The landscape of equine reproduction research dramatically changed May 29, when a University of Idaho (UI) and Utah State University team announced they were the first to clone a member of the equine family--the mule--according to an article to be published in the journal Science. The research team includes Gordon Woods, DVM, MS, PhD, Dipl. ACT, UI professor of animal and veterinary science; Kenneth L. White, PhD, Utah State University professor of animal science; and Dirk Vanderwall, DVM, PhD, Dipl. ACT, UI assistant professor of animal and veterinary science.

"Idaho Gem," a mule colt, was born on May 4 at UI's animal and veterinary science department in Moscow, Idaho, but researchers had to wait to announce his arrival. They first had to verify whether he was genetically identical to DNA from a fetal cell culture first established at UI in 1998. After genetic verification, researches submitted a manuscript summarizing the project to Science. It was fast-tracked to publication, and the foal's announcement was made to coincide with the journal's online publication date of the article.

In cloning, using microsurgery techniques, scientists remove the nucleus from an egg cell, which contains the cell's genetic material, creating an enucleated egg. A somatic cell (which is any body cell other than an egg or sperm cell) is placed adjacent to the enucleated egg, making sure the membranes of the two cells are in contact. An electric pulse fuses the egg cell and the somatic cell with the new genetic material. The cell is then treated in media that allows it to develop into an embryo that can be implanted in a surrogate mare's womb and carried to term.

Researchers hold that this technology will not replace natural sexual reproduction in animals, but allows scientists to increase the impact of important genetic traits in populations of animals.

It's Not Just the DNA

From 1998-2000, the research team had little success in their cloning effort--after transferring the nuclei from mule cells into 134 horse eggs and implanting them into mares, two pregnancies resulted, but both failed to proceed past four weeks.

In 2001, the team shifted their focus to the calcium levels in the fluid surrounding the eggs during cloning. White explained that equine cells have much lower intracellular (within the cell) calcium levels than extracellular (outside the cell) calcium levels, suggesting that horse cells probably regulate calcium in a different way than other species.

White said, "When an egg is fertilized by a sperm, 'activation' occurs, which defines a whole cascade of events that tell the egg to begin to develop. One of the very key factors is intracellular calcium peaks that occur in a very precise manner--intracellular calcium plays a very important role in turning on or activating the egg and other communication within the cell." This understanding, discovered by the company CancEr2 (more on this later), spurred the scientists to manipulate the calcium concentration of the medium in which the eggs were held.

The change led to the first fetal heartbeat in a cloned equid, which alerted them that they had made significant progress. That year, the scientists transferred 84 eggs, establishing five pregnancies (none made it to term).

In 2002, the team continued to adjust the calcium levels in the fluid surrounding the egg during the cloning procedure. The scientists transferred 113 cloned mule embryos--each surrogate mare would generally receive three to five cloned embryos, since the likelihood of success was much lower than in conventional embryo transfers. Fourteen pregnancies resulted, and eight made it past the 40-day stage when heartbeats are detected. Three pregnancies continued, one of which resulted in Idaho Gem. The other two pregnancies are proceeding normally, according to researchers, and the surrogate mares will hopefully deliver this summer.

Vanderwall said, "We really had questions about whether we'd see problems with the placenta and/
or fetal viability during gestation. We monitored all (pregnancies) very closely with transrectal palpation and transrectal ultrasound to evaluate placental health and fetal viability.

"We never saw any indication of any sort of aberrations or reasons for concern with the health and well-being of the fetus," Vanderwall continued. "And we've seen no evidence of health concerns with Idaho Gem. He's a very active foal and robust since the time of delivery. The birth of Idaho Gem was completely a spontaneous, natural, and unassisted delivery."

What About Cloned Horses?

The researchers aren't expecting any cloned horse foals as a part of their research this year. "We actually made the attempts with the horse DNA after we'd made the change in the calcium concentration. We didn't transfer enough of the horse DNA-cloned embryos," said Vanderwall. "We transferred many more mule embryos (113 mule vs. only 61 horse embryos). It boils down to numbers, and the team holds that the method developed by the researchers to successfully clone a mule should work equally well with a horse."

