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REPRODUCTION
REVIEW
Wildlife conservation and reproductive cloning
William V Holt, Amanda R Pickard and Randall S Prather
1
ZSL Institute of Zoology, Regent’s Park, London NW1 4RY, UK and
1
University of Missouri-Columbia, Columbia,
Missouri 65211, USA
Correspondence should be addressed to W V Holt; Email: Bill.Holt@ioz.ac.uk
Abstract
Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving
endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application
of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may con-
tribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which
stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources
could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the
population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical indi-
viduals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conven-
tional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may
be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In
this respect species with external embryonic development may have an advantage over mammals as developmental abnormal-
ities associated with inappropriate embryonic reprogramming would not be relevant.
Reproduction (2004) 127 317–324
Introduction
Nuclear transfer technology, known popularly as cloning,
whereby new individuals are created in the laboratory
from the nuclear DNA of other individuals, has a history
that extends back to the late nineteenth century when
Driesch (1892; cited by Di Berardino 2001) produced sea
urchin larvae from isolated blastomeres. Although nuclear
transfer did not create these larvae, they represented
‘proof of principle’ that early-stage embryonic cells had
the capacity to develop autonomously into a whole indi-
vidual. Di Berardino (2001) describes this and other
examples in a review that covers the development of
embryological and associated studies in cloning technol-
ogy from those significant early beginnings to the pro-
duction of the first cloned mammals. Interestingly, the
review also covers the development of cloning technology
in insects, amphibians and fishes, species that rarely enter
the public debate on cloning in species conservation.
The public and media often equate conservation solely
with saving endangered mammals such as the giant
panda, forgetting that mammals are an ‘ethnic minority’
among vertebrates. Reid & Hall (2003) recently compared
the number of described fish species (25 157) with the
equivalent numbers belonging to other vertebrate classes
(i.e. birds (9040), reptiles (6458), mammals (4629)
and amphibians (4222)). Thus mammals comprise just
under 10% of the total number of vertebrates, but seem to
gain 90% of the attention. Any assessment of the potential
role of reproductive cloning in species conservation
should consider this discrepancy. Factors that govern the
desirability, feasibility and practicality of cloning vary
among different classes of vertebrates, depend upon the
peculiarities of the biological systems themselves, the type
of species under threat and even the chances of obtaining
suitable funding.
Objectives of conservation and the role of cloning
This review does not aim to examine the technical details
involved in cloning methodology as plenty of such
reviews have already been written. Instead, it considers
the conservation-related goals that this technology would,
or could, serve. As the detailed biology of cloning in most
species is unknown at the present time, the authors
do not aim to consider the detailed advantages, disadvan-
tages and biological implications of every potential
cloning application; that would require virtually unlimited
amounts of text. Moreover, in line with the objectives of
review papers in this journal, this review is aimed at a
wide audience who are not necessarily experts in either
wildlife conservation or cloning; species-specific details
q2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00074
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
of cloning are therefore less relevant in this context than
the general principles.
Simplistically, cloning is one of several ways of increas-
ing the number of individuals within a population. Clearly,
natural breeding is the preferred method for thriving
populations; but by definition these are not of conserva-
tion concern. However, when populations of free-living
species are found to be in decline, conservation biologists
begin to seek methods of slowing or reversing the threat-
ening processes. Many such threats exist, including habitat
loss through human activity, hunting and over-fishing,
effects of pollution on fertility and fecundity, predation by
introduced species or, indeed, poor diet through loss of
prey species. In a few cases these threats can be alle-
viated, but this may require the development of national
and international policies that support the conservation
goals. Reproductive technologies may then provide sup-
port, usually by assisting with genetic management. An
important common aim of conservation breeding pro-
grammes, with or without the use of assisted reproduction,
is the avoidance of inbreeding depression and the associ-
ated exposure of rare, and often deleterious, alleles. The
Mauritius kestrel provides a good example of successful
reintroduction without assisted breeding; the population
declined to about nine individuals in the early 1970s, four
were reintroduced to the island of Mauritius, and the
population is now estimated at 700–800 (Groombridge
et al. 2000). Genetic analyses have revealed that, com-
pared with pre-crash individuals, the population is now
extremely homogeneous and therefore poorly equipped to
adapt to environmental changes. Nevertheless, this has so
far not proved to be a problem. The black-footed ferret
represents an example where assisted reproduction played
a major part in the recovery and survival of a species. This
species declined almost to the point of extinction in the
1970s–1980s, but 18 animals were captured by the
Wyoming Game and Fish Department (Thorne & Oakleaf
1991). A species recovery strategy was developed in
which assisted reproduction within a captive-breeding
group played a key role. The captive-breeding programme
proved such a success that reintroductions have now been
possible in several states of the USA (for review see
Howard et al. 2003).
