Helping endangered donkeys and zebras from the laboratory
Extinction is not something that necessarily springs to mind when considering donkeys and other equids such as horses and zebras. But for different species of wild donkey, zebra and wild horse, there are wildly different situations regarding their population health, as The University of Queensland’s Andrés Gambini and Nicole Duncan explain.
Profile: Andrés Gambini
Andrés Gambini graduated with honours as a veterinarian at the University of Río Cuarto, Argentina, in 2008.
He produced the first cloned horses born in South America in 2010 and in Australia in 2018. He is a senior lecturer in Animal Science, School of Agriculture and Food Sciences at The University of Queensland, Australia.
Profile: Nicole Duncan
Nicole Duncan is Bachelor of Equine Science and a Bachelor of Agribusiness from School of Agriculture and Food Sciences at The University of Queensland.
She is currently performing her Bachelor of Science (Honours) at UQ in horse reproduction and endocrinology under the supervision of Dr Andrés Gambini and Associate Professor François-René Bertin.
Top image: Alert mountain zebras in Karoo National Park, South Africa. © flowcomm (CC BY-SA 2.0) https://creativecommons.org/licenses/by-sa/2.0/legalcode
Donkeys, horses, and zebras: same but different
Donkeys (also known as ‘asses’), horses, and zebras have a shared evolutionary history as a part of the Equidae family. The common ancestor of all three species existed around four million years ago in what is now North America. However, despite their similarities, they have vastly different traits.
Horses are known for their speed and endurance, having been used throughout history for warfare, transportation, and recreation. Domestic donkeys are known for their hardiness and are used as pack animals, for farming, and for transportation. In contrast to these domestic animals, all species of zebras and some species of donkeys are primarily wild, undomesticated, animals. However, there are some individuals who have been successfully trained for riding and other uses.
Overall, the three species are distinct from each other, but they can interbreed to create hybrid animals such as mules (the offspring of a female horse and a male donkey), hinnies (the offspring of a female donkey and a male horse), zebroid (offspring of a horse and a zebra) or a zonkey (donkey and zebra) offspring. Most of the time these hybrids are sterile and cannot reproduce, but the genetic plasticity within these animals is fascinating.
It is worth mentioning that feral donkeys and horses, also known as wild asses or brumbies, are a common sight in many parts of Australia. These are domestic animals that were abandoned or escaped and have since thrived in the wild. Feral donkeys can be found in various habitats, including deserts, woodlands, and savannas. They are hardy animals and can survive in harsh conditions, making them well-adapted to the Australian landscape. Despite their negative impact on the environment, feral donkeys hold a certain cultural significance in Australia. They are admired for their resilience and adaptability and have become an iconic symbol of the Australian outback.
The relationship between donkeys and humans dates back thousands of years. Donkeys are known to be hardy and reliable animals, making them valuable assets to many communities. Donkey domestication is thought to have occurred around 4000-3000 BCE in Egypt and the Near East, where they were initially captured and tamed for use as beasts of burden, as well as a source of meat and milk (Rossel et al., 2008). Over time, donkeys were selectively bred for certain traits such as strength, size, and endurance, leading to the development of different breeds. Today, donkeys continue to play important roles in many communities around the world.
Modern-day donkeys are used for a variety of purposes, including transportation, agriculture, livestock herding, and milk production. They are also used in therapy programmes for children and adults with special needs and as companions for people of all ages.
A captive Somali wild ass. The species is considered critically endangered. © cuatrok77 (CC BY-SA 2.0) https://creativecommons.org/licenses/by-sa/2.0/legalcode
Are donkeys and zebras endangered?
Although most people are aware of the existence of the domestic donkey, most will not know that there are six
surviving species of donkeys, including the Somali wild asses (Equus africanus somaliensis) and Nubian wild asses (Equus africanus africanus) both of which are considered to be critically endangered, and the Asiatic Wild Ass, represented by Hemiones (Equus hemionus) and Tibetan Kiangs (Equus kiang), which are considered as near threatened and less concern respectively on the International Union for Conservation of Nature’s Red List (IUNC). A similar situation happens with the zebras. The general public commonly do not think of zebras as endangered animals as there are usually ‘too many’ in most of the documentaries on TV. However, there are three different species of zebras: Plains zebras (Equus quagga), Mountain zebras (Equus zebra) and the Grevy’s zebra (Equus fregyi). The Grevy’s and mountain zebra are listed as endangered and vulnerable by IUNC respectively.
