Cloning via nuclear transfer involves the transfer of a donor diploid cell into an enucleated oocyte. The oocyte-cell complex is then artificially activated to develop into an embryo, which shares a nuclear genome identical to that of the donor animal. This technique, known as somatic cell nuclear transfer (SCNT), enables the production of genetically identical clones of the donor.
The cloned embryo typically develops in the laboratory to the blastocyst stage, after which it can be either cryopreserved or transferred into a surrogate female for gestation, resulting in an offspring that is genetically identical to the donor.
Although the nuclear genome of a clone is identical to the donor animal's genome, factors such as mitochondrial DNA and epigenetic modifications can influence the clone’s final phenotype.
Cloning in domestic animals is most commonly applied in horses, cattle, dogs, and camels. In veterinary practice, it is used primarily to reproduce elite breeding animals; to edit genes; to preserve the genetics of high-performing, valuable, or companion individuals; or to support conservation efforts for rare or endangered breeds.
Donor Cell Selection and Cloning Techniques
Selection of the donor cell has been one of the most extensively explored areas in cloning research. Cells derived from early embryos, up to the morula stage, are highly efficient as donor cells. Cloning using embryonic cells was successfully conducted for more than a decade before the landmark birth of the first mammal produced via nuclear transfer from adult somatic cells—Dolly, a sheep cloned by the Roslin Institute in Scotland in 1996.
In contrast to cloning from embryonic stem cells, SCNT, which uses differentiated somatic cells as nuclear donors, offers the distinct advantage of duplicating proven genetics from adult animals.
Among somatic cells, subcutaneous connective tissue is the most common source used for cloning adult animals. The tissue is mechanically disaggregated and cultured in vitro, where fibroblasts outgrow and are harvested for passaging in new culture dishes to promote proliferation. This process is repeated until millions of cells are produced, which are then cryopreserved for future cloning applications.
Studies have suggested that the use of stem cells could potentially improve the efficiency of animal cloning. This remains a subject of debate; however, commercial cloning companies are increasingly adopting bone marrow–derived stem cells for horse cloning.
For nuclear transfer to be successful, mature oocytes from the same species or closely related subspecies are required. Although the genetic value of oocytes is not critical, their mitochondrial composition might influence the outcome, because the resulting clone inherits the mitochondria of the host oocyte.
Oocytes are typically recovered from slaughterhouse material or, in some species, collected from live animals through ovum pickup (OPU) and subsequently matured in vitro. In some species, such as dogs, mature oocytes must be collected either from preovulatory follicles or directly from the oviduct after ovulation.
Nuclear transfer is performed under a microscope using micromanipulation tools as follows:
The chromosomes (genomic DNA) of the oocyte are removed, creating an enucleated oocyte, or ooplast. The somatic cell selected for cloning must be synchronized to an early phase of the cell cycle, typically before DNA synthesis (G0–G1 phase).
The somatic cell is fused with the ooplast either through membrane fusion via an electrical pulse or by direct injection.
The recombined oocyte, now containing the donor cell’s nucleus, is artificially activated to mimic the fertilization signal, prompting the oocyte to begin embryonic development.
These steps can also be performed with oocytes from which the zona pellucida has been removed after in vitro maturation. This technique, known as "zona-free" cloning, simplifies some of the cloning steps; however, it requires a special culture system to keep the embryo cells close to each other during division.
Nuclear transfer technology has been used successfully in cats, dogs, and most large domestic species. After oocyte activation, the developing embryo can be transferred surgically into the oviduct of a recipient female (a common practice for species like dogs), or it can be cultured in vitro until it reaches a suitable stage for cryopreservation or transcervical (nonsurgical) transfer to the uterus (the standard practice for large domestic mammals).
Health and Phenotype in Donor Cell Selection and Cloning Techniques
Several factors—including epigenetic effects, mitochondrial DNA, uterine and postnatal environment, mutations, and individual variation—influence the health and phenotype of cloned individuals.
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Epigenetic Effects
After nuclear transfer, the ooplast must reprogram the DNA from the somatic cell so that it functions like that of a zygote. This reprogramming is regulated predominantly through epigenetic mechanisms, such as DNA methylation and histone modification. These processes collectively control gene transcription without altering the DNA sequence itself—a phenomenon known as epigenetic regulation.
