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Horse cloning is the process of obtaining a horse with genes identical to that of another horse, using an artificial fertilization technique. Interest in this technique began in the 1980s. The Haflinger foal Prometea, the first living cloned horse, was obtained in 2003 in an Italian laboratory. Over the years, the technique has improved. It is mainly used on high-performance but castrated or infertile animals, for reproductive cloning. These horses are then used as breeding stock. Horse cloning is only mastered by a handful of laboratories worldwide, notably in France, Argentina, North America and China. The technique is limited by the fact that some differences remain between the original and its clone, due to the influence of mitochondrial DNA.

Reproductive cloning of the Pieraz and Quidam de Revel horses began in 2005. The International Federation for Equestrian Sports (FEI by its acronym in French) decided to ban clones from competition in 2007, before authorizing them in 2012. A few clones are used in equestrian sports, winning major titles such as the Argentine polo championship in 2013. The majority of studbooks and associations for the various horse breeds refuse to accept clones, with the European Union completely banning the trade in cloned animals in 2015. Nevertheless, the number of cloned horses is growing every year. The practice is highly controversial, particularly for bioethical reasons, since it involves a high failure rate on embryos. It also raises questions about the management of horses' genetic diversity, the future of the horse breeding profession, and the outbreak of new genetic disorders or fraud.

History

The Zangersheide stud farm (shown here with their branding) has been a driving force behind the development of horse cloning.

Horse cloning has undergone a rapid qualitative and quantitative evolution.[1] While Italian professor Cesare Galli believes that horse cloning has aroused less interest than that of other large mammals,[2] other scientists believe that the high commercial value attained by some horses has created immediate interest, unlike in the case of less valuable agricultural animal species.[3] Equine cloning owes much of its development to the Belgian stud farm of Zangersheide, one of the pioneers of artificial insemination and embryo transfer. According to Éric Palmer, a French biologist specializing in horse reproduction (who introduced ultrasound to mares and produced the first foal by in vitro fertilization),[4] the way for the use of cloning was initiated in the 1980s by veterinarian surgeon Dr. Leo de Backer. He was in contact with some of the world's leading sports stables. According to Palmer, the ones that are interested are Alwin Schockemöhle, Jan Tops, Thomas Fruhman, John and Michael Whitaker, Willi Melliger, Jean-Claude Van Geensbergen and La Silla (in Mexico), among many others.[5] The value of cloning high lineage horses was recognized as early as 1998, with the Westhusin study.[6] Research to this end was publicly announced in 2001.[7] That same year, with the support of Genopole, Éric Palmer founded Cryozootech, a company dedicated to preserving the genes of horses with exceptional performance, with a view to future cloning. The horse is not the first large mammal to be cloned, as Dolly the sheep and other animals precede it, making it the seventh mammal to be cloned.[8]

Birth of Prometea and Pieraz

The birth of three cloned mules in the United States on May 4, 2003, came just before that of the first horse.[9] The first successful attempt to produce a viable clone was made by the Italian laboratory LTR-CIZ, which in 2003 gave birth to Prometea, a Haflinger foal carried to term by her mother, whose genetic copy she is.[8] These scientists worked under the guidance of Professor Cesare Galli.[8] In 2002, LTR-CIZ merged with Cryozootech. In Italy, they produced the world's second cloned horse, Pieraz-Cryozootech-Stallion. This is a purely commercial clone, aimed at obtaining a fertile genetic copy of a successful but castrated horse. According to Bernard Debré, the birth of Pieraz-étalon heralded the commercial direction that equine cloning would later take.[10] Prometea and Pieraz were obtained using the same method, that of Professor Galli.[9][11]

