Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryo cryopreservation allows preservation of genetics from both male and female and is the fastest method to restore a breed. Embryo production in vivo is limited in equids. We recently established a technique of ovum pick up (OPU) in the donkey. Conditions of in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture of zygotes have been evaluated. Equine abattoir–derived oocytes were used as controls. Donkey cumulus-oocyte complexes (COCs) collected by OPU were matured in vitro in TCM199 with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. Forty-four percent were in metaphase 2 after 34 hours. In our conditions, IVM of donkey oocytes was slower than that of equine oocytes and the optimal duration for donkey oocytes IVM may be 34 hours. Oocytes we co-incubated with frozen-thawed donkey semen treated with procaine for 18 hours and cultured for 30 hours in a Dulbecco Modified Eagle Medium-F12-based medium. Only 15% of jennies oocytes contained 2 pronuclei after co-incubation, and none of them developed further after 48 hours after IVF. Treatment of donkey sperm with procaine may not be efficient for IVF. Some parthenogenetic activation occurred. In conclusion, we confirm that our conditions for OPU in jennies yielded high recovery rates that improved with operator experience. Maturation rates of 44% can be achieved using the IVM medium routinely used for equine oocytes in our lab. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.