Overview of Cloning of

Domestic Animals
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The basic concept of cloning via nuclear transfer is that the nucleus of a cell, taken from a
tissue sample of a donor individual, is transferred to an enucleated oocyte, and then the
oocyte is stimulated to develop into an embryo. The embryo thus produced has the same
genotype as the original donor individual.
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Technical Aspects of Cloning

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Cells recovered from early embryos, up to the morula stage, work efficiently as donor cells
for cloning. Cloning from embryonic cells was conducted successfully for more than a
decade before the birth of the first mammal produced via nuclear transfer using adult
somatic cells (“Dolly,” a sheep reported by the Roslin Institute, Scotland, in 1997).

The most common tissue used for cloning from adult animals is subcutaneous connective
tissue. The tissue is minced and cultured in vitro. Outgrowing fibroblasts are harvested and
replated (passaged) in new dishes to continue proliferation until millions of cells have been
produced. These are then typically cryopreserved for future use.

Mature oocytes from the same or closely related species are required for the nuclear transfer.
The genetic value of the oocyte is not important, although their mitochondrial makeup may
be, because the resulting clone will carry the mitochondria of the host oocyte (see Health
and Phenotype of the Cloned Animal). Oocytes are typically recovered from slaughterhouse
material and then matured in vitro, although in some species, meiotically mature oocytes
(from preovulatory follicles or from the oviduct after ovulation) are collected.

Nuclear transfer is typically performed using a microscope with micromanipulators. The

chromosomes of the oocyte are removed, creating an enucleated oocyte, or ooplast. The
somatic cell used for cloning must be synchronized to early in the cell cycle (before DNA
synthesis). The somatic cell is combined with the ooplast, either by membrane fusion using
an electric pulse or by direct injection of the donor cell into the ooplast via
micromanipulation.
The recombined oocyte, now containing the nucleus of the donor cell, is treated to mimic the
activation signal of fertilization, which stimulates it to develop into an embryo. After activation,
the developing embryo may be transferred surgically to the oviduct of a recipient female or may
be cultured in vitro to a stage at which it can be transferred to the uterus transcervically
(nonsurgically), as for standard embryo transfer.

Health and Phenotype of the Cloned Animal

Several factors influence the health and phenotype of the cloned individual, including epigenetic
effects, mitochondrial DNA, uterine and postnatal environment, mutations, and individual
variation.

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 is controlled largely by methylation and demethylation of
bases in the DNA and by modification of the histones, the proteins around which the DNA is
wrapped. Controlling the transcription of DNA in this manner, without altering the structure of
the DNA itself, is termed epigenetic control. The oocyte must reprogram the DNA of the donor
cell initially at the time of cloning, and then maintain the normal patterns of epigenetic
modification through the different stages of development. The amount and accuracy of
reprogramming of the donor DNA is probably the major reason for success or failure of fetal
development in cloning. Minor changes in epigenetic status may not affect the general health of
the cloned individual but may still cause it to vary in phenotype from the donor animal. A visible
example of this is seen in the first cloned cat, CC. CC expresses only brown coat color, whereas
her genetic donor expresses both orange and brown (eg, calico). Because the X chromosome
carries the gene for coat color in

cats

, this indicates that in CC, X chromosome inactivation is not random; rather, the same X chromosome is
inactivated in all cells, presumably because of failure to reactivate the inactive X at the time of cloning.

Epigenetic effects may also influence the phenotype of the nuclear transfer–derived animal after
birth; eg, an animal with growth-factor genes transcribed at high levels may grow larger than its
cloned sibling with less active genes, even though the actual number and makeup of the genes
are the same. However, it has been shown in all species studied that major epigenetic anomalies
are not passed on to the offspring of clones because of resetting of epigenetic status during sperm
and oocyte development.

Mitochondrial DNA

The nuclear transfer embryo will have the nuclear DNA of the genetic donor but have the
mitochondrial DNA of the recipient oocyte. A small number of mitochondria from the donor cell
may also be present, but proportionately few. The impact of the source of mitochondria, or a
mixture of mitochondria, on the traits of the progeny is currently unknown. Because
mitochondria are the source of energy for the cell, differences in mitochondria could potentially
have an effect on production, stamina, or other physical or behavioral traits.

The heterogeneous mitochondria present in a female produced by nuclear transfer will be passed
down to the female's offspring, because they will be present in her oocytes. However, although
sperm from a male produced by nuclear transfer will carry the heterogeneous mitochondria, these
mitochondria will be eliminated after the sperm fertilize an egg, so a male clone can be
considered to produce progeny that are genetically identical to those that the original donor
animal would have produced.