Entering the commercial phase

Shortly afterwards, on 13 March 2005, Dr Katrin Hinrichs gave birth to Paris-Texas, a clone of Quidam de Revel, in a laboratory at Texas A&M University in Texas (USA). The clone foal is also produced commercially for breeding purposes, at the request of Quidam's owner. The technique used is slightly different from that of the Italians.[12] As a result, the number of clones produced has increased over the years. In 2009, the E.T. FRH clone became the first cloned show jumping horse authorized for breeding by a studbook (the Zangersheide studbook), while endurance champion Pieraz's clone entered its third breeding season.[13] In Argentina, polo player Adolfo Cambiaso uses Crestview Genetics to clone his polo ponies. At the end of 2010, a clone of his polo mare Cuartetera was sold at auction for a record $800,000.[14][15] In May 2013, a non-clone foal was born for the first time from two parents cloned by embryo transfer.[16][17] On 7 December 2013, a cloned polo pony won a major sporting competition for the first time. It was the Argentine polo championship.[18] In 2018, equine cloning was widely used in Argentina's polo scene.[19] The Argentine polo horse has become the most cloned animal in the world.[20]

Technique

Cloning research is often carried out in secret, due to poor public acceptance.[16] Commercial cloning companies sometimes reveal the births of these horses, but the techniques employed remain mostly secret.[21] According to the French national stud farm, the cloning technique used, known as "somatic" cloning, involves taking cells by biopsy, usually from the breast of an adult animal. Fibroblasts are extracted and cultured in vitro until a sufficient number is obtained, then stored in liquid nitrogen. Oocytes are harvested from a mare, either living or dead. The DNA is removed by enucleation, then in vitro culture makes it suitable for receiving fibroblast DNA from the animal to be cloned.[22][23] Due to the high demand for mare oocytes, these are usually obtained from slaughterhouses.[2]

After a week or so of in vitro culture, the resulting embryo is implanted into the uterus of a carrier mare, using the embryo transfer technique. After eleven months' gestation, the mare gives birth to the cloned foal. However, this type of gestation is much riskier than a conventional one.[22]

Success rate

According to a Belgian researcher interviewed by Le Vif, the failure rate is the main reason for opposition to cloning, for bioethical reasons due to the mortality of embryos, fetuses and newborn foals.[24] This rate is high, but is gradually decreasing thanks to better-controlled techniques. Professor Galli obtained 15% viable embryos for his second clone, Pieraz, compared with only 3% for Prometea,[25] the first horse, which required 328 attempts.[26] The first mule trials, in 2003, involved 118 embryos, 13 of which produced a gestation, resulting in 3 live mules.[21] To clone Calvaro V in 2006, Cryozootech used over 2,000 oocytes, which produced 22 embryos, only one of which was carried to term.[27]

Estimates of this rate vary from source to source. In 2012, according to a Belgian researcher, the average success rate for animal cloning was around 5%.[24] Argentine researchers estimate that 6 or 7 embryos are needed out of 20 trials (in 2013).[28] In 2010, according to a French source, around 2,500 mare oocytes had to be used to obtain a single viable foal.[23] There are many abortions as well. Despite increased susceptibility to neonatal disease, a clone has the same life expectancy and robustness as a conventional horse.[29] There is nothing to differentiate a clone from a conventionally bred horse.[3]

Usage

The cost of equine cloning varies between €200,000[29] and €300,000,[30] depending on the source. In 2010, clones intended for sporting competitions represented just 22% of operations.[31] Cloning is therefore mainly carried out in Europe for the purpose of breeding high-performance horses. A gelding can be copied to ensure its progeny.[32] The same applies to a stallion that has become too old to reproduce, or to a mare whose number of foals is naturally limited.[22] The use of cloning relies heavily on the belief that DNA is the most important factor in competition performance.[33] Anne Ricard's study estimates that, in equestrian disciplines (show jumping, dressage and endurance) where geldings represent around 40% of competitors, the use of reproductive clones will enable a genetic improvement of 4% per generation. Once their fertility has been established, their semen is frozen as for any other stallion.[31][34]

Effective cloning remains very marginal[31] due to its cost.[28] In the US and Argentina, requests for equine cloning come mainly from polo players (who allow their mares to play all the seasons) and Arabian horse breeders.[9] Cloning can also be used to preserve rare breeds threatened with extinction,[35][36] but customers' motivations are essentially commercial.[31] Nevertheless, the discovery of a perfectly preserved prehistoric foal in Siberia (in 2018) augurs well for cloning trials by Russian and Korean researchers, to resurrect extinct equine breeds or species.[37]