How could nuclear transfer have helped with these two
examples? Populations with low numbers of individuals
possess minimal genetic variation. It is therefore desirable
to avoid further losses of diversity. A subsequent gener-
ation resulting from natural breeding or artificial insemina-
tion (AI) would contain some, but not all, of the genetic
variability of its parents. Losses would occur if any of the
individuals failed to breed, a strong possibility with small
populations. If cloning was guaranteed to be 100% suc-
cessful, a good strategy might be to clone every individual
(not impossible if the population size is only 9 –18),
then allow the offspring to mature and breed naturally.
The probability of losing genetic diversity would then be
reduced, especially if each parent gave rise to more
than two identical copies of itself. Thus, an interesting
and novel theoretical principle in animal conservation
emerges, where individuals are effectively induced to
reproduce asexually, somewhat like plants, thereby
improving the long-term fitness of the species through the
retention of genetic diversity.
It is important to ask, however, to what extent this could
be achieved, if at all? Current success rates with nuclear
transfer in mammals are very low (less than 0.1 –5% of
reconstructed embryos result in a live birth (Di Berardino
2001, Wakayama & Yanagimachi 2001). Therefore,
between 20 and 1000 nuclear transfers would need to be
performed to achieve one viable offspring. Assuming, for
example, that oocyte recovery from two black-footed
ferret females could be justified, it is most likely that
two or three oocytes might be recovered, rendering the
realistic chance of obtaining a single offspring somewhere
between 0.0006 and 0.3%; vanishingly small. To date the
cloning of birds has not been accomplished; therefore the
Mauritius kestrel example could not have been addressed
at all with this technology.
This suggests that applying cloning technology to
highly endangered species is hopelessly optimistic given
current efficiencies. However, should the idea of cloning
be ruled out altogether? More embryos and births could
be expected when dealing with larger populations.
The best way to maximise success might therefore be to
concentrate on poly-ovulatory, litter-bearing species pro-
vided that a minimum number of viable concepti were
available to sustain pregnancy to term. This would
immediately exclude many of the larger mammals, includ-
ing the giant panda that only ovulates one or two oocytes
per year (Kleiman 1983, Hodges et al. 1984). Neverthe-
less, this is such a popular choice of candidate species
that a special research programme has been initiated in
China. Paradoxically, this argument leads towards using
cloning technologies for endangered rodents, where other
more traditional assisted reproductive technologies have
been largely overlooked. Although there are 330 endan-
gered rodent species (International Union of Conservation
of Nature and Natural Resources (IUCN) 2002), tech-
niques such as AI, semen freezing and embryo transfer
have not been applied successfully to any of them. It
would be feasible to collect and cryopreserve ovarian
slices from many individuals of such species, and prepare
fibroblast cell lines from muscle or skin, with the expec-
tation that cloning might be ultimately successful. Off-
spring representing the genetic variability of the founder
populations could then be regenerated using methods
allied to those currently being developed for the labora-
tory mouse. This implies that any attempt to clone such
species should be approached on a grand scale, where
sufficient numbers of offspring could be generated to
maintain a genetically diverse population. In this case the
level of genetic diversity could never exceed that of the
original population and would undoubtedly be less.