Environment
Uterine size and health; milk
production of the dam; nutritional,
exercise, and training programs; and
handling to which the neonate is
exposed may all influence the animal
as an adult. For example, the cloned
cat, CC, is outgoing and gregarious,
whereas her genetic donor was
reserved. However, the genetic donor
was a research cat, raised in a cage and
unused to attention, whereas CC was
raised with an overabundance of
attention and stimulation.

Mutations

Genetic mutations are potentially more

likely in cloned animals, because
cultured cells are used as the DNA
source. The donor cells are grown and
passaged in vitro, and this is associated
with an increasing number of
chromosomal abnormalities with
increasing passages. However, cells
with chromosomal abnormalities are
unlikely to produce viable embryos.

Individual Variation

Cell differentiation occurs in cascades,

as 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 will occur in the
makeup of tissues, even in individuals
of the same genetic background. A
visual example of this is in the
markings of cloned animals;
individuals cloned from the same cell
line tend to have white in similar
places, but the markings can vary
dramatically in size and shape.

Last full review/revision November

2013 by Katrin Hinrichs, DVM, PhD,
DACT

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al_aspects_of_cloning.html

Consumer FAQs
The responses to the questions provided in this document represent the FDA's view in light of the
conclusions and recommendations outlined in the Animal Cloning Risk Assessment, Risk
Management Plan, and Guidance for Industry #179.

What is the Animal Cloning Risk Assessment?

What is the Risk Management Plan?
What is the Guidance for Industry?
What are animal clones?
Are there long-term studies on the consumption of food from clones?
If there are no long-term studies on the consumption of food from clones, why is FDA
concluding that it is safe to consume these cloned animals and their offspring?
Are there any substantive differences between the conclusions in the draft and final versions of
the cloning risk assessment regarding the consumption of food from clones?
Now that FDA has concluded that food from clones from specified species and from clone
progeny is as safe as food from conventionally-bred species, is FDA lifting the voluntary
moratorium?
If FDA thinks food from cloned livestock is safe, why does FDA continue to recommend that
food from sheep clones be kept out of the food supply?
How soon will meat or milk from clones be on the market?
Is meat or milk from clones safe for pets?
Is cloning the same as genetic engineering?
Why are livestock producers interested in cloning? Don't the more conventional means of
producing animals work?
Is FDA considering the ethics of animal cloning?
Would food from clones be labeled?
Is animal cloning allowed in other countries? Can food products from these animals be sold for
human consumption in other countries?

What is the Animal Cloning Risk Assessment?

It's a report written by scientists in the Food and Drug Administration's Center for Veterinary
Medicine. Agency scientists analyzed data from hundreds of published reports and other detailed
information about clones of livestock animals. The report provides FDA's conclusions on the
risks to the health of animals involved in the cloning process, and on the safety of food from
animal clones and their offspring.

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What is the Risk Management Plan?

The Risk Management Plan takes into account the risks to animals identified in the Risk
Assessment, and suggests how those risks could be managed.

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What is the Guidance for Industry?

It explains FDA's recommendations for industry on the use of clones and their offspring for
human food and animal feed.

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What are animal clones?

An animal clone is a genetic copy of a donor animal. Clones are similar to identical twins, just
born at a different time. Cloning could be thought of as an extension of the assisted reproductive
technologies that livestock breeders have been using for centuries. These include artificial
insemination, and more recently, embryo transfer, embryo splitting, and in vitro fertilization.

Cloning is the newest and most complex form of assisted reproductive technology, and has been
around for more than 20 years in various forms. The form used most frequently today is known
as Somatic Cell Nuclear Transfer, or SCNT. With SCNT, the genes of the donor animal are
inserted into an egg cell that has had its nucleus removed, and after a few steps in the lab is
implanted into a surrogate dam where it develops just like any other embryo. (Dam is a term that
livestock breeders use to refer to the female parent of an animal.)
You can learn more about the cloning process by going to our "Cloning Primer." You may also
ask for single printed copies of these documents from the Communications Staff (HFV-12),
Center for Veterinary Medicine, Food and Drug Administration, 7519 Standish Pl., Rockville,
MD 20855. If you make this request, please enclose a self-addressed, adhesive label. Doing so
will help that office respond to your request more effectively.

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Are there long-term studies on the consumption of food from clones?

