1. Sperm is either thawed from a cryopreserved stock or extracted fresh from the cauda epipidymis of the male mouse and incubated for 30 min. at 37 °C in TYH medium.
2. Cumuli containing oocytes are transferred to a 90 µL HTF medium drop on a petri dish, covered by mineral oil (NidOil).
3. 10 µl sperm suspension are added to the HTF drop and the IVF dish is incubated at 37 °C for 4-5 hours. Afterwards, fertilization is assessed by second polar body formation.
4. Sperm thawing and IVF procedures as well as TYH and HTF media preparation are performed according to the protocols described at
https://www.infrafrontier.eu/knowledgebase/protocols/cryopreservation-protocols
5. Following successful IVF, the zygotes are supplemented with fresh KSOM Embryomax media everyday until E5.5