"Five years ago, presenting 'A Review of Cloning' in the horse was almost unimaginable," began Katrin Hinrichs, DVM, PhD, professor of veterinary physiology and pharmacology and Patsy Link Chair in reproductive studies at Texas A&M University. "But the field has taken off very quickly, partly because of advances in other species."
At the 52nd annual American Association of Equine Practitioners Convention, held Dec. 2-6 in San Antonio, Texas, she discussed the successes, failures, and lessons learned in equine cloning to date. She also covered concerns about the health of clones and how this technology can and should be used in the equine industry.
What is Cloning?
Cloning is also called nuclear transfer (NT) or somatic cell nuclear transfer (SCNT), Hinrichs explained. "Cloning as most people think of it is SCNT," she said. Either term refers to removing a cell's nucleus, which holds its DNA, and placing it inside an oocyte (egg cell) that will grow into a cloned animal. Plain "nuclear transfer" refers to taking the nucleus from any cell--even an undifferentiated embryonic cell (one that has not yet become a specialized skin, heart, hoof, or whatever type of cell)--while SCNT refers to taking the nucleus from an adult's differentiated somatic or body cell (a specialized skin cell, for example).
"There is a higher chance of viable offspring with embryonic cells than with a differentiated adult line," Hinrichs said. "Before Dolly (the sheep) was produced, people had been doing nuclear transfer with cattle and sheep embryonic cells for a decade, but using differentiated cells was new."
The donor animal has a very small role to play--a small incision is made and a small amount of subcutaneous (just beneath the skin) connective tissue is removed. "This can be done in the field by a practitioner, as long as the sample is quickly put into proper holding media," she explained. In the lab, the sample is cut into smaller samples and fibroblasts are grown from it to increase the number of donor cells (up to 10 million or so).
The oocyte (egg) that will receive the donor nucleus must be mature (ready to be fertilized), then its own DNA is removed. "In our lab, we inject the donor nucleus into the oocyte's cytoplasm," Hinrichs said. "Another way to do NT is to place the donor cell under the zona pellucida (oocyte's outer membrane) and fuse them with an electrical pulse. After the cell and oocyte are combined, we use sperm extract to stimulate the oocyte to divide and start forming an embryo; the sperm extract causes calcium oscillations in the oocyte. That's the trigger to set embryonic development in motion so the transferred DNA will start replicating."
Hinrichs offered the following timeline of cloning milestones:
2000: The first equine nuclear transfer embryo, with five cells, was produced.
2002: Woods, et al., announced cloned mule pregnancies at the 8th International Equine Reproduction Symposium.
2003: Three mules cloned from fetal cells were born through the efforts of the University of Idaho and Utah State University. Their DNA came from an embryo that was a full sibling to a famed racing mule.
Also in 2003, one foal cloned from adult cells was born in Italy. The mare that carried the foal to term was the same mare from which donor cells were taken, so she essentially carried her identical twin to term.
2005: The Italian research group announced the birth of a second cloned horse, and another died at about 48 hours of age from septicemia. Two cloned horses were born in Texas--the third and fourth live cloned foals.
"Italy got one live foal from nine pregnancies resulting from 101 embryo transfers," reported Hinrichs. "Texas A&M got two live foals from three pregnancies out of 11 embryos transferred. Why such a difference? Both labs had only about 5% blastocyst rate (survival of the cloned embryos to the early blastocyst stage), so the difference is in the quality of the blastocysts."
2006: Texas A&M announced the births of seven viable cloned foals. From one donor, 13 embryo transfers resulted in nine pregnancies and five cloned foals. The sixth and seventh foals were from two other donors.
Also in 2006, the commercial company ViaGen announced the birth of three live cloned foals.
"So we know cloning is possible, and that its efficiency varies by the laboratory," Hinrichs summed up. "Unfortunately, there are currently only two labs I know of working on equine cloning--Texas A&M and ViaGen. With that little amount of work being done, it will be very hard to see what protocols affect the health of foals."
Equine Cloning Concerns
Will cloned foals be healthy and normal?
The cattle and sheep industries see huge abnormalities with clones, said Hinrichs--such as abnormally large offspring, fluid swelling, abnormal placentae, and heart and lung abnormalities. Luckily, cloned foals don't seem to have that level of health problems.