Cloning doesn't put any new substances into an animal, so there's no "new" substance to test.
Feeding milk or meat from clones to lab animals as part of a regular diet wouldn't let us tell
whether any negative outcomes observed were due to the food from clones or from something
else the lab animals were exposed to. It isn't possible to have someone (or even lab animals) eat
only meat or drink only milk. Doing so would not provide a healthful diet and would likely cause
illness.

Food scientists, toxicologists, and regulators have faced this problem before and decided that
long term feeding trials of whole foods don't give meaningful results.

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If there are no long term studies on the consumption of food from clones, why is FDA
concluding that it is safe to consume these cloned animals and their offspring?

Cloning doesn't put any new substances into an animal, so there's no "new" substance to test.
Feeding milk or meat from clones to lab animals as part of a regular diet wouldn't let us tell
whether any negative outcomes observed were due to the food from clones or from something
else the lab animals came across. It isn't possible to have someone (or even lab animals) eat only
meat or drink only milk. Doing so would not provide a healthful diet and would likely cause
illness.

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Are there any substantive differences between the conclusions in the draft and final versions of
the cloning risk assessment regarding the consumption of food from clones?

Not really. As we stated in the Draft Risk Assessment, FDA has no concerns about the safety of
food from cattle, swine (pig) or goat clones, or food from the progeny of a clone of a species
traditionally consumed as food. Our conclusions were strengthened by the data that we evaluated
since the publication of the draft. We did not, however, get more information that would allow us
to make decisions regarding food from sheep clones or other species, and we do ask that
producers continue to voluntarily keep food from clones of other species (e.g., sheep) out of the
food supply.

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Now that FDA has concluded that food from clones from specified species and from clone
progeny is as safe as food from conventionally-bred species, is FDA lifting the voluntary
moratorium?

In 2001, U.S. producers agreed to refrain from introducing meat or milk from clones or their
progeny into the food supply until FDA could further evaluate the issue. The U.S. Department of
Agriculture will convene stakeholders to discuss efforts to provide a smooth and orderly market
transition, as industry determines next steps with respect to the existing voluntary moratorium.

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If FDA thinks food from cloned livestock is safe, why does FDA continue to recommend that food
from sheep clones be kept out of the food supply?

At this time, the agency does not have sufficient information to make a decision on food
consumption risks from clones of species other than cattle, swine, and goats. Although we have
no specific concerns related to sheep or other species, because of the uncertainty we continue to
recommend that edible products from cloned sheep or other species not be introduced into the
human food supply.

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How soon will meat or milk from clones be on the market?

It's highly unlikely that you will see meat from clones at the supermarket any time soon. We
anticipate that clones would be used as elite breeding animals rather than as food themselves.
Instead, the sexually reproduced offspring of animal clones would be the animals intended to
produce food. Milk from cow clones may enter the food supply once clones are bred and have
their calves (cows don't make milk until after they have calves). It's important to remember,
however, that at this time there are only a few hundred cattle clones, most of which are not dairy
cows, so again, it's highly unlikely that there will be much milk from dairy cow clones in the
food supply.

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Is meat or milk from clones safe for pets?

Yes. We believe it is safe for meat or milk from clones to be used in making pet food.

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Is cloning the same as genetic engineering?

No, cloning is not the same as genetic engineering. Genetic engineering involves adding, taking
away, or modifying genes, while cloning does not change the gene sequence.
Back to the top

Why are livestock producers interested in cloning? Don't the more conventional means of
producing animals work?

Livestock producers will continue to use the more conventional means of breeding food animals.
The point of cloning is to increase the number of breeding animals with naturally occurring
desirable traits. This will allow for the more rapid spread of these characteristics through the
herd, such as disease resistance or higher quality meat.

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Is FDA considering the ethics of animal cloning?

FDA recognizes that animal cloning for agricultural purposes raises moral, religious, and ethical
issues that are important to some members of the public. The relevant issues in this context for
the agency are limited to the animal health and food safety issues. The agency is not charged
with addressing moral, religious, or ethical issues related to animal cloning for agricultural
purposes. We are also aware that these ethical concerns can become intertwined with, and
amplify concerns about, food safety. We have participated, and will continue to participate in
discussions on the ethical issues to ensure that people have the correct facts in order to make
informed decisions about clones.

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Would food from clones be labeled?