Donkeys and zebras face endangerment due to a combination of factors including habitat loss, over-exploitation, and disease. Wild donkeys and zebras are losing suitable habitats due to human development and land use changes, such as urbanisation and agriculture expansion. With an increasing human population, wild donkeys and zebras compete with domestic livestock for food, water, and other resources, leading to overgrazing and habitat destruction. Particularly in wild donkeys, the population has declined drastically in recent years due to poaching and the illegal trade in donkey skins used for traditional medicine and cosmetics.
Dr Andres Gambini (UQ) and DVM. Olinda Briski (University of Buenos Aires, Argentina) with a male donkey.
What can we do to help endangered donkeys and zebras?
- Increase conservation efforts: One of the most important ways to prevent these animals from extinction is to increase conservation efforts. This includes creating protected areas and implementing new technologies in breeding programmes to preserve genetic variability;
- Raise awareness: Raising awareness about the plight of the donkey and zebras is crucial to saving the species. Educating the public about these animals’ importance can help create a greater sense of urgency to save the species;
- Regulate hunting and trade: It is essential to regulate the hunting and trade of the donkey to ensure that they are not hunted or traded to the point of extinction. Governments should enforce strict laws to protect endangered animals;
- Promote sustainable livelihoods: Encouraging sustainable liveli hoods that do not harm wild animals is important. This includes promoting sustainable agriculture and forestry practices; and
- Encourage research: Research is crucial to understanding the threats facing the equids and finding ways to save the species. Scientists and conservationists should be encouraged to study these wild species to under- stand their developmental biology and habitat more.
Image: Horse egg under the microscope about to be enucleated to perform cloning. Left: holding pipette; right, enucleation pipette.
Our contribution: helping donkeys and zebras from the laboratory
Assisted reproductive technologies, commonly known as ARTs, are methods used to help endangered species reproduce. They include artificial insemination, in vitro fertilisation (IVF), embryo transfer, animal cloning, and the cryopreservation, or freezing of sperm, eggs and embryos. These techniques can be used to increase the genetic diversity of populations and reduce the risk of inbreeding. ARTs have the potential to play a significant role in the conservation of endangered species. Still, they should be used alongside other conservation measures, such as habitat protection and reducing human impact.
In the last ten years, the implementation of ARTs has made significant progress in horse breeding practices (with the big exception of the thoroughbred industry which has strict regulations over what can be used). Hundreds of the horses that we see now in competitions have been produced in a laboratory. Our research group reported the first cloned horse in Argentina in 2010 (Gambini, Jarazo, Olivera, & Salamone, 2012) and in Australia when one was born in 2018 (Damasceno Teixeira et al., 2019).
All the efforts investigating and elucidating the best procedures to successfully produce horses in the laboratory have allowed us to move towards applying these technologies to related species such as zebras and donkeys. In vitro embryo production in donkeys refers to the process of creating and growing embryos outside of the animal’s body, using, something like IVF techniques. In our research, we used a special technique known as ‘Intracytoplasmic sperm injection’ (ICSI). ICSI is typically used when there is a limitation in the number of available eggs and sperm, as is the case for endangered species. The procedure begins by collecting eggs from the jenny (female donkey) using a procedure called ovum pick-up (OPU). The eggs are then placed in a dish under a microscope and carefully examined for any abnormalities. Once a suitable egg is found, a single sperm is selected and injected into the egg using a glass pipette.
The egg is then placed in a dish with special growth media to encourage division into the multiple cells which form an embryo. Once the embryos reach a certain stage of development, they can be transferred into a donkey’s uterus or can be frozen. Frozen embryos can be stored for an indefinite period, allowing for the preservation of valuable animals’ genetics for future use. Our group reported the production of the first viable embryo in donkeys produced in the laboratory last year (Flores Bragulat et al., 2023). Our previous experience in producing embryos in the laboratory for horses, allowed us to identify the differences needed to be successful in donkeys. After several attempts, we identified better conditions for the donkey egg to maintain its viability.
