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. 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.
The horse is the seventh species to be cloned yet.(See Cloning for a list and names)
History
Horse cloning has undergone a rapid qualitative and quantitative evolution. While Italian professor Cesare Galli believes that horse cloning has aroused less interest than that of other large mammals, 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. 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), 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. The value of cloning high lineage horses was recognized as early as 1998, with the Westhusin study. Research to this end was publicly announced in 2001. 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.
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. The first successful attempt to produce a viable clone was made by the Italian laboratory LTR-CIZ, which gave birth to Prometea on May 28, 2003, a Haflinger foal carried to term by her mother, whose genetic copy she is. Her birth was announced publicly on August 6, 2003. The name "Prometea" is the feminine form of Prometeo ("Prometheus" in Greek). These scientists worked under the guidance of Professor Cesare Galli.
Dr. Cesare Galli and others at the lab experimented with 841 reconstructed embryos; of the 14 viable embryos, four were implanted in surrogate mothers - only that of Prometea succeeded in being born. Prometea was born to her twin mother who her cloning cells originated from. Texas A&M University was also undertaking a horse-cloning project when the Italian team first succeeded.
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. Prometea and Pieraz were obtained using the same method, that of Professor Galli.
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. 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. 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. In May 2013, a non-clone foal was born for the first time from two parents cloned by embryo transfer. On 7 December 2013, a cloned polo pony won a major sporting competition for the first time. It was the Argentine polo championship. In 2018, equine cloning was widely used in Argentina's polo scene. The Argentine polo horse has become the most cloned animal in the world.
Technique
Cloning research is often carried out in secret, due to poor public acceptance. Commercial cloning companies sometimes reveal the births of these horses, but the techniques employed remain mostly secret. 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. Due to the high demand for mare oocytes, these are usually obtained from slaughterhouses.
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.
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. 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, the first horse, which required 328 attempts. The first mule trials, in 2003, involved 118 embryos, 13 of which produced a gestation, resulting in 3 live mules. To clone Calvaro V in 2006, Cryozootech used over 2,000 oocytes, which produced 22 embryos, only one of which was carried to term.
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%. Argentine researchers estimate that 6 or 7 embryos are needed out of 20 trials (in 2013). In 2010, according to a French source, around 2,500 mare oocytes had to be used to obtain a single viable foal. 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. There is nothing to differentiate a clone from a conventionally bred horse.
Usage
The cost of equine cloning varies between €200,000 and €300,000, depending on the source. In 2010, clones intended for sporting competitions represented just 22% of operations. 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. The same applies to a stallion that has become too old to reproduce, or to a mare whose number of foals is naturally limited. The use of cloning relies heavily on the belief that DNA is the most important factor in competition performance. 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.
Effective cloning remains very marginal due to its cost. 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. Cloning can also be used to preserve rare breeds threatened with extinction, but customers' motivations are essentially commercial. 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. The foal's DNA matched the Lena horse (Equus lenensis), which went extinct around 5,000 years ago.
After the last Abaco Barb, a mare named Nunki, died in 2015, her cells were frozen for cloning purposes. In 2017, Viagen and other partners pledged services valued at approximately $2 million to support the project. Viagen nurtured enough genetic material to create two clones of Nunki, but it is unclear if the project was successful. One of the main issues with the Abaco Barb was inbreeding, with a population of 30 horses descended from just three individuals noted in 1992, and no new foals were born after 1999. The Wild Horses of Abaco Preservation Society, the nonprofit that manages the cloning project, proposed crossing Nunki's clones with selected Spanish Mustang or Colonial Spanish Horse stallions to revive the breed, as well as add genetic diversity.
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. Mitochondria represent only 1 or 2% of the genome, but could influence the clone's sporting performance. They are more important in the case of a mare than a stallion, since the mare transmits her mitochondria during reproduction, unlike the stallion. 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. 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. The ban on clones in a large number of stud books and in certain competitions also limits interest.
Specialized companies