In Vitro Maturation and Fertilization of Donkey Oocytes

Many wild and domestic donkey breeds are now at risk of extinction, therefore there is an urgent need for genetic conservation of these breeds. The development of in vitro conditions that support oocyte maturation, fertilization, and embryo development may improve in vitro production of donkey embryos. This work was designed to identify the optimal condition for in vitro production of donkey embryos. Two experiments were conducted. In experiment 1, grade 1 and 2 cumulus-oocyte complexes were subjected to in vitro maturation in the following media: tissue culture medium 199 (TCM199), Christopher Rozenkrans 1aa medium (CR1aa), Christopher Rozenkrans 2aa medium (CR2aa), potassium simplex optimized medium (KSOMaa), centrifuged follicular fluid (CFF), or non-CFF from preovulatory follicles. In experiment 2, in vitro–matured donkey oocytes were subjected to in vitro fertilization (IVF) in Fert-TALP medium supplemented with 0%, 25%, 50%, 75%, or 100% CFF. After fertilization, the cleavage rates and embryo development were assessed across groups. The results of experiment 1 revealed that non-CFF and KSOMaa significantly increase cumulus cell expansion. The percentage of oocytes reaching the metaphase II stage nuclear maturation was significantly higher for donkey oocytes matured in non-CFF. Also, the percentage of degenerated oocytes was lower for donkey oocytes matured in vitro in non-CFF compared with those in TCM199, CR1aa, CR2aa, KSOMaa, or CFF. In experiment 2, IVF of in vitro–matured donkey oocytes using 100% CFF as IVF medium produced a significantly higher fertilization rate with respect to the number of fertilized oocytes extruding the second polar body. In conclusion, non-CFF from preovulatory follicles gave the highest cumulus cell expansion and nuclear maturation rates for in vitro–matured donkey oocytes. Hundred percent CFF was the best medium for IVF of matured donkey oocytes; however, cleavage rate and embryo development are still very low and need further investigations.