History of Horses and the Biotechnologies Applied to Its Reproduction

Abstract

The history and domestication of horses date back to thousands of years, making these animals an integral part of human life. Over time, selective crossbreeding led to the creation of different breeds with specific characteristics that made them suitable for various tasks. The advent of modern technology, such as the automobile and the railroad, reduced the dependence on horses as a means of transportation, but their importance persists in sports, recreational, and therapeutic activities. The special interest in this animal species and the need to improve its qualities have led to the development of different reproductive technologies. In recent decades, significant advances have been made, providing new opportunities to improve equine breeding and preserve valuable genetic lines. Artificial insemination, embryo transfer, in vitro fertilization, and gamete freezing, among others, have revolutionized the equine industry by facilitating selective breeding, genetic preservation, and improvement of animal quality. In this chapter, we present a historical description of the evolution of the horse and at the same time a review of the most relevant aspects of its domestication. Likewise, we make a review and description of the reproductive technologies that have marked an important advance in the knowledge of the physiology and reproductive improvement in this animal species.

1. Introduction

The history of horses can be traced back to over 50 million years ago when they first evolved in North America. According to a study published by Prothero in 2017 [1], horses’ evolution can be traced through their teeth, and they found that the earliest known horse, called Hyracotherium or Eohippus, was about the size of a small dog and had four toes on its front feet and three toes on its back feet. Over time, horses evolved into larger and more powerful animals with a single toe on each foot.

Evolution in equids is not represented linearly, but in a branched way. Therefore, there are no evolutionary similarities in all lines of horses. In general, horses became progressively larger. However, some genera, such as Eohippus, became smaller. Horses from 5 to 10 million years ago had three toes, instead of one like today’s horses. These one-toed individuals prevailed as the three-toed lines became extinct. Moreover, these traits did not necessarily evolve together or at the same rate. Several structural aspects changed in a series of modifications. For example, during the Eocene, only the teeth evolved, and the feet had little change. However, in the Miocene, both feet and teeth evolved at a dizzying rate. The rate at which horses evolved depended on the pressures faced by the equids. Along with today’s horse, other species of the genus Equus also evolved, such as the donkey, the onager, and the zebra. If we trace a line of descent from Eohippus to Equus, the fossils found show four guidelines: 1.- Reduction in the number of toes, 2.- Increase in the size of the teeth, 3.- Lengthening of the face, and 4.- Increase in body size [2].

2. Domestication of horses

Around 10,000 B.C., humans began domesticating horses for transportation, agriculture, and warfare. The domestication of horses revolutionized human society, as it allowed for faster and more efficient travel, making trade and communication easier. According to a paper published in the Journal of Anthropological Archaeology, the domestication of horses occurred in multiple regions of the world, including the Middle East, Central Asia, and China [3]. Horse domestication refers to the process by which horses were tamed and trained for human use. The domestication of horses is believed to have occurred around 4000–3500 B.C. in the Eurasian Steppe, with evidence of horse domestication found in archeological sites in Kazakhstan and Ukraine [4].

One of the key factors in the domestication of horses was their use in transportation. Domesticated horses were used to pull carts and wagons, as well as for riding. This greatly improved human mobility and allowed for the expansion of trade and communication networks. Horses were also used in warfare, providing speed and mobility on the battlefield [5].

Another factor that contributed to horse domestication was their use in hunting. Domesticated horses were used to track and pursue game, particularly in the Eurasian Steppe, where hunting was an important source of food for early humans [5]. Domesticated horses were also used in herding, allowing humans to manage and control large herds of livestock [6].

The process of horse domestication involved selectively breeding horses for certain traits, such as docility, strength, and speed. Over time, this led to the development of different horse breeds with specific characteristics suited for different tasks, such as racing, riding, and draft work. Domestication also involved the training and socialization of horses, including the use of bits, bridles, and saddles [7].

Despite the many benefits of horse domestication, there were also negative consequences. The use of horses in warfare led to the development of new forms of violence and the expansion of empires through conquest [6]. Horses have been used in battles throughout history, providing soldiers with greater mobility and enabling the rapid movement of troops and supplies. According to the book “War Horse: A History of the Military Horse and Rider,” written by Louis A. DiMarco, horses were used in battles for thousands of years, and their contribution to warfare continued until the development of modern technology made them less necessary [8]. The domestication of horses also had environmental repercussions, such as overgrazing and deforestation, as humans cleared land for pasture and feed [7].

