Italy made world head-lines when researchers there produced the first successfully cloned horse in 2003. And the United States followed suit on March 13, 2005, when a cloned horse was born at Texas A&M University. His name is Paris Texas, and with his birth, Texas A&M is the first academic institution in the world to have cloned six different species.

Previously, researchers at the College of Veterinary Medicine and Biomedical Sciences had cloned cattle, goats, pigs, a deer and a cat.

Paris Texas came to be through a program directed by Katrin Hinrichs, DVM, Ph.D., of the department of veterinary physiology and pharmacology. With cells provided by a French reproductive sciences laboratory called Cryozo-otech, the researchers produced a bay foal.

“The way we produced our foal, with an in vitro system, is similar to what they used in Italy, to produce a cloned horse foal in 2003,” said Hinrichs.

Hinrichs’ program had actually produced a pregnancy in 2003, but the host mare lost the foal at nine months’ gestation, when she developed placentitis.

While the loss was a blow to their program, Hinrichs was thrilled that the mare had carried the foal almost to birth.

“We knew the procedure would work, because we’d gotten that far along with it,” she said.

The following year Hinrichs decided to start again on a cloning project. Then, a colleague of hers from France, Eric Palmer, contacted her. He was considering cloning as a commercial venture because so many quality geldings in Europe, both show jumpers or dressage horses, can’t leave offspring.

“I told him we were going to do some more cloning research, and he said, ‘Why not use cells that would produce a valuable foal, rather than using cells from any old horse?’ ” said Hinrichs.

Paris Texas was cloned from skin cells of a European sport horse. Palmer, at Cryozootech, collected the cells, cultured them, froze the cultured cells and sent them to Hinrichs’ lab at Texas A&M.

The frozen cells were thawed and cultured until they were ready for the nuclear transfer procedure. The resultant embryo was then transferred (at seven days) into the recipient mare—a Quarter Horse-type named Greta, who had been in Hinrichs’ research herd for four years.

The owner of the donor horse wants the horse to remain anonymous. The donor may have been a stallion or a gelding; the foal is a male.

“We know the foal is a clone, because we’ve done all the genetic markers on him, and he’s identical to the donor animal’s cells, genetically,” said Hinrichs.

Give Me Some Skin

To produce a cloned foal from a valuable gelding, all you have to do is take a small skin sample from that horse, about the size of a pea.

That sample is placed in culture medium, in an incubator. Then out from the skin sample will grow cells, which are called fibroblasts. These are cells of connective tissue that would normally hold the skin together.

Then the skin tissue is removed, and the new cells growing in culture are the ones used for cloning. The cells in the culture grow rapidly, and soon there are thousands of them, and they can be frozen and later thawed to produce a cloned foal.

“One of the things that’s exciting about this is that there are frozen fibroblasts from endangered equids [certain species of zebras, Przewalski Horses] at many of the zoos throughout the world,” said Hinrichs. “At the San Diego Zoo [Calif.] they have frozen cells from as far back as 1975. Some of those animals are under-represented in the gene pool. [How] valuable it would be to be able to go back and get cells from an animal that might have died in 1975 and produce a new individual with those valuable genetics.”

The process for producing the cloned embryo is called nuclear transfer.

“We take a mare’s oocyte [egg] and culture it until it’s ready to be fertilized. That’s why it’s such a big cell–it’s responsible for providing all the materials to make the embryo,” she explained.

The researchers then remove the DNA (deoxyribonucleic acid, the carrier of genetic information) of the oocyte so it contributes no chromosomal DNA to the genetics of the foal—it’s now a receptacle, primed and ready for new DNA.

The clone is essentially a twin to the donor. “The hero is the oocyte–that it’s able to do this with a skin cell nucleus from a completely different individual,” said Hinrichs. “The oocyte is programmed to work with foreign DNA, because the sperm is always foreign.”

But what’s miraculous is that the oocyte can reprogram the skin cell and can “turn on” its entire DNA. The nucleus of the skin cell contains the entire DNA to make the whole horse, but when that nucleus was in a skin cell, most of that DNA was turned off.

Not Another Secretariat

Some people are uneasy about cloning because they believe someone would clone a superstar horse and return it to the show ring. Researchers insist that the purpose of producing a clone is not to take this horse back into competition.

A cloned foal, however, is unlikely to be the performer the original horse was, for several reasons. First of all, it spent its first seven days in a Petri dish.

“We know that even with fertilized embryos, this can affect their growth when they’re born. They don’t have the optimum conditions of a normal foal that starts out in the uterus,” said Hinrichs.

