When people think of cloning, a vision of hundreds of identical blonde haired, blue-eyed children taking over the country may come to mind. When thinking of cloning horses, many people will envision a sea of identically marked sorrels filling the trails.
 
This is not quite accurate, according to Dr. Katrin Hinrichs, DVM, PhD, veterinarian and professor at the Texas A&M University College of Veterinary Medicine. Hinrichs started TAMU’s Equine Embryo Laboratory in 1999, and has specialized in equine reproduction since her residency at the University of Pennsylvania in 1982.  
 
In 2005, Hinrichs and Dr. Young-Ho Choi, an associate research scientist at Texas A&M worked in Hinrichs’ laboratory. The team became responsible for the first cloned horse in North America. The cells used for cloning were supplied by Dr. Eric Palmer, founder of Cryozootech, a French company dedicated to preserving the genes of exceptional horses for their use in producing cloned offspring.
 
The bay colt, named Paris Texas, was cloned using skin cells from Quidam de Revel, a famous European jumping stallion. Paris Texas was foaled at Texas A&M University, making it the first academic institution in the United States to clone six species of animals.
 
Hinrichs laughed at the attention she received after Paris Texas’ birth. “We are an equine oocyte, fertilization and embryo transfer lab. We are only interested in cloning as a way to explore the biology of the horses at fertilization,” Hinrichs explained. “The procedures we use for cloning are some of the same procedures we’ve been using for years with embryo transfer and artificial insemination.
 
“Theoretically, cloning is a very simple process,” she said. “Cloning uses many of the same processes we use to look at fertilization and embryo development. You have to obtain oocytes from the horse and mature them in an incubator to the point where they are ready to be fertilized. At this point, we either fertilize them for our fertilization studies or do nuclear transfer for our cloning studies.  
 
“Then we culture the embryos for seven days in the incubator and evaluate them to see how many have developed to the point where they can be transferred.” Hinrichs has been doing research on maturing the eggs (the first step) since 1990, on fertilization since 1999 and research on nuclear transfer since the year 2000.”
 
Hinrichs explained that an egg is normally fertilized by a sperm. Because the egg and the sperm have to combine to form a cell, they each only have half the chromosomes you would find in a regular cell. As soon as the sperm enters the egg, it tells the egg to start making the embryo.  
 
Cloning a cell is a similar process. The scientist takes out the egg’s chromosomes and puts chromosomes in from the donor’s skin cell. Once the chromosomes are placed in the egg, the scientist activates the egg by sending it the same signal the sperm would normally send the egg to begin forming an embryo. Once the egg has received the signal, Hinrichs grows the resulting embryo in a Petri dish in the laboratory for seven days. Then, the embryo is transferred trans-cervically (non-surgically) into a recipient mare.
 
Skin cells are used for cloning because thisprocedure is the least invasive way to get the cells. A tiny cut is made in the skin and small chunk of cells (approximately the size of a pea) is taken. The skin sample is placed in culture and individual skin cells grow from it. The cells are then frozen, and will be thawed when they are to be used.
 
Hinrichs’ team first began equine cloning research in 1999, but their studies were experimental. Hinrichs’ team produced their first cloned horse embryo late in 1999. The embryo had only five cells, but it was a big step for the clinic. According to Hinrichs, the process was fairly inefficient, but the team kept trying different methods to get better results. By 2004, the team was able to transfer approximately two to three percent of their embryos into recipient mares. One of these embryos eventually became Paris Texas.
 
Some of the first cells Hinrichs’ lab used were from her daughter’s show gelding. The embryo transfer was successful, but the foal was unfortunately lost at nine months.
 
“We were so excited to have such success with the cells from my daughter’s horse, but I was starting to get worried about what we would do with the horse if it was carried full-term,” Hinrichs said. “I felt terrible when we lost the foal; it really started me thinking that we needed to use quality cells, even in our research work, if we were going to produce foals.”
 
At this point in time, breed registries will not allow clones to be registered. According to the American Quarter Horse Association rule 227, “Horses produced by any cloning process are not eligible for registration.” Cloning is defined in the AQHA rule book as “any method by which genetic material of an unfertilized egg or an embryo is removed, replaced by genetic material taken from another organism, added with genetic material from another organism or otherwise modified by any means in order to produce a live foal.”
 
Paris Texas is of impeccable breeding and Hinrichs said he would only be used as a breeding horse, not for performance. “Cloning is not a way to produce competitors,” Hinrichs explained. “There are so many factors that go into a performance horse that will affect it. For example, Paris Texas spent his first seven days in an incubator in a Petri dish, during embryo culture before transfer to the recipient mare. That can affect his growth rate and size even after birth, which can affect his performance as an adult. As a sire, however, Paris Texas will pass on exactly the same genes as his donor horse.”
 
Paris Texas is still doing well, according to Hinrichs. He was recently shipped to Europe and is a healthy yearling. His owners will not use him for performance, but plan to eventually use him in their breeding program.
 
Hinrichs did not stop after her success with Paris Texas; in 2005, she began more cloning research. Currently she has four mares in foal with clones of anonymous stallions, as well as seven cloned embryos from the AQHA stallion, Smart Little Lena. Smart Little Lena, also known as ‘The Legend,’ was shown eight times, winning over $700,000. He is also the NCHA All Time Leading Stallion, with his get earning an excess of $29 million.
 
The seven clones of Smart Little Lena will begin foaling in February, with the last foal due in April. “Cloning Smart Little Lena has been a mutually beneficial research agreement,” Hinrichs said. “The Smart Little Lena Syndicate was interested in cloning the horse and we’re always interested in doing research on the biology of the horse egg.”
Although these clones will be genetic replicas of Smart Little Lena, that does not mean they will look or perform the same as ‘The Legend.’ “Color is genetically determined, so it will be the same between donor and offspring,” Hinrichs said. “The presence of leg and face markings are also genetically determined, but the size and shape of the white markings are due to random migration of the white cells during development, so the size, shape and placement of white markings will be different.” Hinrichs pointed out that identical twin foals follow the same rules.  
 
“Size and conformation also will be varied,” Hinrichs said. “The environment of the embryo, health of the placenta and amount of milk the mare produces all affect the size and build of the horse. For this reason, cloning is not a good way to produce a performance horse.”
 
Although clones will share the same chromosomal DNA with the donor horse, their mitochondrial DNA will be different, according to Dr. Hinrichs. Mitochondrial DNA typing will be able to identify the cloned animal the original and any other clones.
 
Hinrichs also pointed out that horses have more individual identity than some other species that have been cloned in the past. Therefore, horse owners will have an easier time distinguishing the differences in appearance between clones and their originals. “They will have a different look to their face and eye,” Hinrichs said.
 
Texas A&M practices cloning for research and educational purposes only; the university does not perform cloning as a commercial business. Hinrichs estimated that it could cost between $100,000 and $200,000 to commercially clone a horse at this point in time.
 
Although cloning is a controversial topic across the globe, there is no doubt that Hinrichs and her students are learning massive amounts about fertilization in the horse, due in part to their cloning research. While many may debate the usefulness of a clone, there are few that will debate the benefits of the research.