The role of horses in transportation has also been significant throughout history. In the 1800s, horses were the primary means of transportation in urban areas, pulling streetcars, omnibuses, and carriages. They also performed an important function in the development of the Wild West, as they were used for transportation and hauling goods during the Gold Rush. According to a paper published in the journal Transfers, the use of horses in transportation declined significantly in the 20th century due to the rise of the automobile [8].

Today, horses continue to play an important role in human society, though their uses have shifted from transportation and warfare to recreation and sport such as jumping, dressage, rodeo, horse racing, and polo, which are popular around the world, and many people enjoy horseback riding for leisure. According to the Food and Agriculture Organization of the United Nations, there were approximately 57 million horses in the world as of 2022 [9].

2.1 The use of the horse in Spain

Horses have also had an important place in Spanish history and culture for centuries. The Iberian Peninsula, which includes modern-day Spain and Portugal, is considered to be the birthplace of the horse breed known as the Andalusian, or Pure Spanish Horse. The Andalusian breed has been used for a variety of purposes, including war, bullfighting, and classical dressage.

Horse riding is a popular sport in Spain, and the country is well-known for its equestrian traditions. The Spanish Riding School, for example, is one of the oldest and most prestigious equestrian schools in the world and has been training horses and riders for over 450 years. Spain is also famous for its bullfighting events, which often involve skilled horsemen called picadors who use horses to control the bull [10].

In addition to bullfighting, horses were in Spanish military history. The Spanish cavalry was known for its skill and bravery in battle, and horses were used extensively in the Spanish conquest of the New World. The horses used by the Spanish cavalry were typically Andalusians or other Iberian breeds, which were prized for their agility, endurance, and courage [10].

Today, horses remain an important part of Spanish culture and heritage. The Andalusian breed is still popular for its beauty and versatility and is used for everything from dressage to pleasure riding. Horse doma clasica, or classical dressage, is a form of equestrianism that has its roots in ancient Greek horsemanship. It involves training horses to perform a series of precise and controlled movements, such as pirouettes, piaffes, and passages, all done in harmony with the rider [11].

3. Evolution of equine reproductive technologies

Reproductive technologies have been extensively studied and developed in horses to improve breeding efficiency and genetic diversity. The application of reproductive technologies in horses has evolved, from traditional breeding methods to advanced techniques that allow for the preservation and manipulation of genetic material.

3.1 History of artificial insemination in horses

One of the earliest reproductive technologies used in horses was artificial insemination (AI). AI involves the collection and storage of semen from a stallion and its subsequent use to artificially inseminate a mare. This technique allowed for the distribution of genetic material from highly prized stallions to a greater number of mares, thus increasing the genetic diversity of the population [12].

The first recorded successful artificial insemination in horses was in 1901 by the Russian biologist I. Ivanov. He was a landmark in the development of the technique. Ivanov organized the first equine AI center; improved methods of collecting, diluting, and transporting semen; and introduced a technique for inseminating mares. Later, in the 1930s, the development of artificial vagina techniques allowed for the collection of semen from stallions with greater ease and efficiency [13]. Although this was a significant achievement at the time, it took several decades before AI became a widely accepted practice in the equine industry [14].

In the 1930s, researchers at the University of Kentucky began to investigate the use of AI in horses, and by the 1940s, they had developed a technique for collecting and processing equine semen for AI. However, it was not until the 1960s that AI became widely accepted in the equine industry, and it is now commonly used for both breeding and reproductive management purposes [14].

One of the breakthroughs in the history of AI in horses was the development of cryopreservation techniques for semen. In the 1950s, researchers began experimenting with freezing and storing stallion semen, which made it possible to extend the availability of high-quality semen for use in AI [15]. The introduction of liquid nitrogen for long-term storage further revolutionized the use of frozen semen in horse breeding [16].

Freezing equine semen involves cryopreservation, a process in which the semen is cooled to very low temperatures, typically using liquid nitrogen, to preserve its viability. This technique has revolutionized the horse breeding industry by allowing breeders to utilize the genetic material of high-quality stallions worldwide. Freezing semen also provides a practical solution for transporting semen to mares that are geographically distant from the stallion, expanding the range of breeding possibilities [17].

To freeze equine semen, it first needs to be collected from the stallion using artificial vagina techniques. After evaluation, the semen is processed to remove the seminal plasma and dilute it with an appropriate extender to protect the sperm cells during the freezing process. The extender often contains cryoprotectants, which help safeguard the sperm against damage caused by ice crystal formation [18].