"Of 16 known live foals cloned from adult cells, three died," she reported. "At 48 hours, one died of septicemia, one died at 48 hours from pneumonia, and a third died at four days from complications during anesthesia--he went under for correction of a possible bladder tear (he ended up not having one), had a low blood pressure episode while being induced, and never recovered."
The three cloned mules appear healthy and have normal blood chemistry, and two raced this year, Hinrichs reported. However, remember that they were cloned from fetal cells, and as such are different from foals cloned from adult cells--they tend to have fewer problems.
"About 50% of foals cloned from adult cells seem to have problems at birth requiring neonatal care, such as a variable amount of tendon contraction in the front legs and enlarged umbilical remnants," said Hinrichs. "We've had to ligate (tie off) some of those and remove some surgically. This problem seems to go along with contracted tendons and might be part of the same syndrome. We also see weakness or maladjustment, but that seems to be a different syndrome. There might be some difficulty in oxygenation right after birth that contributes to maladjustment.
"They seem to get over problems pretty well," she went on. "All known foals viable past one week are currently healthy."
Why might clones suffer from more health problems than other foals? Hinrichs explained that each cell in a horse's (or human's) body has the exact same DNA, but only those genes required by that particular cell type are turned on while all other genes are turned off (this is called epigenetics). "A skin cell has 'skin cell' genes on and all others off," she said. "After nuclear transfer, the oocyte must reprogram the DNA so the genes needed for embryo development are turned on and all others are turned off. Improper or incomplete reprogramming of the cell to do all functions is thought to be the reason for poor viability of pregnancies from cloned embryos. Much research is being done on what constitutes 'reprogramming' and what affects its efficiency."
What about premature aging?
Many people are worried about premature aging in equine clones after widespread media discussion of Dolly the sheep's supposed premature aging. Hinrichs explained: "Telomeres are noncoding caps on the ends of chromosomes that shorten each time that cell replicates, so they are shorter in older animals. Dolly had short telomeres for her age because she was cloned from a mammary cell, which is one of the few cell types that doesn't regenerate telomeres well (at least in cattle). We never really got to see if she'd age prematurely, because she was euthanized due to a transmissible lung tumor at six years of age."
It remains to be seen if equine clones will age prematurely, but since they are cloned from skin cells and not mammary cells, it is likely that they will age normally.
How closely will cloned foals resemble the donor?
"A clone will not be an exact duplicate," stated Hinrichs flatly. "The shape and amounts of markings differ simply because of random migration of pigment cells during growth. For example, as the embryo matures, genetics call for white at the extremities, not necessarily for white on the left front to halfway up the cannon. So socks mix and match, and a donor might have one white hind sock while a clone gets a blaze.
"It would be interesting to clone an Appaloosa and see what happens," she said with a laugh.
"The best way to think about a clone is as if it were an identical twin," she added. "Twins have minor differences, their teeth might be different and their baby teeth will fall out on different days. And nutrition and environment are going to affect the way their genes are expressed."
She showed the audience a photo of Smart Little Lena, a Quarter Horse stallion from whose cells five clones were produced at Texas A&M. All five clones have the same basic coloring as the donor, but all five have different markings. And they're different heights, with slightly different conformation.
"It's just the way things happen in utero," said Hinrichs. "That's what makes foals resemble more closely or not. The biggest environmental impact is from the uterus. For example, with these natural equine twins (she showed a photo), this one was 80 pounds at birth while the other was 29 pounds. The larger one had more placental attachment area and thus more nutrition from the mother, while the small one had about one-third the placental area of the large one. The big foal was 16.1 hands high at maturity; the other was 14.2. So the uterine environment can affect phenotype (physical characteristics) at birth and at adulthood. The recipient mare will put her own influence on the foal.
"And placenta seems to be the most affected part of cloning," she noted. "Tthe placenta is not as good as that of a non-cloned individual.
"Then there's the postnatal environment: Milk production in the dam, character of the dam, nutrition, exercise, deworming programs, and handling!" she explained. "The biggest example of this is CC (CopyCat) from A&M--the big news was that it doesn't behave at all like the original cat. However, the original cat was a lab cat--she got very little attention, so she was shy and scared. CC was played with, given a rich environment, handled, played with toys--she is a happy, healthy, social cat. Does that mean behavior can't be inherited and clones are not like the donor? No! It just means environment has an impact."