No. FDA is not requiring any additional measures relating to food derived from adult clones of
cattle, swine, and goats, and the offspring of clones of any species traditionally consumed as
food, including labeling. Under our current laws, FDA may require specific food labeling if there
are any safety concerns, or if there is a material difference in the composition of food. We have
not identified any food safety concerns, and we have found no material difference in food from
clones as compared to food from conventionally bred animals. For example, FDA scientists
found that the milk components from dairy clones were of the same type and present in the same
amounts as milk sold every day. Therefore, there is no science-based reason to use labels to
distinguish between milk derived from clones and that from conventional animals. Should a
producer express a desire for voluntary labeling (e.g., "this product is clone-free"), it will be
considered on a case-by-case basis to ensure compliance with statutory requirements that
labeling be truthful and not misleading.

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Is animal cloning allowed in other countries? Can food products from these animals be sold for
human consumption in other countries?

Scientists in many other countries are using cloning technology. "Dolly the sheep," the first
mammal to be successfully cloned from an adult cell, was from Scotland. Several countries have
been actively engaged in agricultural cloning, including Argentina, Australia, Brazil, Canada,
Chile, China, France, Germany, Japan, New Zealand, South Korea, and the United Kingdom. At
this time, we can not confirm whether or not food from clones is being sold in other countries.

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Additional Information

 A Primer on Cloning and Its Use in Livestock Operations

 CVM GFI #179 Use of Edible Products from Animal Clones/Progeny for Human Food/Animal
Feed- draft (PDF - 46KB)

Page Last Updated: 07/23/2014

http://www.fda.gov/AnimalVeterinary/SafetyHealth/AnimalCloning/ucm055516.htm

Status of Cloning Domestic Animals

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Live young have been produced by nuclear transfer in all major domestic mammalian
species:

cats
,
dogs
, horses, cattle, goats, sheep, and pigs. Cloning in cats appears to be repeatably successful;
oocytes may be obtained using tissue recovered from clinical ovariohysterectomies.
Interspecies cloning of closely related nondomestic cat species using domestic cat oocytes
has produced live young.

Cloning in dogs is complicated by several factors. Mature canine oocytes must be obtained
from the oviduct after ovulation, because effective methods for in vitro maturation of canine
oocytes have not been developed; additionally, bitches are in estrus only about once every 6
mo, so oocyte availability is low and synchronization of recipients is problematic. Cloning
in horses results in a low blastocyst development rate (3%–10%), but viability after transfer
is among the highest reported; ~30% of transferred embryos produce live foals, and
 postnatal survival is high (>85%).

Cloning in food animal species is facilitated by the ready availability of oocytes from
slaughterhouse tissue. Cloning in sheep and cattle is inefficient (5%–15% of transferred
embryos result in live young) and 30%–50% of live neonates die by 4 yr of age. Cloning in
sheep and cattle is associated with frequent placental abnormalities, especially low numbers
of atypically large cotyledons. There is a high incidence of large offspring syndrome in
cloned calves and lambs (ie, fetal overgrowth with related abnormalities), and, in live
calves, of transient metabolic abnormalities such as hypoglycemia or poor renal function.
Cloning in goats has similar efficiency in production of live young per embryo transferred,
but cloned kids tend to have greater viability. Goat oocytes are typically obtained by follicle
aspiration ex vivo, and this may increase viability of resulting clones. In pigs, 1%–5% of
transferred recombined oocytes produce live young, but cloning is made practicable by the
ready availability of large numbers of oocytes and the ability to transfer hundreds of
recombined oocytes to the oviducts of a single recipient. Cloned piglets are generally
healthy; they have a higher than normal incidence of stillbirth and of some abnormalities
but do not develop large offspring syndrome.

Last full review/revision November 2013 by Katrin Hinrichs, DVM, PhD, DACT
http://www.merckmanuals.com/vet/management_and_nutrition/cloning_of_domestic_animals/status_
of_cloning_domestic_animals.html

Rationale for Cloning

Companion animals may be cloned for emotional reasons or as models for endangered
species. Horses are cloned mainly to allow continued production of offspring from
individuals with valuable genotypes or to retain a genetic type (eg, geldings that are
exceptional performers). Cloning of farm animals may be performed for agricultural or
biomedical applications. Agricultural applications include production of animals with
valuable production traits, such as high-producing dairy cows, animals with superior carcass
quality at slaughter, or production of additional sires of an established valuable genotype.

Biomedical applications of cloning are largely related to the ability to perform nuclear
transfer using a cell line that is genetically altered (transgenic) and, thus, to produce an
animal with those characteristics. Biomedical applications include generation of animals of
specific genotypes as disease models, animals carrying genes for the production of
medically important proteins that may be harvested from the milk or tissues, and animals
having genetically altered organs (of low immunogenicity) for transplantation to people.