The oocyte's ability to accurately reprogram the donor cell's DNA during cloning is crucial. The proper establishment and maintenance of epigenetic mark throughout the various stages of embryonic development are essential for successful fetal development.
Errors in epigenetic reprogramming are thought to be important to the success or failure of cloning. Minor inaccuracies in reprogramming might not severely affect the health of the cloned individual, but they can lead to phenotypic differences from the donor.
The first cloned cat, CC, offers an example of epigenetic variation in cloning. Although CC’s genetic donor was a calico cat, CC expressed only brown coat color. In calico cats, coat color is determined by X chromosome inactivation, a process in which one of the two X chromosomes in female cells is randomly inactivated. In CC, X chromosome inactivation was nonrandom, likely because of incomplete epigenetic reprogramming, leading to activation of only the brown color gene.
The placenta is one of the tissues most susceptible to abnormal epigenetic reprogramming after SCNT. Many pregnancy losses of cloned embryos have been attributed to abnormal placental function, which has been linked to improper epigenetic regulation during early development. Therefore, strategies aimed at improving epigenetic regulation during cloning could greatly enhance the overall efficiency of the procedure. However, many aspects of the epigenetic reprogramming process remain complex and not fully understood.
Epigenetic differences can also influence the phenotype of a cloned animal after birth. For example, changes in how growth-related genes are expressed can lead some cloned animals to grow larger than others, even though they share the same DNA. However, research across multiple species has shown that most of the epigenetic abnormalities observed in cloned animals are not passed on to their offspring. This is because during the formation of sperm and eggs, most epigenetic marks are erased and reset, helping to ensure that the next generation starts with a clean slate. Some epigenetic marks can escape this reprogramming and be passed on, but most are reset, so cloning remains an attractive tool for breeding, because the risk of passing epigenetic defects to offspring is generally low.
Mitochondrial DNA
A cloned embryo inherits its nuclear DNA from the donor cell but its mitochondrial DNA from the ooplast. Although a small number of mitochondria from the donor cell might persist, they are typically few compared to the mitochondria from the recipient oocyte.
The role of mitochondrial DNA in determining traits such as stamina and metabolic efficiency remains unclear; however, it is a potential area of interest, particularly in production animals. In some cases, mismatches between mitochondrial and nuclear DNA can disrupt processes like placental development, contributing to abnormal pregnancies.
Female clones pass on their mitochondria to their offspring, possibly resulting in a mixture of donor and host mitochondria. However, the mitochondrial bottleneck during oocyte development means that the proportion of each type of mitochondria inherited by offspring can vary. In male clones, mitochondrial DNA is not transmitted to offspring, because paternal mitochondria are typically eliminated after fertilization.
Producing cloned animals with mitochondrial DNA that is genetically identical to that of the donor animal is possible if the oocytes used in the process are from the maternal line genetically related to the donor animal. This can be achieved by collecting eggs through OPU.
Environment
Environmental factors such as uterine health, nutrition, exercise, and handling during development can influence the phenotype of cloned animals. Such influence is particularly evident when the cloned animal's phenotype is expected to match that of the donor but exhibits variability that is due to differences in the environment. For example, CC, the cloned cat, displayed behavioral traits distinct from those of her genetic donor likely because she was raised in a more stimulating environment.
Such observations emphasize that although genetic identity is retained in cloned animals, environmental factors can lead to phenotypic differences.
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This phenomenon is also apparent in horses, whose white markings on their coat can vary in quantity and location among cloned individuals, despite their shared genetic makeup. These variations in white markings across different cloned horses highlight the influence of uterine environmental factors on phenotype (see cloned horses image).
Courtesy of Clonargen Biotech.
Cell differentiation occurs in cascades, because differentiation of one cell type affects the status of the cells around it. During development, cell multiplication and apoptosis occur in response to many environmental and internal stimuli. Thus, random individual variations occur in the makeup of tissues, even in individuals of the same genetic background.
Genetic Mutations
Cells used for cloning, particularly those cultured in vitro, can accumulate chromosomal abnormalities during passaging. As cells grow and divide in culture, the risk of mutations increases, potentially affecting the viability of the cloned embryo. However, most embryos derived from cells with substantial chromosomal abnormalities fail to develop to term, limiting the impact of such mutations on live-born clones.
One female cloned horse was reported to have been born from a male donor. Subsequent studies revealed loss of the Y chromosome during the cell culture or cloning process, resulting in a female with an XO genotype and phenotype. This rare occurrence highlights the potential chromosomal instability that can arise during cloning, particularly with respect to sex chromosomes.