The research group hasn't transferred any horse clone embryos in 2003, as the mule's birth and maintaining the other two mule clone pregnancies has kept them busy.

The next phase in cloning research would be the successful birth of a cloned horse, something that Italian researchers are close to achieving, according to Vanderwall. "It was literally a race down to the wire (to have the first successful equine cloning)," said Vanderwall. "We knew that the Italian group, based on the word on the street, had a cloned foal due to be delivered pretty much as we speak (in late May)."

But the race extends beyond Idaho, Utah, and Italy. Several universities, including Texas A&M University, Louisiana State University, and the University of Cambridge (United Kingdom), all have equine cloning projects.

While the process of cloning is far from efficient at this point, it is ready for commercial clinical application. "It's a matter of extending (the technology) to the horse, and continuing to refine the efficiency of the system," said Vanderwall.

The researchers say that while breed or sport organizations have not contacted them about the prospect of cloning, individual horse owners have contacted the researchers about the possibility of cloning specific horses. The company ClonE2 has spun out from the project and will handle the commercial side of cloning stemming from the research team.

In the meantime, Idaho Gem stands alone as the only announced product of a successful equine cloning. "He's special and he knows it," said Woods. "He's really animated--he was ducking, dodging, and kicking" during his official debut.

Equine Cloning: Helping Human Health

Beyond the commercial applications, Woods sees other hopeful prospects in what he's learned from the cloning success. For example, researchers have noticed a drastic difference in the occurrence of cancers (such as melanomas) and other age-onset diseases in humans in comparison to horses.

"Today is the 50th anniversary of (Edmund) Hillary first summitting Mount Everest," said Woods on May 29. "It's interesting, because from that point, everything was downhill. I see this (cloning success) not as a summit, but I seek to use this information to investigate age-onset diseases in humans. That, for me, is the main direction I'd like to see this go."

Woods says that past research has proven that aged horses with white-colored skin have a high incidence of melanomas, but the melanomas do not metastasize (spread) in horses as readily as in humans. There is a high mortality rate for cancers overall in humans (24%), whereas it is not as high in horses (8%).

"Why do horses have melanomas that don't metastasize and humans do?" asked Woods. "We think that with work, we have a key answer to that question, and we think the horse would make a great model for age-onset disease in humans."

Woods also pointed out that stallions have never been identified as having prostate cancer, one of the more common cancers in men. "I think we have a comparative model," he said, but admitted that it is difficult to recruit government funding since horses are not the typical research subjects for human disease research.

"You have a thousand-pound mouse that kicks," Woods said, which might make some scientists leery of their usefulness as study models. But, "that is where my heart really is--in using the horse as a model to study these (human) diseases."

The horse's basic metabolism is "slow" compared to humans and other mammals, said Woods. He speculated that the difference in cellular activity might play a role in both cancer development and reproduction.

Woods formed an outside company (CancEr2) in 1997 to investigate his observations with private investors' backing. Their studies have shown a fundamental difference between men and stallions in the calcium concentrations within cells and surrounding fluid, which led to advances that resulted in the mule clone.

Groups Performing Equine Cloning Research:

  • Louisiana State University's Department of Pathobiological Sciences in the School of Veterinary Medicine;
  • The Link Equine Reproductive Biology Program in Texas A&M University's College of Veterinary Medicine;
  • The Equine Fertility Unit at the University of Cambridge's Department of Clinical Veterinary Medicine in the United Kingdom; and
  • Laboratorio di Tecnologie della Riproduzione (LTR) in Cremona, Italy.

About the Author

Stephanie L. Church, Editor-in-Chief

Stephanie L. Church, Editor-in-Chief, received a B.A. in Journalism and Equestrian Studies from Averett College in Danville, Virginia. A Pony Club and 4-H graduate, her background is in eventing, and she is schooling her recently retired Thoroughbred racehorse, Happy, toward a career in that discipline. She also enjoys traveling, photography, cycling, and cooking in her free time.

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