664) donor female mice (age 3 weeks) were superovulated to maximize embryo yield. Each donor female received five International Units (IU) of Pregnant Mare Serum Gonadotropin (PMSG, ProSpec HOR-272) intraperitoneally (IP), followed 47 hours later by 5 IU of human chorionic gonadotropin (hCG, ProSpec HOR-250), IP. Immediately post-administration of hCG, the female was mated with a single C57BL/6J stud male and was checked 22 hours later for the presence of a copulation plug. Females displaying a copulation plug were euthanized and the oviducts excised and placed into M2 medium. Prior to clutch collection the oviducts were placed in M2 medium containing hyaluronidase (Sigma H3506, 0.3 mg/mL). The oocyte clutch was removed by mechanically lysing the ampulla and the clutch was allowed to incubate in the hyaluronidase-containing M2 medium until the cumulus mass had disintegrated to the point of exposing the oocytes/prospective zygotes. The oocytes/prospective zygotes were then transferred through several washes of fresh M2 medium and then, through the process of visual grading, individual identified zygotes were separated and transferred to microdrops of K-RCVL (COOK K-RVCL) medium that were equilibrated under mineral oil (Sigma M8410) for 24 hours in a COOK MINC benchtop incubator (37°C, 5%CO₂/5%O₂/N₂).