Status of Cloning of Domestic Animals
Live young have been successfully produced by nuclear transfer across all major domestic mammalian species, including cats, dogs, horses, cattle, buffalo, goats, sheep, pigs, and camels.
Status of Cat Cloning
Cloning in cats has been relatively successful, with oocytes often obtained from tissue recovered during ovariohysterectomies. Domestic cat cloning is used primarily for replicating pets, and its application has expanded to include conservation efforts for endangered wild cats through interspecies cloning.
Although the success rate of producing live cloned kittens remains modest, it continues to improve with technological advancements.
Status of Dog Cloning
Since the birth of the world's first cloned dog, Snuppy, in 2005, SCNT has been extensively used to clone various types of dogs for a range of purposes. Dog cloning, however, faces major biological challenges.
Canine reproductive biology is unique, with mature oocytes needing to be harvested from the oviduct after ovulation because in vitro maturation methods are not yet reliable. Furthermore, estrous cycles in dogs occur only approximately twice a year, limiting the availability of oocytes and complicating synchronization with recipients.
Despite these challenges, commercial dog cloning has seen success, and several companies have produced cloned dogs for specific purposes, such as preserving elite working dogs or beloved companion animals and supporting research in animal models. The process has also been applied to conservation efforts for endangered species, using SCNT technology to preserve genetic material.
Although oocyte synchronization remains a hurdle, continued advancements in reproductive technologies are making SCNT in dogs more accessible across various fields.
Status of Horse Cloning
Horses are frequently cloned because of the high value of individuals in sports such as polo and show jumping. The reproduction of castrated animals through SCNT is also an attractive option for some horse owners.
This author's experience with cloning companies in Argentina, as well as recent studies, suggests that cloning efficiency in horses remains low. However, blastocyst development rates are now up to 20–30%, and the number of cloned foals has risen, especially in Argentina and the US. The overall efficiency of cloning horses is approximately 5% (1, 2, 3).
Horse clones are valued for competitive performance and have shown notable success in elite competitions. Breed associations differ on whether cloned horses may be registered for competition. Up to 15 clones of the same donor have been produced in Argentina. Concerns about genetic variability will persist if cloning is not managed wisely.
Notably, domestic horse oocytes have been used to clone endangered species, such as Przewalski’s horse, in the US. Successful cloning of zebra blastocysts using horse oocytes demonstrates the genetic flexibility within the Equus genus.
Status of Ruminant Cloning
Cloning in cattle and sheep is less efficient than in horses; only 5–15% of transferred embryos result in live offspring (4). A large proportion of cloned calves and lambs (30–50%) die within the first 4 years of life.
Cloning in cattle and sheep is often associated with placental abnormalities, including decreased numbers of cotyledons, and large offspring syndrome (fetal overgrowth). Although changes that have been made in culture conditions, such as decreasing serum, have improved outcomes, these problems have not been fully resolved.
Cloned cattle might require extensive medical care in the neonatal period because of metabolic abnormalities; however, survivors perform similarly to noncloned counterparts in milk production and agriculture.
The efficiency of goat cloning is similar to that of cattle cloning; approximately 10% of transferred embryos result in live births (5, 6). However, cloned goat kids tend to exhibit higher viability compared to cloned calves and lambs, possibly because follicle aspiration is used for oocyte recovery.
Status of Pig Cloning
Pig cloning has become increasingly important in the biomedical field, particularly for its applications in disease modeling and organ transplantation research.
Cloned pigs are genetically engineered to replicate human diseases, providing valuable models for studying conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders. Pigs are also used in xenotransplantation research, in which their organs are explored as potential candidates for human transplants.
However, pig cloning faces several challenges, including low cloning efficiency, high rates of stillbirth, and developmental defects, which hinder widespread use and require further refinement.
Cloning in pigs remains challenging; only 1–5% of recombined oocytes produce live young (7). However, cloning is feasible because large numbers of oocytes are available, enabling the transfer of hundreds of recombined oocytes to a single recipient.
Cloned piglets are generally healthy; however, they experience higher rates of stillbirth and other abnormalities.
Status of Camel Cloning
Camel cloning has advanced since the first successful clone in 2009, and there is great potential for enhancing genetic gain, particularly in communities in arid regions that rely on camels for economic and sociocultural reasons.