318 W V Holt and others
Reproduction (2004) 127 317–324 www.reproduction-online.org
Does cloning have a place in conservation?
The low conception rates currently associated with clon-
ing cannot be cited as justification for not embracing this
technology; while pragmatic, to do so is disingenuous and
misleading. Dramatic improvements in the success rate
achieved with, for example, ungulates would remove that
particular argument, but would conservation biologists
then rush to include cloned gazelles, oryx and addax in
their breeding programmes? As many conservationists are
still suspicious of reproductive technologies, it is unlikely
that cloning techniques would be easily accepted. Individ-
uals involved in field conservation often harbour suspi-
cions that hi-tech approaches, backed by high-profile
publicity, would divert funding away from their own
efforts. This may be true, but often the sources of funding
would not necessarily compete with each other.
A major practical objection to using cloning technology
in wildlife conservation is a fundamental lack of infor-
mation about the basic physiology of endangered species.
While it is obvious that the species requiring most urgent
protection and conservation are those that are considered
‘endangered’, it may be less obvious to some that these
are the very same species for which the least background
physiological knowledge exists. Reliable protocols for
inducing oocyte recruitment, development and maturation
simply do not exist in most cases. Where intensive efforts
have been made to develop such protocols, these are
often far more complex projects than originally envisaged.
For example, approximately a decade ago a focused
research programme was initiated that aimed at develop-
ing AI techniques for snow leopard and clouded leopard
(Barone et al. 1994, Brown et al. 1995, Swanson et al.
1996). Breeding leopards by the use of in vitro fertilisation
and embryo transfer (IVF–ET), or simply by AI, is of inter-
est because behavioural incompatibilities often preclude
the use of natural breeding. To date, using exogenous hor-
mones to control the timing of ovulation and the quality
of oocytes in leopards has presented a major problem.
However, while progress has undoubtedly been made, the
research can only be undertaken slowly and on a modest
scale because, being endangered, research animals are
not readily available. In fact, the regulatory authorities in
the United Kingdom frown on research projects that use
insufficient subjects, because animal welfare is then sacri-
ficed for the sake of experimental data that may be
invalid.
The same principles would apply to a cloning project.
However, if suitable oocytes were eventually obtained for
nuclear transfer experiments, questions about the viability
and fitness of resultant offspring would still need investi-
gation. Therefore, the acquisition of scholarly knowledge
about a species’ reproductive system, together with a
detailed understanding of cellular and developmental
processes peculiar to that species, is a valuable exercise in
itself but can only be undertaken within focused research
programmes. Irrespective of any cloning objectives,
scientific benefits are obtained from such studies. In most
cases, however, these projects are difficult to fund unless
the species in question is particularly unusual and the
study would lead to novel scientific insights.
A significant shortcoming of nuclear transfer technology
in its current state is the prospect that resultant offspring
will suffer from some degree of abnormality. Since the first
sheep were produced by nuclear transplantation using
cultured cells as sources of nuclei (Campbell et al. 1996,
Wells et al. 1997, Wilmut et al. 1997) many studies have
revealed that cloned mammals suffer from developmental
abnormalities. These include extended gestation, large
birthweight, inadequate placental formation and histologi-
cal defects in most organs, including kidney, brain, the
cardiovascular system and muscle (Hill et al. 1999, Barnes
2000, Chavatte-Palmer et al. 2000, De Sousa et al. 2001,
Hammer et al. 2001, Renard et al. 2002). These effects
have been attributed to inefficient reprogramming and
imprinting of nuclear DNA (Young & Fairburn 2000,
Humpherys et al. 2001, Chung et al. 2003), a process that
occurs naturally during gametogenesis and early develop-
ment, and governs whether certain genes are expressed
from the maternal or paternal chromosomes.
