Sperm preparation, in vitro fertilization (IVF) and in vitro culture of embryos (IVC)
Motile spermatozoa were selected by the Percoll gradient method. Briefly, frozen-thawed cattle semen at 35 °C was filtered by centrifugation on a Percoll discontinuous gradient (45–90%) at 1680 rpm for 15 min. To produce the 45% Percoll solution, 1 mL of capacitation-TALP medium was added to 1 mL of 90% Percoll. The sperm pellet was washed two times by the addition of 3 mL of capacitation-TALP medium and subsequent centrifugation at 1680 rpm for 5 min. Washed, motile spermatozoa were used for IVF. Spermatozoa (1–2 × 106 sperm/mL) were incubated with mature oocytes for 18 h in 50 µl microdrops of IVF-TALP medium covered with mineral oil (Nidacon, Cat. no. NO-100) in humidified atmosphere of 5% CO2 at 38.5 °C. After 18 h of co-incubation, cumulus cells were removed from presumptive zygotes. The zygotes were cultured in a two-step chemically defined culture media [reference] that was covered in mineral oil in an atmosphere of 5% O2, 5% CO2 and 90% N2 at 38.5 °C.