Limits

If the foal is the genetic copy of its donor, the question of the influence of the mitochondria that remain present in the recipient oocyte is still open.[38] Mitochondria represent only 1 or 2% of the genome, but could influence the clone's sporting performance.[22] They are more important in the case of a mare than a stallion, since the mare transmits her mitochondria during reproduction, unlike the stallion.[39] Similarly, the cloned horse is not necessarily a perfect copy of the donor in terms of phenotype and character. The horse markings may vary, and the character, depending less on genetics than on the influence of the mother and upbringing, may also turn out to be very different. The technique also has its limits when it comes to breeding, as the pattern sought in horses evolves over time. There is therefore little point in cloning a horse clone.[29] It also takes a long time to implement, and the number of specialized laboratories and companies is limited. The Kheiron company in Argentina, for example, estimates its waiting time at eighteen years, with demand far outstripping supply.[28] The ban on clones in a large number of stud books and in certain competitions also limits interest.[23]

Specialized companies

color photo of a company building
Genopole d'Évry supported the creation of Cryozootech, France's first commercial horse cloning company.

A few companies are known for their specialization in commercial equine cloning: ViaGen, Replica Farms, Crestview Genetics, Kheiron and Cryozootech. Competition between these laboratories is fierce.[28] French company Cryozootech is a pioneer in the field, having produced the first commercial clone in 2005.[31] It has made the production of famous horse clones its specialty.[40] ViaGen was originally based in Texas in the United States, but the laboratory moved to Canada after the last American slaughterhouses closed in 2007, to source mare oocytes.[41] Kheiron was set up in Argentina in 2009 with a team of eight people. Equine cloning has developed strongly in this country, thanks in particular to demand from polo players, the profusion of mare oocytes available for research (the country exports a lot of horse meat, and has numerous slaughterhouses supplying oocytes) and the easy breeding conditions in the Pampas.[28] In 2012, Argentina was estimated to be the country producing the most horse clones in the world.[42] In Texas, more than 900 clones were born between the creation of the first laboratory and 2014.[28] In 2019, equine cloning companies are expected to open in China.[43]

Cloning laboratories
Laboratory Country Creation date Number of clones Sources
Cryozootech France 2001 (2005 for cloning activity) 28 (2001 – January 2014) [40]
ViaGen Texas, USA

Canada since 2007[41]

January 2002 World leader in equine cloning in North America[1] [44]
Crestview Genetics USA

Argentina

2009 70 (creation – December 2013), capacity from 20 to 30 each year [18][15][45]
Kheiron Argentina 2009 Capacity of 10 to 20 each year [46][15]
Replica Farms USA

Canada

2005 [47][48]

Reception in competitions and breeding registers

Allegory of the triumph of capitalism over communism at Texas A&M University, the first research institution on the American continent to successfully clone a horse.

The use of clones for the genetic improvement of horses' sporting performance is recognized, including by veterinarians, although skepticism remains high among some professionals.[1] According to Éric Palmer, acceptance of horse cloning is growing, and attitudes are changing, in the same way as the gradual acceptance of in vitro fertilization and artificial insemination techniques in horses.[29] Fears of the birth of malformed or monstrous animals diminished when clone owners realized that their animals were in good health.[49]

Competitions

In 2007, the International Federation for Equestrian Sports ruled that cloned horses should be banned from the official competitions it organizes, believing that opening up participation to clones would be unjust and unfair to the competition. It revised its opinion in July 2012. Horse clones are now allowed in all FEI competitions.[50] This reversal is seen as an important sign of recognition of the usefulness of clones in sport horse breeding.[34]

In the United States, the National Cutting Horse Association and the National Barrel Horse Association allow clones in cutting and barrel racing competitions.[31] The American Quarter Horse Association was taken to court by owners and riders of cloned horses in 2012, for refusing to allow these horses to take part in official breed competitions.[18][51] The initial ruling ordered the association to amend its bylaws to allow clones to compete.[52]