Cloning as a conservation support tool
Various arguments in favour of cloning programmes for
various groups of extant and extinct species have been
proposed (for reviews and proposals see Ryder &
Benirschke 1997, Lanza et al. 2000, Wells 2000, Stone
2001, Ryder 2002, Critser et al. 2003). Proponents often
cite the massive species declines that are currently occur-
ring and recommend taking any suitable actions that
might reverse the trend. These arguments mirror the view
that established reproductive technologies, such as AI and
IVF–ET, are useful for supporting living populations (Holt
et al. 1996, Holt & Pickard 1999, Watson & Holt 2001).
Certainly, focused programmes could help with genetic
management and the maintenance of genetic diversity.
Semen banks could be used for AI, although the detailed
methodologies for insemination timing and semen cryo-
preservation must be optimised for each species. These
banks of frozen semen should be considered as supporting
genetic management strategies whose primary role is to
maintain genetic diversity within populations of limited
size. In principle, frozen semen can be transported within
and between countries, thus removing part of the physical
barrier causing the fragmentation and isolation of small
populations. This function of a genetic resource bank
(GRB) provides an additional genetic benefit in the sense
that a genetically important male can still be used within
a breeding programme long after his death.
The theoretical benefits of this approach have yet to be
matched in practice but progress is being made. Useful
GRBs for a number of species are being established across
the world and some breeding programmes incorporate
frozen semen into their strategies. The black-footed ferret
Cloning and wildlife conservation 319
www.reproduction-online.org Reproduction (2004) 127 317–324
is the best example, but a cheetah breeding programme in
Namibia is now using frozen semen (http://www.cals.ncsu.
edu/agcomm/magazine/winter03/catsdogs.htm)while GRBs
for koalas in Australia (Johnston & Holt 2001) and gazelles
in Spain and Saudi Arabia (Pickard et al. 2001, Holt et al.
2002) are being established.
Applying similar logic, several international groups
have established banks of frozen tissues and cell lines (for
example, the Center for Reproduction of Endangered
Species, San Diego), in anticipation that these will provide
the genetic resources for cloning programmes aimed at
supporting declining populations. This differs from
restoring an extinct species and is, in principle, a sensible
idea. At the very least, banks of cells and tissues are useful
resources for molecular studies of evolution and phy-
logeny. Considerable caution should be exercised, how-
ever, before animals produced from such materials are
considered to make a positive and direct contribution
to the genetic well-being of a population. Inadequate
nuclear reprogramming and phenotypic abnormalities
may reduce, rather than support, the fitness of the whole
population. However, in some species these abnormal
phenotypes are not transferred to subsequent offspring
(mice, Shimozawa et al. 2002; pig, Prather et al. 2003).
This observation leads to one defence of cloning as a
potential conservation tool; provided the limitations of
these first-generation effects are recognised, it should be
possible to breed a second generation of healthy individ-
uals (Fig. 1). Using this argument raises a number of wel-
fare and ethical concerns that may eventually take
precedence over the biological arguments. Some of the
first generation of cloned offspring would have to be kept
in a managed facility or zoo, to meet their husbandry and
veterinary needs. This facility would almost certainly
attract adverse public attention, and even material damage
from animal rights activists, incurring additional costs by
virtue of its good intentions. The occurrence of first-gener-
ation abnormalities differs between species, fewer occur-
rences in pigs and mice having been noted than in cattle
and sheep, therefore the potential value of cloning for
Figure 1 Schematic diagram showing the steps by which a genetically valuable animal (nucleus donor) could be cloned. In this example, a
female of the same species serves as an oocyte donor, thereby eliminating problems of mitochondrial heteroplasmy in the resultant reconstructed
embryo. The diagram also points out that while a cloned offspring may exhibit phenotypic abnormalities, these are not necessarily evident in a
subsequent generation produced through natural breeding.
