History of Equine Cloning

History of Equine Cloning

Animal cloning has progressed significantly over the past decade. In particular, equine cloning has taken off quickly due to the remarkable advances in other species. According to Hinrichs (2014), subsequent trials have resulted in failures and important lessons that improve the genetic replication technique for future mainstream adoption. Still, there are multiple concerns regarding the health of such experimental animals and the overall effect to the equine industry.

The mammal cloning science grabbed worldwide news headlines in July1996 when Dolly was born. It was in the spirit of landing a man on the moon that numerous laboratories across the Western world pushed for analysis, duplication, and refining of the Scottish cloning outcome. The desire to seek an identical offspring of a talented horse is very tempting, especially for an owner of an extremely successful but rare gelded breed. Consequently, many look for cloning services for selective breeding purposes to solve a potential genetic stalemate. The owners castrate male horses for a good reason. However, when a gelding becomes a champion, people have second guesses. What they fail to understand is that if it was not gelded, chances are that he may have become a star. Essentially, studies indicate that gelding makes sports horses easier to train and safer to handle.

Cloning is similar to oocyte or semen freezing because it is a genetic banking technique. Therefore, a specific genetic line may provide the progeny years after the original horse is dead or no longer fertile (Lagutina et al., 2012). Similarly, such a school of thought is easily transferable to sterile horses that cannot produce offspring. The major motivating factor is a gelding that has succeeded as a sports champion but is unable to reproduce his own offspring. Cloning eliminates this concern because it restores the horse’s fertility and allows it to pass on the same nucleic DNA.

Cloning (also referred to as nuclear transfer) entails the removal of the cell nucleus hosting a distinctive DNA sequence. Then, the scientists place it inside an egg cell (oocyte) so that it can grow into a cloned animal. In advanced cases, the researchers will transfer the cell nucleus from any part of the body. Notably, the cell is yet to be specialized in its functions to a heart, skin, or hoof. Despite the latest development in this field, there is a lower chance of a viable offspring with a differentiated adult line than with embryonic cells. What many people do not know is that even before the lab production of Dolly the sheep, the pioneer researchers in genetic cloning had transferred the nucleus of cattle’s embryonic cells for more than a decade, although the use of cell differentiation was at an initial stage.

Essentially, the cell donor plays an insignificant role. Universally, scientists make a small incision on the subject for removal of a small amount of subcutaneous connective tissue beneath their top skin layer. Experienced practitioners can execute the procedure as long as they put the sample immediately into a non-defective holding media. Thereafter in the lab, scientists cut the samples into smaller fibroblasts to encourage donor cell growth. It is imperative for the oocyte to be mature and ready for fertilization prior to its reception of donor nucleus. Yet, the egg’s nucleus must be removed first. Rather than injecting donor nucleus into the oocyte cytoplasm, the modern technological advancement in nucleus transfer allows the fusion of donor cell with zona pellucida using an electrical pulse. In the case of equine cloning, a sperm extract is used to stimulate oocyte’s division and the eventual formation of an embryo (McCue, 2013). In fact, the horse sperm extract initiates calcium oscillations in the oocyte. Mainly, the transferred DNA starts to replicate since the sperm trigger sets embryonic development in motion.

            Equine cloning has attained breakthroughs and advances since the year 2000. Researchers made history at the dawn of the century by transferring the first equine nuclear, where the embryo replicated five cells. In the year 2002, Dr. Woods announced a success in mule cloning in his speech at the IERS (International Equine Reproduction Symposium. Within a year since the declaration, Utah State University’s efforts bore fruits as a surrogate horse gave birth to three healthy mules cloned from fetal cells (Woods et al., 2013). The students gathered DNA from a full sibling embryo. Eventually, one of them became one of the fastest racing mules. Furthermore, a cloned foal was born in the Italian Laboratory of Reproductive Technology (ILRT). Originally, ILRT obtained 840 Haflinger mare’s (Prometea) reconstructed embryos and implanted 15 of them. Interestingly, the mare that carried the foal embryo produced the adult donor cells, thus she carried an identical embryo twin to term (GAlli et al., 2013). Two years later, an Italian research group announced the delivery of a cloned horse, but septicemia killed him after 48 hours. The short-lived success inspired Texas researchers to copy two horses genetically that became third and fourth live cloned animals. Similar to mammal clowning pursuit, the births of three cloned mules in Russia’s NWERL (North West Equine Reproduction Laboratory) and the Italian Prometea clone ignited interests in horse cloning.