Sporting training of the Levisto Alpha Z clone makes it possible, even probable, for a clone horse to win an Olympic title in the future.[53]

Studbook entries

Clones are generally not entered in the stud books of their respective breeds. The American Jockey Club refuses to allow cloned horses to race. In France, clones are also banned from trotting and galloping races.[18] Two European sporthorse studbooks accept clones: the Zangersheide and the Anglo-European.[22] France's national stud farms advise against banning clones from the various studbooks, arguing that this will ultimately drive the best gene pool abroad.[31]

Cloned horses

The existence of clones is not always made public, due to the poor reception they receive.[9] Although in Belgium, Isabelle Donnay believes that commercial cloning of horses has not been very successful,[24] on a global scale, their numbers have clearly increased over time. Equidia Life's 2013 survey describes the practice as "booming".[16] In winter 2010, 56 clones were counted worldwide, produced by laboratories in Europe, the United States and South America. Americans clone more mares than Europeans.[31] Between 2006 and 2011, at least twenty American Quarter Horses were cloned.[54] In early 2014, there were an estimated 900 clones in the state of Texas alone.[9] In Europe, the Belgian Zangersheide stable regularly uses this technique, with four horses cloned between 2006 and 2013.[30] The stallion Salute, one of Quarter Horse Smart Little Lena's clones, was exported to Australia in 2010 for breeding.[41]

Cloned horses
Cloned horse (breed) Name of the clone(s) Laboratory Date of birth Sources
Stella Cometa (Haflinger) Prometea Prof. Galli Cesare, LTR-CIZ, Italy 28 May 2003 [55]
Pieraz (Arabian) Pieraz-Cryozootech-Stallion Prof. Galli Cesare, LTR-CIZ, Italy

Dr. Éric Palmer, Cryozootech, France

26 February 2005 [56]
Quidam de Revel (Selle Français) Paris-Texas Dr. Katrin Hinrichs, Texas A&MUniversity, USA

Dr. Éric Palmer, Cryozootech, France

13 March 2005 [57]
Royal Blue Boon (Quarter Horse) – Replica Farms, USA 19 February 2006 [9]
E.T. FRH (Hanoverian) E.T.-Cryozootech-Stallion Cryozootech, France 2 June 2006 [5][58]
Scamper (Quarter Horse) Clayton ViaGen, USA 8 August 2006 [59]
Smart Little Lena (Quarter Horse) Thirteen clones since 2006, including the stallion Salute Dr. Katrin Hinrichs, Texas A&M University, USA 2006 [1][41]
Poetin 2 Poetin 1

Poetin 2

Cryozootech, France 30 March 2007 [60]
Chellano Z – Cryozootech, France 25 July 2008 [60]
435 Airwolf Wolfie ViaGen, USA 4 August 2008 [38]
Gem Twist (Thoroughbred) Gemini Murka's Gem Cryozootech, France September 2008

2011

[9][61]
Calvaro V (Holsteiner) Calvaro-Cryozootech-Stallion Cryozootech, France July 2006 (stillborn foal)

2008 (success)

[9][62]
Ratina Z Ratina Z alpha

Ratina Z bêta

Ratina Z gamma

Cryozootech, France 2009 [5][22]
Air Jordan Z Air Jordan Alpha Z April 2009 [63]
Levisto Z (Holsteiner) Levisto Alpha Z May 2009
Sapphire (Holsteiner) Saphir Replica Farms, USA 11 February 2010 [64]
Top Gun La Silla (Hanoverian) Top Gun Cryozootech Cryozootech, France 16 March 2010 [40]
– Nandubay Bicentenario

South America's first cloned horse

Bio Sidus, Argentina 2010 [65]
Cuartelera (Polo pony) – Crestview Genetics, USA 2010 [15]
Frenchmans Guy (Quarter Horse) Three clones ViaGen, Canada Spring, 2011 [39]
Zandor Z Zandor Alpha Z Born in November 2011, existence revealed in February 2012 [66]
Rusty (Letton) Two clones Cryozootech, France Born in 2012, existence revealed in 2013 [67]
Tamarillo Tomatillo Replica Farms, USA Existence revealed in 2013 [68]
Cruising (Irish Sport Horse) Cruising Arish

