First lion embryos from frozen egg cells

Lion eggs

Egg cells from an African lioness. (Image: Jennifer Zahmel / Leibniz-IZW)

In order to save endangered animal species from extinction, researchers are trying to preserve the egg cells of the females and later artificially fertilize them. For the first time, eggs from African lions that have been frozen and stored for several days have survived. Seven embryos developed from this. However, since they only survived to the 4-cell stage, more research is needed to improve freeze-preservation.

For cryopreservation, egg cells are removed, rinsed and either slowly or immediately frozen in liquid nitrogen at minus 196 degrees Celsius. In this state, they can be stored for an unlimited period of time and eventually fertilized artificially. In the case of human egg cells, freeze preservation now has a similar chance of success as in vitro fertilization of “fresh” egg cells and is used to determine the time of pregnancy itself.

Is freezing successful in lions too?

Freezing could also be a way to store the eggs of endangered species like the Asiatic lion. However, because of their high fat content, feline oocytes are considered to be relatively sensitive to freezing, especially if it happens slowly. Researchers led by Jennifer Zahmel from the Leibniz Institute for Zoo and Wildlife Research on African lions (Panthera leo) have now investigated whether cryopreservation achieves reproductive successes similar to that of fertilization with fresh egg cells. Previous studies with domestic cats had already shown that rapid vitrification of cat egg cells in liquid nitrogen is the most promising.

On this basis, the scientists have now removed egg cells from four African lionesses from the Danish Givskud Zoo – Zootopia after these animals had to be euthanized there. 60 egg cells were immediately vitrified on site and stored in nitrogen for six days. The researchers then heated them up and matured them in vitro for a total of 32 to 34 hours at 39 degrees Celsius in an incubator. The other half of the egg cells were ripened directly as a control group without prior freezing. Zahmel and her colleagues fertilized the mature egg cells of the two groups with frozen and then thawed sperm from African lions and finally observed their development.

Egg cells survive vitrification

The result: Although lion’s egg cells are particularly sensitive to cooling due to their high fat content, most of the egg cells survived vitrification. “After thawing the vitrified egg cells again, we were able to determine a high proportion of surviving and matured egg cells,” says Zahmel. “Almost half of the egg cells had matured, a similarly high proportion as in the control group.” The difference was only about six percent. The vitrified egg cells eventually even developed into seven embryos; in the control group, this was achieved for three embryos. However, none of the embryos grew beyond the 4-cell stage.

The results show that lion ova can indeed be successfully cryopreserved, matured and fertilized for the conservation of genetic resources. “To the best of our knowledge, in-vitro embryos have never been created from vitrified egg cells from African lions or any other wild cat species. This is the first time, ”says Martina Colombo from the University of Milan. “Although embryonic development is still impaired as things stand, our results give reason to hope that wildcat egg cells can be stored quickly and safely in biobanks in the future,” adds Zahmel’s colleague Katarina Jewgenow.

Future research should now clarify which cellular processes are impaired during freezing in order to understand which specific conditions the vitrified egg cells need after heating. “Our goal is to continuously improve these methods using model species such as the domestic cat and the African lion, so that one day they can be used for the assisted reproduction of endangered cat species such as the Asian lion,” added Jewgenow.

Source: Leibniz Institute for Zoo and Wildlife Research (IZW) in the Forschungsverbund Berlin eV, article: Cryobiology, doi: 10.1016 / j.cryobiol.2020.11.011

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