Over time, improvements in semen processing techniques have contributed to the success of AI in horses, and both fresh semen and thawed frozen semen AI are widely used in the equine industry. The typical dose of fresh semen for insemination into the mare’s uterine corpus uteri is >300×10⁶ progressively motile sperm (PMS). The minimum recommended dose has been set at 250×10⁶ PMS for frozen-thawed semen [19]. The use of centrifugation and filtration methods to separate and concentrate sperm cells from the seminal fluid has enhanced the viability and motility of the spermatozoa, increasing the chances of successful pregnancies through AI [20].

The use of artificial insemination in horse breeding has had a significant impact on genetic selection. By enabling breeders to access stallions located in different regions or countries, AI has facilitated the spread of desirable traits and genetic diversity within various horse breeds. It has also provided opportunities for breed improvement and the preservation of rare or endangered equine populations [12]. The ability to select stallions based on their performance, conformation, and genetic profiles has contributed to the overall advancement and quality of horse breeds [21].

Today, AI is a standard breeding method used in many equine operations worldwide, and advances in technology have led to improvements in semen collection, processing, and storage techniques. Additionally, the use of AI has facilitated the widespread use of frozen semen, which allows stallions to breed mares across long distances or even posthumously. Despite these advances, AI is not without its limitations, and researchers continue to explore new techniques to improve the success rates of AI in horses [22].

However, AI has its limitations, especially in cases where the semen quality is poor, or the mare has a history of reproductive problems. In the case of mares with a risk of uterine complication following conventional inseminations, the low-dose intrauterine insemination technique (LDIUI) was developed and evaluated by different research groups [23, 24, 25]. This technique is based on depositing in the uterine horn a smaller number of spermatozoa compared to normal AI to achieve pregnancy [25].

In the hysteroscopic insemination technique, a pre-warmed 1.2 or 1.6 m flexible video endoscope is inserted vaginally into the mare’s uterine body. The endoscope operator inserts his hand into the previously evacuated rectum and identifies the tip of the endoscope by rectal manipulation and advances the endoscope toward the tip of the uterine horn ipsilateral to the ovary containing the preovulatory follicle [26].

On the other hand, in the rectally guided deep horn insemination technique, the placement of a flexible catheter about 65 cm long is performed in two phases. First, vaginally, it is introduced into the uterus through the cervix. Next, the inseminator’s hand is removed from the vagina and inserted into the previously evacuated rectum. The tip of the pipette is identified and advanced by rectal manipulation and gentle pressure toward the tip of the horn ipsilateral to the ovary that has the preovulatory follicle, and the dose of semen is deposited [26].

Results suggest that LDIUI is beneficial for mares with a history of breeding challenges and had comparable pregnancy rates with normal AI [25]. Beyond what has been previously described, each AI technique has its advantages and limitations, and the choice of method depends on various factors, including the mare’s reproductive health, the stallion’s fertility, and the breeding goals. To overcome these limitations, advanced reproductive technologies, such as in vitro fertilization (IVF) and embryo transfer (ET), were developed.

3.2 Embryo transfer

Embryo transfer in horses is a reproductive technology that has been used for several decades to facilitate the reproduction of high-value mares or stallions, preserve genetic diversity, and accelerate genetic progress. The technique involves the removal of an embryo from the uterus of a donor mare and transferring it into the uterus of a recipient mare that will carry the pregnancy to term [27].

The first successful embryo transfer in horses was reported in the late 1960s by a team of researchers led by Dr. Gordon Woods at Colorado State University. They collected embryos from donor mares using a surgical technique and transferred them into recipient mares. Although the success rate was low, the technique showed promise and paved the way for further research [22].

In the 1980s, embryo transfer in horses became a commercial enterprise, with several companies offering services to horse breeders. The technique was particularly useful for breeding high-performance mares that were competing in equestrian sports, as it allowed them to continue competing while their embryos were being transferred to recipient mares. It has also been employed in mares with reproductive pathologies or orthopedic conditions that prevent them from successful gestation or foaling a foal [28].