Cruising Encore

Born in 2012, existence revealed in 2015 [69]
Otterongo Z Otterongo Alpha Z 2014
Storm Cat (Thoroughbred) Two clones Crestview Genetics, USA Born in April 2015, existence revealed in July 2015 [70]
As Cold As Ice Z As Cold As Ice Alpha

As Cold As Ice Bêta

2015
Cocaïne Z Cocaïne Alpha Z

Cocaïne Bêta Z

2015

2016

Cumano Z Cumano Alpha Z 2017

Opposition

color photo of a horse on a farm
One of the fears introduced by cloning is that it will jeopardize traditional horse breeding and reproduction techniques.

According to various surveys, including one carried out by Cheval Savoir in 2009, horse cloning is generally poorly accepted by riders and horse professionals. They believe it introduces unfair competition to "normal" horse breeders, while constituting a highly lucrative and ethically unacceptable activity. For French scientist Éric Palmer, the technique is demonized due to misunderstandings.[29] The American Quarter Horse Association has stated that "[...] clones have no parents, cloning is not breeding. It's just photocopies of the same horse", pointing to its low success rate and the risk of as yet unknown genetic disorder developing.[71] The Jockey Club was also strongly opposed.[34] Dr Thomas Reed, who owns the private stud Morningside in Ireland (where Hickstead was born), is also publicly opposed to cloning after the accidental death of his stallion in competition at the end of 2011.[48] In September 2015, the European Union banned all trade in cloned animals on its territory.[72]

Bioethics

Horse cloning, like that of other animal species, raises bioethical issues, since it involves a high mortality rate of embryos, fetuses and young foals. The Swiss National Stud's ethics study reports "massive loss during gestation", with less than 1% of oocytes obtained resulting in a live foal. Furthermore, foals born from cloning suffer from frequent health problems. An American study looked at 14 clones born between 2004 and 2008.[73] Six (43%) were normal, while the other eight suffered from neonatal disorders, umbilical problems and limb deformities.[73] There are a large number of stillborn foals, deaths in the first few days after birth, immune deficiencies, and muscle and bone deformities. Problems at foaling are common for both carrier mare and foal, with cesarean section being a common option. If foals survive their postnatal period, they do not appear to be more susceptible to disease thereafter. The question of their longevity remains unknown, as the first clones are still too young to draw any statistics.[74]

In the UK, researcher William (Twink) Allen was refused permission to continue his cloning trials in 2004 for these ethical reasons, as the cloned animals could present malformations, anomalies and diseases, according to the British authorities.[75] Dr. Natasha Lane, of the Society for the Prevention of Cruelty to Animals (SPCA by its acronym in French), said it was not acceptable to clone animals by sacrificing embryos "just to get a gold medal".[76] Allen spoke out against the decision, saying that the British government had "caved in to the animal protection lobby".[25]

Loss of genetic diversity and confiscation of living matter

On 8 June 2005, a number of French farmers belonging to the Confédération Paysanne demonstrated in front of the Genopole d'Évry, Cryozootech's headquarters, to denounce the "seizure of living matter" and a future loss of genetic diversity, arguing that the development of cloning will eventually lead to the disappearance of the breeding profession.[9] A number of specialists warn against the widespread use of cloning, believing that it will seriously damage the equine breeding industry, particularly in equestrian sport, by reducing demand for naturally-born foals. Cloning would also drastically reduce genetic diversity, as the same genes "could be reproduced over and over again".[77][78]

Fraud

One fear that has arisen with cloning is that of new forms of fraud. Ill-intentioned people will be able to steal mane from a prize horse that doesn't belong to them, and have it cloned. Current cloning technology makes it impossible to clone from mane, but it's evolving fast.[78][79] In studbooks that refuse to accept clones, particularly the Thoroughbred studbook, it would be possible to pass off a horse cloned from a champion as another animal, by falsifying its identification documents.[77]

See also

References

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Bibliography

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