Cloning offers the ability to replicate elite animals, regardless of how old they are or whether they are alive or dead, producing a large number of offspring with predetermined sex and genotype. However, the current low efficiency of the process, including embryo survival and cost, severely limits its commercial viability, despite its clear benefits for breeding and genetic preservation.
Although reports exist of cloned camel calves reaching full term, little is known about their long-term health status. Further research is needed to evaluate the viability and potential complications of cloning in this species.
Controversies and Outlook of Cloning of Domestic Animals
Despite its benefits, cloning presents ethical concerns. More standard breeding procedures, such as conventional embryo transfer and in vitro fertilization, share some risks. These risks are heightened with cloning, and they must be carefully weighed against the potential benefits of the technology.
In agricultural cloning, concerns have been raised about the potential impact on genetic diversity, particularly in species like dairy cattle, in which a single cloned bull could sire thousands of offspring. Although the same concerns apply to artificial insemination and semen distribution, cloning can amplify these risks.
Some horse owners have produced up to 15 genetically identical copies of the same animal, potentially decreasing genetic variation when cloning is combined with other reproductive technologies. However, cloning can also maintain genetic diversity when applied, for example, to castrated animals, which would otherwise be absent from the gene pool.
Animal and breed associations should consider regulations to ensure the responsible use of cloning, weighing both the advantages and risks to genetic diversity.
Concerns about the effect of animal cloning on human health focus mainly on the consumption of food produced from cloned animals. After years of study, the FDA and the European Food Safety Authority concluded that consumption of meat or milk from cloned animals poses no public health risk.
In the EU, although the lack of evidence of a human health risk is recognized, marketing food from clones requires authorization. There are calls for EU rules to prohibit cloning for farming purposes and to ban the marketing of food from clones.
Counterarguments to these ethical concerns are that cloning occurs in nature in the form of identical twins, that people have been producing plants and animals by “unnatural” means from the first time they planted a seed in a new area or bred a cow to a selected bull, and that this is simply a new development within the field of domestic animal breeding.
Embryonic cloning was performed for more than 10 years before the birth of Dolly, with essentially no public attention, and even the birth of two lambs cloned from cultured cells of embryonic origin, announced a year before Dolly, had no public impact. Thus, it appears that the main ethical issue of public concern is not the production of embryos without fertilization, but the production of embryos from cells of an existing, known animal.
The cost of cloning, especially in companion animals, continues to generate debate. Some view this cost as a personal choice, but others see it as unjustifiable, particularly given the many shelter animals needing homes. Cultural and religious perspectives also influence opinions, with some considering cloning acceptable and others raising concerns.
These debates often intersect with broader ethical issues in modern reproductive technologies that similarly involve the manipulation of animals for human purposes.
Commercialization of cloning brings with it the possibility of fraud and of preying on the emotions of bereaved pet owners. Cloning companies should state clearly that although the technique will produce another individual with the same genetics as the original animal, it will not “resurrect” an animal or create an animal identical to the donor (eg, with the same coat pattern or personality). The best analogy to a cloned animal is an identical twin born later in time; just as with naturally occurring identical twins, they will be very similar but also different in many ways.
In the near future, induced pluripotent stem cell (iPSC) and other stem cell technologies could revolutionize cloning by enabling the production of embryos via in vitro methods involving stem cell culture. Stem cells can be reprogrammed from adult cells and used to create "cloned" embryos without the need for traditional SCNT. Stem cell technologies hold the potential to bypass many ethical concerns and inefficiencies of SCNT. As research advances, these technologies could enable more efficient and ethically acceptable approaches to cloning.
For More Information
Klinger B, Schnieke A. Twenty-five years after Dolly: how far have we come?Reproduction. 2021;162(1):F1-F10.
Keefer CL. Artificial cloning of domestic animals. Proc Natl Acad Sci U S A. 2015;112(29):8874-8878.
Loi P, Palazzese L, Scapolo PA, Fulka J, Fulka H, Czernik M. Scientific and technological approaches to improve SCNT efficiency in farm animals and pets. Reproduction. 2021;162(1):F33-F43.
References
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Martins LT, Neto SG, Tavares KC, et al. Developmental outcome and related abnormalities in goats: comparison between somatic cell nuclear transfer- and in vivo-derived concepti during pregnancy through term. Cell Reprogram. 2016;18(4):264-279. doi:10.1089/cell.2015.0082
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