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THE PRZEWALSKI’S HORSE PROJECT

The world’s first successfully cloned endangered Przewalski’s horse (Equus przewalskii) was born on August 6, 2020. Revive & Restore, San Diego Zoo Global (SDZG), and ViaGen Equine collaborated to clone from a cell line of a genetically important stallion that had been cryopreserved since 1980 at the SDZG Frozen Zoo. This groundbreaking achievement was conceived as a new strategy to help restore genetic diversity to the Przewalski’s horse species.

OVERCOMING A GENETIC BOTTLENECK
The Przewalski’s horse (pronounced “shuh-VAL-skees”) population faces the same struggle that many endangered species face—recovering from a severe historic bottleneck. Today’s Przewalski’s horses, of which there are now approximately 2,000, are descendants of just 12 individuals saved from extinction in the early 1900s. Learn more about the species.
Genetic bottlenecks like this can lead to inbreeding and loss of fitness. These negative impacts may emerge long after a species seems to have recovered in numbers. This is because the genetic diversity of endangered species continues to decline over the many generations it takes to repopulate the species, due to a process known as genetic drift. A seemingly healthy recovered population can quickly decline due to inbreeding depression. Or the species can struggle to cope with disease or environmental changes—all because the population has lost the genetic variation it needs to adapt. Additionally, larger species like the Przewalski’s horse are more vulnerable to environmental disruption, habitat loss, and climate change than are smaller animal species. Increasing their genetic variation now may help the Przewalkski’s horse species survive in a changing world.
Genetic rescue is one strategy to reduce the negative impact of inbreeding by introducing individuals from unrelated populations. But for the Przewalski’s horse, there are no unrelated populations to draw from. However, forward-thinking conservationists saved living cells from over a dozen Przewalski’s horses and cryopreserved them at the SDZG Frozen Zoo. These cell lines contain genetic diversity that has been lost to recent generations.
CLONING FOR CONSERVATION
Now a portion of this lost genetic diversity may be recovered by cloning historic Przewalski’s horse from frozen cells. Successful breeding can increase genetic diversity by reintroducing lost variants to the surviving population. This is the hope for the new foal, Kurt, who was cloned from cells that had been cryopreserved at the SDZG Frozen Zoo in 1980. These were cells from astallion that was born in 1975 in the UK, was transferred to the US in 1978, and lived until 1998. He was recorded as Stud Book number 615 (SB615) and known as “Kuporovic” by his zookeepers. Learn more about this cloning process.
The SB615 cell line was chosen for genetic rescue cloning because an analysis of the captive breeding pedigree revealed that the genome offers significantly more genetic variation than any living Przewalski’s horse. Now that the genetic variation from Kuporovic “lives” again in Kurt, Kurt may become the most important horse in the North American captive breeding population. He may also become the first cloned animal to restore lost genetic variation to its species.

THE IMPORTANCE OF GENETIC VARIATION AND SB615

This is the second time that SB615 has changed the course of Przewalski’s horse conservation. In the early 1900s, there were very few Przewalski’s horses left in captivity. To save the species, several zoos interbred their Przewalski’s horses with domestic horses. This created a clash amongst zookeepers. Some felt that “hybrid” horses were inferior to “pure” Przewalski’s horses, and so lineages containing domestic horse ancestry were intentionally segregated for decades. (Here “pure” and “hybrid” are meant as animal husbandry terms.) Due to this, different Przewalski’s horses today have ancestry from only a few of the 12 founders. Separating these lines created two additional bottlenecks for the species. Later, in an effort to purge domestic horse genetics from the “hybrid” line, zoos in North America selectively bred only those males that had “pure” wild horse ancestry. Stallions with domestic horse genes were not bred. This practice led to increasingly more severe inbreeding within both lines of Przewalski’s horse. 

By the 1980s, this became a major concern and prompted an extensive pedigree analysis. It was discovered that the stallion SB615, although a descendant of the hybrid horses, actually possessed unique ancestry from two wild founders, SB11 and SB12. In fact, SB615 carried significantly more unique alleles (or variants of genes) from those wild founders than other living Przewalski’s horses.
Due to the discovery of his valuable ancestry, SB615 was bred to pass on his unique genetics. The captive breeding program in North America changed course in 2004. Zookeepers began breeding all viable Przewalski’s horses, mares and stallions, both “pure” and “hybrid” and the genetic diversity of subsequent generations substantially improved. SB615 was one of the first stallions of the “hybrid” line to be bred in North American zoos, and today he has descendants living in several zoos across the US. While the program greatly improved the genetic health of recent generations, genetic drift continues to erode genetic diversity. 

THE BEGINNINGS OF THE PRZEWALSKI’S HORSE GENETIC RESCUE PROJECT

Kurt is named in honor of Dr. Kurt Benirshke, a geneticist at the San Diego Zoo who in 1975 had a prescient idea. Dr. Benirshke began what is now the Frozen Zoo, collecting and cryopreserving the cell lines of endangered species and safely storing away genetic diversity before it was lost. At the time the collection was a bet on cloning and reproductive technologies that did not yet exist. Nearly fifty years later, with the partnership of San Diego Zoo Global Frozen Zoo, Revive & Restore, and ViaGen Pets and Equine, Dr. Benirschke’s plans are quite literally coming to life. 

It was in February 2017 that Revive & Restore co-founders Stewart Brand, Ryan Phelan, and lead scientist Ben Novak first met with Blake Russell, president of ViaGen Pets and Equine to discuss the potential for cloning endangered species. ViaGen’s advancements in the commercial cloning of domestic species—including cattle, horses, pigs, goats, sheep, dogs, and cats—made it clear that the technology was no longer a theoretical or academic endeavor. 

Could ViaGen use their extensive knowledge in cloning domestic species to help save related wild species? Which species might benefit first? Revive & Restore reached out to San Diego Zoo Global to ask. It was Dr. Oliver Ryder who helped identify many viable, decades-old cell lines from multiple, endangered species. Together, Revive & Restore, ViaGen Equine, and San Diego Zoo Global selected the Przewalski’s horse as a most worthy candidate.
After the foal is weaned he will join others of his species at the San Diego Zoo Safari Park. It is our hope that in five to ten years, as Kurt matures into the world’s first cloned Przewalski’s stallion, he will successfully mate and thus contribute to the genetic diversity of his species and to the future of conservation innovation. Reply