Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL^-1 hyaluronan, 0.6 mM cysteine, 10 ng mL^-1 epidermal growth factor (EGF), 0.1 U mL^-1 porcine LH and FSH, and 1 × Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 × 10⁵ sperm mL^-1) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mM bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1 % v. 14.3 %). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.