The process of embryo transfer involves several key steps, including superovulation of the donor mare, artificial insemination, embryo recovery, and embryo transfer. Over the years, advances in technology have made embryo transfer in horses more efficient and effective. Mare superovulation is a reproductive technique used to stimulate the ovaries of mares to produce multiple mature oocytes (eggs) in a single estrous cycle [29]. The age of the mare, as well as the superovulation protocols, can also influence their efficiency [30]. Different hormonal preparations and protocols have been used to superovulate in cyclic mares. Multiovulation of mares during the reproductive season can be achieved via treatment with equine pituitary extract (EPE), purified equine FSH (eFSH), or recombinant equine FSH [31, 32, 33, 34, 35], or recombinant eFSH and recombinant equine LH (eLH) [35, 36, 37]. However, great variability in results has been reported.

Regarding EPE preparations, a 5:1 FSH/LH ratio has been verified by radioimmunoassay [38]. When EPE was given twice daily, with the time of initial treatment 5–6 days after ovulation, it increased the number of follicles >35 mm, ovulation rates, and embryo recovery rates when compared with once-daily injections of the same dose: 6.1 versus 2.0 follicles, 7.1 versus 2.4 ovulations, and 3.5 versus 1.6 embryos, respectively [39]. Related to eFSH preparations, in the 2000s Bioniche Animal Health Labs, a semi-purified EPE with an FSH/LH ratio of 10:1 became available. When eFSH was given to mares twice daily (25 mg IM), starting on day 5–6 post-ovulation during the ovulatory season, the number of follicles >35 mm increased from 1.1 to 6.7 [40].

Procedures for nonsurgical embryo collection in mares were developed in the late 1970s [41, 42]. Embryo collection is typically performed around 7 days after ovulation. Transvaginal ultrasound-guided techniques are used to collect the embryos from the donor mare’s uterus. The embryos are flushed out using a catheter and then recovered and examined for quality and viability [43].

The most appropriate day for embryo collection remains controversial. Embryo collections carried out between days 7 and 8 post-ovulation recovered a greater number of embryos [27, 44]. Other authors state that they could achieve similar rates of embryo recovery on days 5 to 8 after ovulation [45].

Embryo transfer in horses is also used as a tool for genetic preservation. In some cases, mares or stallions with valuable genetics may die before they can reproduce naturally, and embryo transfer can be used to produce offspring using their stored genetic material [46]. Other advancements include the use of IVF and intracytoplasmic sperm injection (ICSI) to produce embryos [47].

According to the International Embryo Transfer Society (IETS) in 2021, the total production of in vivo developed was 25,475 transferable embryos. From these numbers, South America produces 24,125, which represents 94.70% of the world’s production. Meanwhile, Europe informs 772, representing 3.03% of the total production [48].

3.3 In vitro fertilization

In vitro fertilization in horses is a reproductive technology that involves the collection of oocytes from a mare, fertilization in the laboratory with semen from a stallion, and subsequent transfer of the resulting embryo to a recipient mare. This technique makes it possible to produce multiple offspring from a single mare or stallion and preserve the genetic material of rare or valuable individuals. The first successful IVF performed in horses was reported in 1975 by the team of Dr. Teruhiko Wakayama and Dr. Katsumi Takahashi in Japan. The pioneering work of Wakayama and Takahashi opened a new era in equine reproduction and laid the foundation for further advances in the field [49, 50].

The first successful application of IVF in horses occurred in the late 1970s at Colorado State University. Using this technique, an effective method for collecting, fertilizing, and culturing equine oocytes in the laboratory was developed [51]. And the first foal produced via in vitro fertilization (IVF) was born in 1990 [52].

Equine in vitro embryo production involves different steps: oocyte collection, maturation, fertilization, and embryo development [53]. Oocyte collection can be done from live mares as well as from ovaries collected from mares postmortem or at the slaughterhouse [22].

3.4 Oocyte collection from live mares

Two methodologies have been described for the collection of oocytes from live mares: the recovery of immature oocytes from all follicles of the ovary by transvaginal follicular aspiration guided by ultrasound also called Ovum Pick-Up (OPU) and the recovery of the mature oocyte from the dominant preovulatory follicle after the administration of hCG or a GnRH analog [22].

3.5 Ovum pick-up

The in vivo collection of oocytes by the technique of Ovum Pick-Up (OPU) is a reproductive technology used in equine breeding to collect immature oocytes from a mare’s ovaries. The procedure involves ultrasound-guided aspiration of follicles containing immature oocytes using a needle attached to a suction pump. OPU has revolutionized the field of equine reproduction, allowing the production of genetically superior horses through IVF and ET techniques [22].

