Porcine Oocyte Maturation and Embryo Collection

In vitro maturation (IVM) was performed according to established methods [26] (link). Briefly, porcine ovaries were collected from a local slaughterhouse and kept in saline at 32–37°C. Antral follicles (3–5 mm in diameter) were aspirated with an 18-gauge needle. Aspirated oocytes with an evenly granulated cytoplasm and three uniform layers of compact cumulus cells were selected and cultured in four-well plates containing 500 µl of TCM199 (Gibco) based medium for 42 h. At the end of IVM, the cumulus cells of cumulus oocyte complexes (COCs) were removed by vortexing for 2 m in TCM199 based medium supplemented with 0.5% hyaluronidase (Sigma).
Oocytes at the germinal vesicle (GV) and MII stages and preimplantation embryos were collected. For preimplantation embryos, one-cell, two-cell, four-cell, 8-cell, morula and blastocyst stage embryos were collected at 6 h, 24 h, 48 h, 72 h, 120 h and 168 h post-activation, respectively. To exclude the possible contamination of remaining cumulus cells or sperm, the zona pellucida was removed by treatment with warm Tyrode's acidic solution for 20 sec.