Also, the placenta in cloned animals doesn’t function as well as the placenta of a normal animal. Paris Texas weighed only 60 pounds when he was born, after being more than a month overdue.

“We would have expected, from the size of the recipient mare, Greta, that she would have produced a foal between 80 and 90 pounds,” said Hinrichs. “The fetus was somewhat compromised during gestation.”

Environment is also important. If a mare has twins and they’re small at birth, even though they have great genetics they’re unlikely to be the performers they’d have been as singletons. As breeding animals, however, they would still have value. A cloned foal should still produce exactly the same offspring the original horse would have produced.

“As a veterinarian, I’ve had people come to me with a 30-year-old mare, telling me she’s the last daughter of the last stallion imported to California from Poland before World War II, and asking if I could help them get one last foal from her,” she said. “But there’s no way she’ll ever produce another foal. But now, here’s one more method, one more possibility, that a reproductive veterinarian can offer clients in situations like this.”

Currently, no breed registries will register a cloned foal. Hinrichs said the American Quarter Horse Association is the most forward-thinking registry (they’ll register foals from oocyte and embryo transfer, for example) but has stated they will not register foals produced by any kind of cloning procedure.

“It’s possible that [protocol] will change, once they see what cloning is used for and realize people aren’t going to produce thousands of animals all the same,” added Hinrichs.

Some breeds that are experiencing genetic diseases due to excessive line breeding and inbreeding may benefit from the cloning process. Breeders could return to some of the older individuals that are no longer represented in the gene pool, horses who don’t carry the recessive genes.

Cloning at this point is inefficient, however, and won’t be practiced on a large scale “Out of more than 400 oocytes on which we performed nuclear transfer, we produced only six embryos that were able to be transferred [seven-day-old embryos]. We transferred those and produced one pregnancy,” said Hinrichs.

The efficiency will improve as researchers gain more experience. “But right now, a veterinarian can tell a client to go ahead and have skin cells frozen. You don’t have to use them, but they’ll be there if you decide to do so, if cloning becomes efficient enough and cost-effective enough.”

Similar But Different

While the first in North America to create a cloned horse, Texas A&M wasn’t the first U.S. university to create a cloned equid. In 2003, the University of Idaho successfully cloned mules. But Katrin Hinrichs, DVM, Ph.D., of Texas A&M University, noted there are major differences between the two cloning processes.

“The mules were cloned from cells from a fetus,” she explained. “They bred the mule’s mare mother to the mule’s jack father. They took cells from the resulting fetus and used those cells for the cloning. The reason they did it that way was that you have a better chance of getting a viable offspring using cells from a fetus, than you do from an adult.”

Another difference is that the University of Idaho researchers used oocytes recovered from the mare’s pre-ovulatory follicles. And they didn’t grow the oocyte in vitro as the Texas A&M group did.

“Most people aren’t interested in cloning a fetus; they want to clone an animal that has already existed,” she added. “That’s the advantage of being able to create a foal from a cell [such as a skin cell] from an adult horse.”

Cloning For Research

At Texas A&M University, researchers there are currently studying how the oocyte is able to make an embryo, and they continue to use cells from valuable and well-known horses.

Katrin Hinrichs, DVM, Ph.D., started researching equine reproduction, then embryo transfer and oocyte transfer, and then looked at ICSI (intracytoplasmic sperm injection).

“I think one reason our lab is successful in equine cloning is because we come to it from a veterinary point of view. We know the biology of the mare and all the aspects of embryo transfer, and more recently the factors in oocyte maturation and embryo culture that we learned from our fertilized embryos.

Everything is built on all the steps that came before,” she said.

Cloning has many possible uses. In addition to preserving valuable genetics, cloning offers a way to look at diseases affected by genetics and environment. For example, OCD (osteochondrosis dissicans) has some genetic predisposition, but there are environmental factors as well. It’s hard to separate those two factors, however, because when a foal is born you don’t know its genetic predisposition.

In order to compare two sets of foals, you need such large numbers that it’s difficult to get any straight information about what the environmental differences might be. If you had five to 10 foals with exactly the same genetics, you could do a study and get some real answers, whereas you can’t get definitive answers with 100 or 200 unrelated foals because they don’t have the same genes.

“If you started with a skin sample from an affected horse you’d know that all the cloned foals have the genetics to be affected,” she said.

Then you could use different feeds or levels of nutrients, different levels of exercise, to study the OCD patterns.

“This could save a lot of effort down the line, being able to do the studies with a much smaller number of horses,” she said.