In the early days of equine OPU, the procedure was only possible through laparotomy, which is a surgical method that involves making an incision in the mare’s abdomen to access the ovaries. The first successful collection of equine oocytes by laparotomy was reported by Hinrichs and Kraemer in 1989. However, laparotomy is an invasive technique that carries the risk of complications such as infection, adhesions, and damage to the reproductive tract. Therefore, researchers began to explore less invasive methods of collecting equine oocytes, which led to the development of ultrasound-guided OPU [54].

Ultrasound-guided OPU was first described in horses by Seidel et al. in 1993. The authors demonstrated that it was possible to collect oocytes from mares using a transvaginal ultrasound-guided needle aspiration technique, which eliminated the need for laparotomy. This technique has since become the gold standard for equine OPU, as it is minimally invasive, safer, and less time-consuming than laparotomy [55].

OPU in the horse is similar in concept to that performed in cattle but differs in several aspects. First, equine oocytes have a strong attachment to the follicle wall [56]. In addition, there are projections of cumulus cells into the underlying theca, which anchor the oocyte-cumulus complex (OCC) to the follicle wall [56]. These factors make equine oocyte aspiration challenging. Therefore, when oocytes are retrieved by aspiration, they are usually detached from the surrounding cumulus and have only the corona radiata attached. OPU method involves the use of ultrasound imaging to visualize the ovaries and identify the dominant follicles. A needle is then introduced through the vaginal wall, guided by the ultrasound, to aspirate the follicular fluid containing the oocyte.

3.6 Oocyte collection from the dominant preovulatory follicle

On the other hand, to retrieve the oocytes from the preovulatory follicle, it is necessary to monitor the mare’s ovarian activity by ultrasonography. When the follicle is LH-sensitive, hCG or GnRH analog is administered. Aspiration can be performed 24–35 h by OPU [57], after gonadotropin treatment so that ovulation does not occur before aspiration [22].

Since the development of OPU, numerous studies have been conducted to improve the efficiency and success rates of the technique. Some of the key areas of research include optimizing the timing of OPU, refining the needle aspiration technique, and improving the quality of the collected oocytes. These studies have led to significant advancements in equine reproductive technology, allowing for the production of high-quality embryos and the preservation of genetic diversity in rare and endangered horse breeds [58].

3.7 Oocyte collection from mares postmortem or at the slaughterhouse

In case of mare death, it is advisable to remove the ovaries in less than 6 to 8 hours to have a better chance of producing embryos [59, 60]. Then, the ovaries are transported at body temperature (37°C) if the transport time is <2 h or at room temperature or slightly lower (15–20°C) for longer transport times [61].

As explained above, due to the characteristics of equine oocytes within the follicle, an effective, albeit slow, method for retrieving oocytes from excised ovaries is to completely open each follicle with a scalpel and scrape the entire inner surface of the follicle with a curette to remove the granulosa cell layer. This procedure is associated with the retrieval of whole COCs and has resulted in good development of the retrieved oocytes [62, 63, 64].

3.8 Oocyte and embryo cryopreservation

Equine oocyte cryopreservation is an important step for preserving genetic material independent of time and geographic location [22, 65]. Oocyte cryopreservation protocols can be divided into two categories: 1.- slow freezing and rapid thawing, also called slow cooling, and 2.- rapid cooling and heating, also called vitrification. The slow cooling protocol requires a programmable freezer, while vitrification does not require specialized equipment. Early attempts at oocyte freezing employed the slow-freezing method [66, 67]. However, these protocols have resulted in relatively low survival and pregnancy rates [68, 69].

Currently, vitrification is the most used cryopreservation technique for equine oocytes [70, 71]. Historically, vitrified equine oocytes have improved their MII rates (28–46%); however, approximately 50% of oocytes that reach metaphase II have spindle abnormalities and low developmental competence [72]. Vitrification requires relatively high concentrations of cryoprotectants (CPA). These can be penetrants such as ethylene glycol (EG), dimethyl sulfoxide (DMSO), or propylene glycol (PG) [73, 74, 75, 76]. and non-penetrants such as sucrose, glucose, trehalose, or fructose [77]. In equine oocyte vitrification protocols, the most common combinations of permeant cryoprotectants are EG/DMSO or EG/PG [70, 71, 72, 78, 79].

