Maturation and fertilization of African lion (Panthera leo) oocytes after vitrification

The African lion is an excellent model species for the highly endangered Asiatic lion. African lions reproduce well in zoos, leading to the fact that occasionally ovaries and testis are available for in-vitro experiments. We previously performed in-vitro maturation (IVM) and fertilization of lion oocytes and were able to produce advanced embryos after intracytoplasmic sperm injection (ICSI) with cryopreserved sperm. Here we examined whether our in-vitro method is also applicable after vitrification of immature oocytes. Oocytes of four lionesses (5-7 years old) were obtained after euthanasia and immediately processed on site. Half of the oocytes (n = 60) were subjected to IVM for a total of 32-34 h at 39 degrees C, 5% CO2 and humidified air atmosphere. The second group (59 oocytes) was vitrified instantly using the Cryotop method. Following 6 days of storage in liquid nitrogen, oocytes were warmed and subjected to IVM as well. Mature oocytes of both groups were fertilized with frozen-thawed African lion sperm using ICSI. Maturation rate was 55% and 49.2% for the control and vitrified group, respectively. In the control group, three oocytes cleaved and another three were arrested at the pronuclei stage. Due to the low fertilization result, a sperm sample of another male was used for the vitrified group. Of the vitrified oocytes 7 cleaved and 9 more oocytes stopped at pronuclei stage. All embryos of the vitrified group did not develop beyond 4 cell stage. This is the first time that African lion in-vitro-derived embryos have been produced following oocyte vitrification.