            Italian scientists conducted more than 100 embryo transfers that led to 9 successful pregnancies and one live foal. On the other hand, the Texas researchers transferred only 11 embryos that led to three pregnancies and two successful births. The difference between the two groups is in the quality of blastocysts because both labs had approximately 6% blastocyst rate.

More success in equine cloning came less than a year later as Texas A&M research group announced flawless births of half-a-dozen viable cloned foals. One donor contributed 12 embryo transfers that led to 9 pregnancies and five foals. The sixth cloned foal was from a different donor. In 2006, ViaGen (a commercial cloning firm) announced a birth of three cloned horses. Texas A&M was a prominent cloning firm and worked closely with ViaGen on several projects. For instance, in the year 1983, they cloned Smart Little Lena, a horse that won 1984 Cutting Horse Triple Crown. Besides, the clown gained prominence as a breeding stallion. Indeed, the Texas A&M scientists foaled five Little Lena clones. Other clones bred in the same year include Doc’s Serendipity, Tap o Lena, and Royal Blue Boon.

ViaGen partnered with Encore Genetics (equine marketing organization) to conduct the first commercial horse cloning operation in the country. In March 2006, the firms celebrated their cooperation and business merger by announcing separate births of two clones. Moreover, they raised hopes of a revival of former horse champions by claiming that dozens of other horses were pregnant with clones. The legendary Royal Blue Boon cutting horse registered by American authorities as a Quarter Horse became the first equine to be cloned commercially after Royal Vista Southwest farm’s recipient mare gave birth to a foal on 19^th February 2006 (Gambini et al., 2012). The health condition of the foal was stable and continues to thrive as at March 2016. Four weeks later, a clone of Tap O Lena mare joined her. Since then, the firm has sold hundreds of cloned foals worldwide.

 The string of cloning successes has proved that it is possible to improve animal species, even though the efficiency of the process varies from one laboratory to the other. Unfortunately, only two US firms have continued equine cloning. Limited research funding has hampered the efforts of an Italian group, thus resulting in its dormancy. Given that only a few firms perform the cloning today, it is difficult to analyze how the lab protocols affect the foal’s health.

The history of horse cloning spans a brief period of time. In this regard, an adequate and definitive research is yet to determine whether the cloned foal will be normal and healthy. Particularly, sheep and cattle industries observe noteworthy abnormalities with clones, for instance, fluid swelling, an abnormally large offspring, and heart issues. Fortunately, cloned foals do not show signs of these health conditions. Over the Equine cloning history, three adult-cell foals have died and there are 14 survivors.

In the year 2006, severe pneumonia killed a 4-day-old foal after it developed chest complications during the administration of anesthesia. Initially, veterinarians had tried to revive the clone’s chances of survival by performing four surgical corrections of its torn bladder (Hinrichs, 2015). However, they later noticed that it had severe extensive complications ranging from low blood pressure and dysfunctional kidneys. Clearly, the animal would not have survived to adulthood.

Moreover, Haflinger stallion Abendfrust and Prometea had a foal in late 2008. It preceded a clone foaled of Lynx Melody, a winner of 1980 NCHA Derby and 1978 Open Fruity Championship. Of keen to note is that Melody died in the year 2004, thus the veterinarians obtained clone tissue from his frozen carcass.  In addition, two clones of Jae Bar Fletch were born in 2008. The cloning activity took another turn when different research facilities produced champion performance horses such as Gem Twist, Califa, Scamper, and Cuartetera. They participated in various games such as polo, jumping, and barrel racing. In the United States alone, more than 64 clones were born in 2009.

The tests conducted after the birth of a Fire from God horse clone confirmed that indeed,  it was a genetic twin of her donor. However, an immune reaction occurred between the fetus and the mother, hence leading to a premature birth and death. Regardless, the team learned that this cloning technique will be useful for castrated male horses, leading to the use of resultant clones as studs (Allen, 2013).

In 2015, three cloned mules were healthy and had normal blood chemistry. In fact, two of them participated in a competitive race. However, it is notable that the trio was cloned from fetal cells, hence the considerable health difference in comparison to those cloned from adult cells. Statistics indicate that up to 50%of adult-cell foals need neonatal care during birth due to health problems. Some of the health complications include enlarged umbilical remnants and tendon contraction in leg joints. In the United States, the veterinarians litigate and surgically remove the defective parts, but the cloned animals never fully recover. There is also another common syndrome of maladjustment and animal weakness attributable to oxygenation difficulties after birth.