Last full review/revision November 2013 by Katrin Hinrichs, DVM, PhD, DACT
http://www.merckmanuals.com/vet/management_and_nutrition/cloning_of_domestic_animals/rationa
le_for_cloning.html

Controversies About Cloning

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Ethical concerns about cloning may be broadly divided into two

categories: concern about the effect of cloning on animal and
human welfare, and objection to the principle of cloning, ie, to
producing an animal by a means other than fertilization.

Currently, cloning is associated with an increase in animal

suffering when compared with production of animals by
standard breeding methods. This is due to surgeries performed to
obtain oocytes or transfer embryos, pregnancy losses, sickness
and death of neonates, low-level abnormalities in surviving
young, and possible distress from disease in animals produced as
disease models. These concerns are somewhat mitigated by the
fact that most of these findings are not unique to cloning; they
are also associated with other procedures that have been
generally accepted as worthwhile, such as in vitro fertilization
and embryo production, oocyte transfer, and embryo transfer. In
addition, the accepted normal fate of many species being cloned
is to be housed for maximal production and then be slaughtered
and eaten. A compelling argument for cloning is that the
potential benefits of the procedure to the understanding of life
processes and animal disease, to human health, and to food VIEW VET STUDENT
production outweigh the cost of the procedure in terms of animal STORIES
welfare.

Additional concerns rest with the effect of cloning on the entire

animal population, most commonly related to the genetic
variation of the species. This is a legitimate concern in some
species and uses, such as in dairy cattle, in which one bull may
sire thousands of offspring. However, this is more related to the
technology of semen freezing and distribution than to the fact
that a bull itself was cloned. In companion animals, it is
improbable that the very few pets likely to be cloned will have
an effect on the population in general. In horses, cloning may in
fact increase genetic variation, because a major proposed use is
to clone geldings that have been found to be superior
competitors, thus rescuing genetic types that would otherwise
have been lost.

Concerns about human health focus mainly on consumption of

food produced from cloned animals. After years of study, the
FDA and the European Food Safety Authority have concluded
that consumption of meat or milk from cloned animals poses no
public health risk. Therefore, remaining concerns about
consumption of food from cloned animals is likely based more
on principle than on actual potential for harm. Because cloning
is used to produce transgenic animals, many perceived concerns
regarding cloning are actually concerns about transgenic
animals, which present a completely different set of potential
hazards to animal and human health and the environment.
European nations have relaxed their position on use of cloned
animals and their progeny in the food chain based on the lack of
evidence of a human health risk and the difficulty in establishing
a tracing or labeling system to identify such meat when coming
from outside countries.

A key ethical question regarding the principle of producing

animals by cloning is whether this technique is violating some
moral prohibition; ie, that people are “playing God” by
producing embryos without using fertilization. Similar questions
have arisen with each new reproductive technique that has been
developed; however, many people feel that cloning is a special
case. This general moral aversion of the public to the concept of
cloning is enhanced by the portrayal of cloning as a malevolent
force in science fiction books and films.

Counter-arguments to these moral 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 in the
same line. Embryonic cloning was being 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 moral issue of
public concern is not the production of embryos without
fertilization, but the production of embryos from cells of an
existing, known animal.

Arguments against cloning of companion animals have focused

on the cost of producing a clone—tens to hundreds of thousands
 of dollars—when millions of unwanted

dogs
and
cats
are killed each year. However, people currently buy purebred
dogs and cats for thousands of dollars when they could get
animals for free. American culture supports the concept that
people can spend their own money on whatever they wish.

A related argument is that cloning turns animals into a

commodity or an object, rather than a sentient being, and that
producing an animal in this way shows a lack of respect for the
animal as an individual. However, animals have been bought
and sold since they were domesticated; currently semen and
embryos are frozen, shipped across the country, and used to
produce desired young. Cloning does not seem to offer any
unique distinction in this area.

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 the technique will
produce another individual with the same genetics as the original
animal; it does not “resurrect” an animal. The best simile to
draw is to that of 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.

Last full review/revision November 2013 by Katrin Hinrichs,

DVM, PhD, DACT

http://www.merckmanuals.com/vet/management_and_nutrition/cloning_of_domestic_animals/contro
versies_about_cloning.html

Topics in Cloning of Domestic Animals

 Overview of Cloning of Domestic  Status of Cloning Domestic
 Controversies Abo
Animals Animals
Cloning
 Technical Aspects of Cloning  Rationale for Cloning