2.4. In vitro culture (IVC)
Embryo culture took place in synthetic oviduct fluid medium (SOF) (Holm et al., 1999) supplemented
with 13mM sodium pyruvate (Sigma–Aldrich) and 5% FCS (Gibco) under mineral oil
in a humidified atmosphere of 5% CO2 at 39 ◦C. Ten to 14 h post insemination (hpi), presumptive
zygotes were denuded of surrounding cumulus cells by repeated pipetting and subsequently washed
three times in SOF medium. Afterwards, the plates with oocytes were placed in a small plastic bag
(25cm×15 cm), where a gaseous mixture was injected (5% O2, 5% CO2 and 90% N2) through an 18-
gauge needle. Cleavage was recorded 36 hpi. Blastocyst formation rates were recorded on day 8 post
insemination while hatching rateswere noted on day 10 (only for Experiment 2). Culturemediumwas
replaced 48 and 120 hpi.