Department of Reproduction Biology, Leibniz-Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, 10315, Berlin, Germany.
Givskud Zoo - Zootopia, Løveparkvej 3, Givskud, DK-7323, Give, Denmark.
Cryobiology. 2021 Feb;98:146-151. doi: 10.1016/j.cryobiol.2020.11.011. Epub 2020 Nov 26.
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 °C, 5% CO 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.
非洲狮是极度濒危的亚洲狮的极佳模式物种。非洲狮在动物园中繁殖良好，导致偶尔有卵巢和睾丸可用于体外实验。我们之前已经对狮子卵母细胞进行了体外成熟（IVM）和受精，并能够使用冷冻保存的精子通过胞质内精子注射（ICSI）产生高级胚胎。在这里，我们研究了在不成熟卵母细胞玻璃化后，我们的体外方法是否也适用。在安乐死后立即在现场获得了四只母狮（5-7 岁）的卵母细胞。一半的卵母细胞（n=60）进行了总共 32-34 小时的 IVM，温度为 39°C，5%CO 和加湿空气环境。第二组（59 个卵母细胞）立即使用 Cryotop 方法进行玻璃化。在液氮中储存 6 天后，解冻并进行 IVM。两组的成熟卵母细胞均使用 ICSI 与冷冻解冻的非洲狮精子受精。对照组和玻璃化组的卵母细胞成熟率分别为 55%和 49.2%。在对照组中，三个卵母细胞分裂，另外三个卵母细胞在原核阶段停滞。由于受精结果较低，使用了另一只雄性的精子样本进行玻璃化组。在玻璃化的卵母细胞中，有 7 个卵母细胞分裂，另外 9 个卵母细胞在原核阶段停止。玻璃化组的所有胚胎都没有发育到 4 细胞阶段以上。这是首次通过卵母细胞玻璃化获得非洲狮体外衍生的胚胎。