Donkey egg is about to be injected with a spermatozoon (the sperm cell is still inside the injection pipette on the right). The egg is about 100 microns large.
We also reported the production of cloned zebra embryos in 2020 (Gambini et al., 2020). Cloning is a technique that can be used to preserve endangered species by creating genetically identical copies of individuals. The process involves taking cells from a living or deceased individual and using them to generate a new organism. This can be done through techniques such as somatic cell nuclear transfer (SCNT), where a somatic cell (any cell in the body except for sperm and egg cells) is transferred into an egg that has had its own DNA removed. The resulting cell is then able to develop into a new organism.
Cloning has been attempted to preserve endangered species (Gambini, Briski, & Canel, 2022), such as the banteng, a type of wild cattle, and the guar, a type of wild ox. However, it is essential to note that cloning alone is not a solution for endangered species conservation and should be used in conjunction with other conservation efforts, such as habitat protection and reintroduction programmes.
Zebra cloned embryo stained for quality analysis. Blue: DNA, Red: protein expressed by future foetal cells.
In our research, we demonstrated that the horse egg could support the development of zebra embryos. With this, we now understand some of the major challenges of producing embryos in this marvellous family species: the equids. We hope to access a wider range of wild donkey and zebra samples to keep producing embryos and storing them in our frozen bank. When the right time comes, if we need it, we can start transferring embryos and producing foals. Interestingly, some previous researchers have shown that the mare (domestic horse) can get pregnant and carry on a healthy pregnancy when a donkey or zebra embryo is transferred (Allen & Short, 1997). Also, mules can get pregnant if they are artificially prepared to receive an embryo. This means we don’t necessarily need recipients of the same species to produce newborns in the future.
Andrés Gambini & Nicole Duncan
School of Agriculture and Food Sciences
The University of Queensland
IUCN: https://www.iucnredlist.org. Int Union Conserv Nature’s Red List 2022:ISSN 2307-8235
Allen, W. R., & Short, R. V. (1997). Interspecific and extraspecific pregnancies in equids: anything goes. J Hered, 88(5), 384-392. doi:10.1093/oxfordjournals.jhered.a023123
Damasceno Teixeira, T. V., Fry, R. C., McKinnon, A., Fry, K. L., Kelly, J. M., Verma, P. J., . . . Gambini, A. (2019). Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos. Reprod Fertil Dev, 31(12), 1885-1893. doi:10.1071/RD19239
Flores Bragulat, A. P., Ortiz, I., Catalan, J., Dorado, J., Hidalgo, M., Losinno, L., . . . Gambini, A. (2023). Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation. Theriogenology, 195, 199-208. doi:10.1016/j.theriogenology.2022.10.030
Gambini, A., Briski, O., & Canel, N. G. (2022). State of the art of nuclear transfer technologies for assisting mammalian reproduction. Mol Reprod Dev, 89(5-6), 230-242. doi:10.1002/mrd.23615
Gambini, A., Duque Rodriguez, M., Rodriguez, M. B., Briski, O., Flores Bragulat, A. P., Demergassi, N., . . . Salamone, D. F. (2020). Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern. PLoS One, 15(9), e0238948. doi:10.1371/journal.pone.0238948
Gambini, A., Jarazo, J., Olivera, R., & Salamone, D. F. (2012). Equine cloning: in vitro and in vivo development of aggregated embryos. Biol Reprod, 87(1), 15, 11-19. doi:10.1095/biolreprod.112.098855
Rossel, S., Marshall, F., Peters, J., Pilgram, T., Adams, M. D., & O’Connor, D. (2008). Domestication of the donkey: timing, processes, and indicators. Proc Natl Acad Sci U S A, 105(10), 3715-3720. doi:10.1073/pnas.0709692105
All images copyright of A Gambini/ The University of Queensland, unless otherwise stated.