Arguably, the clones are highly susceptible to health problems as compared to normal foals because of oocyte’s failure to reprogram the donated DNA genes. In a normal fertilization process, years of evolution allows natural reprogramming so that all the embryo cells are turned differently to perform distinct functions. The main point of contention lies in premature aging that affected Dolly. Ideally, non-coding caps and telomeres on the chromosome tips reduce the time taken by cells during replication, thus are shorter in elderly mammals. Considering this, Dolly’s telomeres were shorter given that scientists cloned her from mammary cells that do not regenerate telomeres.

The majority of US population has not accepted with enthusiasm the effects of horse cloning for breeding purposes. Nobody is aware of the absolute effects of cloning in 2016. It is unclear if the scientific advancement in this field introduces more mutants that can result in generational disasters in case of cross-breeding with normal horses. There are two genetic diseases often associated with line-breeding of horses. While breeders emphasize on particular genetic lineages, the number of horses affected by HERDA and HYPP mutations rise sharply, especially if the line-breeding technique traces back to a mutant sire.

The horse cloning boom in 2010 prompted the American government to pass laws that restrict the practice in US soil. Hundreds of American horsemen source for such services from outside the country. They ship out frozen semen and embryos for cloning in other developed countries. In particular, many of the horsemen are interested in the genes of Arabian endurance champion reproduced successfully for the first time in an Italian lab.

According to Vanderwall et al. (2015), it is ironic that doping carries significant sanctions and heavy penalties while genetic engineering of a sports animal bears no sanction in the EU, the US and the rest of the planet. Black Caviar was an enormously successful horse and was cloned in the year 2014. Yet, no sports regulation authority took a legal action against the cloning firm for unethical practice. Either way, if cloning enlarges the lung capacity or the overall athletic frame, then the success of the next generation of racetrack horses is guaranteed. The owners can use this argument as a justification for obscene expenditures in equine cloning experiments. In 2016, champion stallions are used mainly for breeding purposes because they command a high price for the service. Nevertheless, there is no guarantee of success because of differences in the mare quality. If a prize-winning horse must be cloned, the government should provide a patent protection for the foal so that the scientists can far more closely control the animal’s bloodline than can be done using traditional means.

In summary, it is clear that horse cloning has led to the restoration of bloodlines that would otherwise be lost through gelding. Remarkably, equine cloning has proved successful in comparison to the cloning of other mammal species such as sheep and cattle. Notably, a cloned horse is unlikely to be a competitor or an exact copy of the cell donor, given the vast difference from its father’s situation. Undeniably, cloning is both labor-intensive and costly. Thus, should only be applied whenever the need for genetic preservation arises. Cloned stallions should produce similar foals as a donor. However, mare clones bear slightly different progeny because of diverse mitochondrial DNA.

References

Allen, W. R. (2013). The Development and Application of the Modern Reproductive Technologies to Horse Breeding. Reproduction in Domestic Animals, 40(4), 310-329.

Gambini, A., Jarazo, J., Olivera, R., & Salamone, D. F. (2012). Equine Cloning: In Vitro and in Vivo Development of Aggregated Embryos. Biology of Reproduction, 87(1), 15.

Hinrichs, K. (2014). A Review of Cloning in the Horse. In Proc Am Ass Equine Pract (pp. 398-401).

Hinrichs, K. (2015). Update on Equine ICSI and Cloning. Theriogenology,64(3), 535-541.

Lagutina, I., Lazzari, G., Duchi, R., Turini, P., Tessaro, I., Brunetti, D., ... & Galli, C. (2012). Comparative Aspects of Somatic Cell Nuclear Transfer with Conventional and Zona-Free Method in Cattle, Horse, Pig and Sheep. Theriogenology, 67(1), 90-98.

McCue, P. M. (2013). History of Embryo Transfer. Equine Reproduction Laboratory-Colorado State University, In: http://csucvmbs. colostate. edu/Documents/Learnmares11-ET-history-apr09. pdf, acedido a, 11, 2013.

Vanderwall, D. K., Woods, G. L., Roser, J. F., Schlafer, D. H., Sellon, D. C., Tester, D. F., & White, K. L. (2015). Equine Cloning: Applications and Outcomes. Reproduction, Fertility and Development, 18(2), 91-98.

Woods, G. L., White, K. L., Vanderwall, D. K., Li, G. P., Aston, K. I., Bunch, T. D., ... & Pate, B. J. (2013). A Mule Cloned from Fetal Cells by Nuclear Transfer. Science, 301(5636), 1063-1063.