Chronology of in vitro maturation of donkey (Equus asinus) oocytes collected by ovum pick up

Most wild donkey breeds are currently endangered, such as the Asiatic and the African wild ass. Moreover, many domestic donkey breeds are vulnerable to extinction, such as the Martina Franca donkey in Italy, the Zamorano-Leonés donkey in Spain and the Normand donkey in France. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. However, embryo production in vivo is limited in equids. In vitro production of embryos would allow the production of several embryos per cycle, but no technique has yet been developed in the donkey for in vitro production of embryos. In that context, ovum pick up (OPU) would come as a very helpful tool both for embryo production and research purposes. Our objective was to develop OPU in jennies and to analyze the chronology of in vitro maturation of donkey oocytes.
Donkey cumulus-oocyte complexes (COCs) were collected by OPU from 5 to 25mm follicles in adult cyclic jennies. During OPU, jennies were injected detomidine, butorphanol, and butylscopolamine for sedation, analgesia and antispasmodia. COCs were in vitro matured in TCM199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34 or 38 hours in groups of 10 to 30 in an atmosphere of 5% CO2 in air at 38.5°C.
Five OPU sessions were performed, with 3 to 6 females per session. We collected a total of 92 COCs out of 193 follicles (48%) with an average of 4.2 COCs per jenny. The recovery rate per follicle increased along the OPU sessions: 34%, 43%, 52%, 47% and 56%, probably due to the increasing skill of the staff. Despite a very large experience with OPU in horses, all staff members required some time to adjust to the specificities of the jenny vs mare, in particular to the differences in holding the ovary. All COCs were expanded after over 24 hours in vitro maturation (IVM). At collection, jennies COCs contained a germinal vesicle. After 24 hours IVM, COCs contained either a germinal vesicle or condensed chromatin. Metaphase 1 oocytes were observed after 30 hours IVM and 45% were in metaphase 2 after 34 hours IVM. Half of the COCs were degenerating after 38 hours IVM.
In conclusion, we established for the first time conditions for ovum pick up in jennies and the chronology of in vitro maturation of donkey oocytes. We showed that in vitro maturation of donkey oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture while most equine oocytes reach metaphase 2 after 30 hours in culture.