Why should anyone want to clone animals if the clone might not look or act like the original?
"Cloning is a tool that might help some people in some circumstances to preserve genetics from dead, old, dying, castrated, or otherwise infertile horses," answered Hinrichs. "Cloning is genetic banking. The progeny of clones are the goal."
How will cloning be used in the horse industry?
"When I first started working in this area in 1999, people weren't sure how they felt about cloning," Hinrichs recalled. "Now a lot of people see a use for it one way or another, and see it being used in other species. Now they ask, 'How can it be used to support genetic improvement in our breeds?' "
Remember that even if a clone looks small or weedy, it has the same genetics as a champion donor. "You can breed the worst mouse clones together and get normal offspring," remarked Hinrichs. "They all have the same genetics."
Cloning might be favored as an assisted reproductive technology over other methods because of efficiency. "At Texas A&M, I would have to say cloning is clinically feasible," said Hinrichs. "We also do intracytoplasmic sperm injection (ICSI), but if a mare dies and the owners want a foal from oocytes present on her ovaries, we would have a better chance of getting a foal from a mare with cloning (and breeding the clone) vs. ICSI on oocytes at this point."
Price might or might not be an issue; she said the commercial price tag for a cloned foal runs about $150,000 and that number will likely decrease as efficiency increases. Texas A&M currently only clones for research purposes.
Clones are not eligible for registration in most breed registries, but the association Zangersheide, for example, does allow clones and their progeny to be registered and to compete in FEI events. "Many competitions do not require breed registration (such as cutting, reining, or jumping)," commented Hinrichs. "The American Quarter Horse Association has distinct wording not allowing clones to be registered. But the big money is in cutting and reining competitions."
Should clones compete?
"If a donor was champion, it is unlikely that the clone will match its record," cautioned Hinrichs. "Epigenetics, health, environmental differences, training, rider, cow, dirt, wind, chance, and the competition all can affect the clone's performance. Plus we're not trying to get just an animal that's as good as the original, we're trying to make them better than the last generation. Maybe the original horse had the perfect trainer, rider, and environment, and those aren't around anymore. And what's the competition like this year? It's about more than just genetics."
She offered another sobering thought: "If the clone of a champion horse competes and does not do well, it will not only detract from the value of the clone as a breeding animal, but it will also detract from the value of the original animal. This may be the most potent factor that will limit the campaigning of cloned animals in performance events."
Can cloning be abused, i.e., claiming an animal is an original when in fact it's a clone?
"Clones can be differentiated from donor and each other by age, appearance, and mitochondrial DNA," said Hinrichs. Mitochondria are the parts of a cell that produce energy for carrying out the cell's functions. They come from the oocyte; they generally have nothing at all to do with the donor DNA (although some clones might have a mixture of donor mitochondria and oocyte mitochondria). Thus, to have a clone that was indistinguishable genetically from the donor, you would have to use an oocyte from the same maternal line as the donor, and a cell nucleus from the donor.
And who's to say mitochondria aren't important to performance? "Mitochondria provide energy--maybe the reason Cigar was a great racehorse was because he had great mitochondria," suggested Hinrichs. "That could have effects on production, stamina, or other physical or behavioral traits."
One area where abuse could occur is the offspring of male clones; it's not possible to differentiate them from offspring of the male donor, she noted.
"Cloning is possible in the horse, and efficiency appears better than in other species," Hinrichs concluded. "A cloned foal is unlikely to be an exact copy of the original, or even a competitor, because of vast differences from the donor's situation. So since it's expensive and labor-intensive, cloning should be considered a method to preserve genetics. A cloned stallion should produce the same foals as the donor stallion, but progeny of mare clones will be slightly different because of different mitochondrial DNA."
Get research and health news from the American Association of Equine Practitioners 2006 Convention in The Horse's AAEP 2006 Wrap-Up sponsored by OCD Equine. Files are available as free PDF downloads
About the Author
Christy West has a BS in Equine Science from the University of Kentucky, and an MS in Agricultural Journalism from the University of Wisconsin-Madison.
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