In vitro maturation, in vitro fertilization, and embryo development will be according to procedures widely published in the literature and frequently used in the laboratory. In vitro maturation and fertilization will be conducted in 400 μl drops at 38.5°C and 5% CO2 in a humidified atmosphere, while embryo culture will be carried out in 100 μl drops under mineral oil of KSOM– 0.4% BSA medium (EmbryoMax, Chemicom International, USA) at 38.5°C and 5% CO2, 5% O2 and 90% N2, in a humidified atmosphere. Bovine IVM matured oocytes and sperm will be co-incubated for 18–20 h in IVF-TL- ( a supplement with BSA (EFAF), sodium pyruvate, gentamicin, and heparin to make IVF-TALP). (Parrish et al. 1986, Felmer et al. 2011). Final IVF-TALP will contain, 2 μg heparin and 1 × 106 Percoll-separated frozen-thawed sperm/mL. Presumptive zygotes will be cultured in different culture systems.
Rafael
Fissore
