The Latest on Clones

As scientists continue to unlock cloning's secrets, perfect its processes, and study the way genes express themselves in individuals, their successes will undoubtedly affect demand and future use.

In March 2008 Prometea, a Haflinger mare and world's first cloned horse, foaled Pegaso in Cremona, Italy, making the colt the first offspring of an equine clone. One month later, Pierazade du Vialaret, a filly and the first foal sired by a cloned stallion, was born in France. Although the arrival of these foals does not represent a baby boom among cloned horses, it does signify a generational milestone in the science of equine cloning.

"I'm sure folks are studying the foals closely," says Ken White, PhD, a professor at Utah State University's Department of Animal, Dairy and Veterinary Sciences and associate director for research at the University's Center for Integrated BioSystems, a facility dedicated to agricultural and environmental biotechnology research. He was part of the team that produced the world's first equine clone, a mule, in 2003. "When we put our (cloned mule) foals on the ground, we ran blood tests and every test we could run. But I can't think they'd be anything other than healthy foals."

It is testament to how far science has come since the notion of cloning--equine or otherwise--was mired in controversy five years ago.

White, along with his colleagues at the University of Idaho, Dirk Vanderwall, DVM, PhD, Dipl. ACT, and Gordon Woods, DVM, MS, PhD, Dipl. ACT, first made international headlines with their trio of cloned mules. Produced with genetic material drawn from fetal mule skin cells, Idaho Gem, Idaho Star, and Utah Pioneer made their debuts. The mules were the first equids cloned ever, but they were quickly followed that year by the birth of Prometea in Italy and two more horse clones born at Texas A&M University in 2005.

The process is straightforward enough: in vitro (in the lab), mature or ready-to-be-fertilized eggs (oocytes) are harvested from a donor mare. The oocytes' own genetic material (DNA) is removed and is replaced with DNA drawn from the donor (skin or other organ cells from the "founder" horse) and injected into the oocyte.

"Tissue is collected from two spots: The neck and under the tailhead," says Candace Dobson, marketing associate for ViaGen, an Austin, Texas-based private sector firm that provides livestock cloning services for a range of clients. "You get a million cells from a tissue sample about the size of a pencil eraser."

Once the genetic material has been implanted into the oocyte, sperm extract is introduced to stimulate oocyte division, initiate embryonic development, and begin the DNA replication process.

Oocytes are allowed to divide for about a week in the lab before they are transferred into a surrogate mare. The optimum result is a full-term pregnancy resulting in the birth of an individual with DNA identical to the founder.

"If they make it to 60 days after they were implanted, they're like gold," says White. "So far all that made it to that 60-day mark went full term."

In the Beginning

Since 2003, 20 equines have been cloned worldwide. According to Katrin Hinrichs, DVM, PhD, professor of veterinary physiology and pharmacology and the Patsy Link Chair in reproductive studies at Texas A&M University, those clone foals have presented no physical problems directly connected to the fact that they are clones.

"They're doing just fine," says Hinrichs, who was on the team that has produced several viable cloned foals since 2005. "They are all just being horses. What problems we've seen are not unique to their being clones."

Researchers have, however, seen complications in foals similar to those that might occur in counterparts born as the result of conventional breeding. One clone, Hinrichs says, died from pneumonia while being treated for a suspected bladder rupture. Another suffered some bladder stones, but it recovered. Overall, she says, about 50% are born exhibiting some weakness.

"We can only guess why," Hinrichs says, "but it seems to have to do with the placenta and the way it works. Still, within seven days, they're normal. In fact, 50% of the clone foals hit the ground and start bouncing around just like any foal would."

To determine the source of and remedy for the weakness in some foals--cloned and otherwise--Hinrichs and others who research cloning and aspects of conventional equine reproduction are studying equine placenta development and performance. They are also seeking ways to make the cloning process more efficient and achieve increased pregnancy rates.

Surviving Transplant

"Cloning is an inefficient process," says Vanderwall. "It has a 3-4% success rate."

In practice, according to Vanderwall, scientists allow 100 oocytes to divide for about a week in the lab. Of the original 100, 15 or 20 will start to divide.

"We implant three to five oocytes at the one-cell stage into surrogate mares," he says. "If we transferred to 12 mares, maybe six would be pregnant. In general you take 100 chances for three or four foals."