An experiment conducted early in the 2000s reported rates of 12% blastocysts and 2 foals born after fertilization of in vivo matured oocytes using EG, DMSO, sucrose, and ficoll. In the above protocol, cryoprotectants were loaded in three steps: first; 5% DMSO and 5% EG for 30 s; second; 10% DMSO and 10% EG for 30 s; and third; 20% DMSO, 20% EG, with 0.65 M sucrose for ∼20 s [80]. The three-step vitrification protocol has been reported to result in less damage to the oocyte by both cytotoxicity and osmotic effect [81, 82]. Using a protocol similar to that for bovine embryos, in 1985, Slade et al. [83] reported an 80% pregnancy rate with embryos (morula or early blastocyst) that had been slowly cooled.

As mentioned above, the vitrification procedure contains high concentrations of cryoprotectants and is characterized by ultra-rapid cooling in which the rapidly cooling solution produces a vitreous formation instead of ice. Data reported from two large commercial farms in the United States indicate that pregnancy rates of fresh and chilled embryos in one farm were 75 and 70%, respectively. While the pregnancy rate of vitrified/warmed embryos at the second farm at 45 days was 51% [84].

Over the years, researchers and veterinarians have made improvements to the IVF procedure for horses, with key advances being made in areas such as oocyte retrieval, in vitro maturation, fertilization techniques, and embryo culture [85, 86, 87]. In 2021, the total production of in vitro developed was 11,619 transferable embryos. From these numbers, Europe produces 6775, which represents 58.30% of the world’s production [48].

3.9 Intracytoplasmic sperm injection (ICSI)

Intracytoplasmic sperm injection technique means that one selected sperm is injected directly into the cytoplasm of the mature oocyte. ICSI is a technology that allows obtaining pregnancies from mares that no longer provide embryos in an ET program. Likewise, ICSI is extremely useful in the case of old stallions, with poor semen quality, oligospermic, or for which a limited amount of frozen semen is available or has died. Therefore, it is presented as a procedure of choice in cases of infertility in both mares and stallions [88, 89, 90].

Mature oocytes can be obtained in vivo by aspirating a mare’s preovulatory follicle after stimulation with gonadotropins, or they can be obtained by in vitro maturation of oocytes collected from small immature follicles, either in vivo or postmortem. Then, under the aid of a micromanipulator, a spermatozoon is collected in a micropipette and injected into the cytoplasm of the mature oocyte [62, 89].

Sperm selection for ICSI is performed using different methods [91]. Normally, a morphologically normal and progressively motile sperm is selected. Before or while preparing the semen, the oocyte is separated from the cumulus cells using hyaluronidase by gently pipetting [91].

Once the oocyte has been injected, it is necessary to culture the presumptive zygote to obtain and produce viable blastocysts. The most commonly used system is DMEM/F12 medium with 10% Fetal Bovine Serum (FBS) [62, 85, 92]. In the horse, it is the cleavage time, which occurs between 12 and 24 h after ICSI [91] and, in those embryos that develop normally, between 5 and 7 days after ICSI blastocyst formation is observed [91].

The first ICSI research foal was born in 1996 [93], but the procedure was not used commercially until the early 2000s. Initially, laboratories working with ICSI in the horse had difficulty achieving good embryo development rates; the results were inconsistent, showing cleavage rates ranging from 20 to 65% [94].

Afterward, in 2002, the use of the piezoelectric drill bit for ICSI was reported to increase both activation and cleavage rates [95, 96]. This device causes small vibrations in the sperm injection pipette, which facilitates penetration into the zona pellucida and ensures the rupture of the plasma membranes of the sperm and oocyte. The use of this device on in vitro matured oocytes resulted in more than 80% cleavage, with an average of >8 cells per embryo cleaved at 96 h of in vitro culture [95].

Another development in equine reproductive technologies is cloning. Cloning involves the production of a genetically identical individual to the donor animal. The first successful cloning of a horse was reported in 2003 [26]. Cloning has been used to produce copies of successful performance horses, such as the Olympic gold medalist, Gem Twist, who was cloned in 2008 [27]. However, cloning raises ethical concerns related to animal welfare and genetic diversity, as well as questions about the authenticity of equine sports.

In conclusion, the evolution of reproductive technologies in horses has allowed for the improvement of breeding efficiency and genetic diversity. AI, IVF, ET, OPU, and cloning are some of the reproductive technologies available or used in the equine industry. These techniques have their advantages and limitations, and ethical considerations must be considered when using them.