As a result, White says researchers are working to learn more about equid oocytes toward improving their handling in vitro and beyond.

"Handling may indirectly compromise oocytes," he says. "As folks learn more about horse oocytes, we may be able to increase the numbers that develop in vitro and also increase pregnancy rates. We also want to make oocyte handling more efficient so that they could be re-implanted into the mare more quickly."

A Game of Chance

But not all what researchers have learned has come from the lab. Even before the first equine was cloned, critics envisioned the creation of "super horses" capable of dominating equine sports. But White and Vanderwall's cloned mules have shown that producing champions remains much a matter of chance.

Cloned from the full sibling of a champion racing mule and born in 2003 to three different surrogate mares, Idaho Gem, Idaho Star, and Utah Pioneer were all destined to have racing careers. All three were placed with winning trainers. But only Idaho Gem and Idaho Star went on race--and place--competitively.

"Here we have three individuals, and they are individuals," says Vanderwall. "Utah Pioneer just wasn't cut out to be a racing mule. He's right here just outside my window enjoying the good life."

Hinrichs is not surprised. Cloning is about preserving genes. When it comes to performance, the nature versus nurture factor can't be ignored.

"If I've cloned a foal, I know ... that it has a certain performance capacity."--Dr. Katrin Hinrichs

"If I've cloned a foal, I know what genetics it's going to have, that it has a certain performance capacity," she says. "That means people can start doing things right from the beginning--getting the right trainers; feeding it the right way. It shortcuts the process."

Epigenetics, or the way genes are expressed in individuals, also plays a role in clones' performance, according to Vanderwall. And cloning researchers are paying attention to what their geneticist colleagues are learning as they unlock those secrets.

"Epigenetics is about how which genes are turned off and which are turned on in one individual and not in another," Vanderwall says. "So you can have an individual with the same DNA, but the genetic information is expressed differently. That helps to explain why we see differences in cloned animals."

Although many of ViaGen's clients are hoping the right gene switches are flipped when they clone their favorite horses, they are at least as interested in preserving high performers' genes that would have been otherwise forever lost.

"Many horses were gelded early on, and thanks to cloning, they can now be brought back as stallions to pass along their genetics," says Dobson. "Another client had a champion cutting horse that was in the top five producers of champions. She died three years ago, but her owner gathered samples that were frozen for future use. Her clone foal was born in 2007."


But not only high-performance horses will eventually benefit from genetic preservation via cloning.

"Cloning is a tool for preserving endangered equids," Hinrichs says. "You can take a horse oocyte and by putting, for example, zebra DNA into it, clone a zebra. You can have a gene pool ready to breed 50,000 years from now."

And there are more immediate incentives for perfecting cloning as well. Stem cells available from cloned embryos can be used for tissue regeneration, opening the door to new treatments for injured equines.

"If a horse has a problem with a hock, stem cells could be used to grow cartilage that could be implanted," Dobson says. "Also, if a horse has a tendon problem, the tendon could be repaired by growing cells taken from a cloned embryo."

The prospect of helping heal their horses--or even regenerate them as clones if they contract disease--is why some ViaGen clients have chosen to deposit their horses' genetic material in the company's gene bank.

"You never know when a horse will become injured or present a serious health problem," Dobson says. "Gene banking is kind of an insurance policy."

Take-Home Message

As scientists continue to unlock cloning's secrets, perfect its processes, and study the way genes express themselves in individuals, their successes will certainly affect demand for, and future use of, the technology.

With the exception of the mule racing industry, a sport horse registry in Europe (Studbook Zangersheide), and discipline associations that don't require breed registration, breed and sport registries prohibit the registration of cloned horses. As private companies such as ViaGen currently produce between 30 and 50 clones annually and seek to double that number in the future, horse industry associations might be forced to take another look at their rules.

"There are logistic problems to work out, such as distinguishing which is the founder horse and which is the clone," says White, "but clones are not Xerox copies or robots. I think registering clones needs to be done. And I think it will be done eventually."

About the Author

Pat Raia

Pat Raia is a veteran journalist who enjoys covering equine welfare, industry, and news. In her spare time, she enjoys riding her Tennessee Walking Horse, Sonny.

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