320 W V Holt and others
Reproduction (2004) 127 317–324 www.reproduction-online.org
conservation may also be species-specific. For the most
endangered species this theoretical argument is unsustain-
able in practical terms, because the background research
required to establish the absence of first-generation
abnormalities could not be undertaken. These arguments
should not, however, prevent the establishment of cell
and tissue collections for wildlife species. If techniques
eventually become simpler and more reliable these
resources could be used in the long-term; this strategy
is currently being used in attempts to prevent extinction
of the Northern hairy-nosed wombat in Australia
(Wolvekamp et al. 2001).
Several attempts at cloning exotic or endangered
species have received widespread publicity (e.g. Gaur (Bos
gaurus), Banteng (Bos javanicus) and Bucardo (Capra pyre-
naica pyrenaica)). The distinguishing feature of these
examples is that they employed trans-species cloning
(Fig. 2). In these instances, the oocyte cytoplasm being
used to create embryos was derived from common dom-
esticated species (Bos taurus (cow) or Capra hircus (goat)),
while the cell nucleus was from the species of interest.
Trans-species clones inevitably differ from either of the par-
ental species in their nucleo-cytoplasmic characteristics.
At the very least, mitochondria inherited from the recipient
oocyte would have a major influence over functions, such
as muscle development and physiology, that depend on
mitochondrial gene expression. Animals resulting from
these trans-specific cloning efforts would be scientifically
valuable for their insights into the functional relationships
involved in nucleo-mitochondria dialogue, but would not
be directly useful for supporting the endangered
populations.
However, when debating the relative value of first- and
second-generation clones, it is useful to remember that
male clones of breeding age would not transfer their mito-
chondria to the next generation (Sutovsky et al. 2000).
This knowledge, together with the ability to identify those
species that do not pass phenotypic abnormalities to
the next generation, leads to an interesting conclusion. In
theory, it should be possible to use trans-species cloning
strategies to restore the nuclear genome of a male genetic
line of particular value. This would be a highly specialised
and targeted use of cloning, and would contribute directly
to the maintenance of genetic diversity (Fig. 3). The same
argument does not apply to the female line because
mitochondria are inherited via the maternal cytoplasm.
Figure 2 This example highlights the importance of considering the fate of mitochondria in relation to trans-species cloning. Here, it is envisaged
that a domestic cow provides the oocyte cytoplasm, while the donor nucleus, and its accompanying cytoplasm, is derived from a female of a
different species. The resultant reconstructed embryo would contain mitochondria from both species, thus being an unusual type of hybrid. If
such a cloned female were to breed naturally with a male that shared its nuclear genome, the offspring would still possess mitochondria that
descended from the original oocyte donor cow.
Cloning and wildlife conservation 321
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As mentioned earlier, it is worth considering whether
cloning technologies might be applicable to non-mam-
malian vertebrates. This possibility seems to have been
overlooked by conservation biologists, despite the fact
that amphibians (Rana pipiens and Xenopus laevis) were
first cloned in pioneering experimental work during
the1960s (Briggs & King 1960, Gurdon 1962). Amphibians
are globally experiencing significant decline, possibly due
to emerging diseases such as chytridiomycosis and
ranaviruses (Daszak et al. 1999). This phenomenon, popu-
larly known as the ‘amphibian extinction crisis’ signals the
urgent need for implementing protective measures. It is
therefore surprising that there is a dearth of studies on
methods of assisted reproduction in these species. Some
research on semen collection and freezing has recently
been published (Beesley et al. 1998, Browne et al. 1998,
Mugnano et al. 1998), but most of this work has
commenced within the last 5 years. Nuclear transfer in
fish has been studied for about 40 years in China by T C
Tung (quoted by Yan 2000), with two recent papers report-
ing successful reproductive cloning in fish. Wakamatsu
et al. (2001) obtained fertile, diploid Medaka (Oryzias
latipes) by transferring embryonic cell nuclei into
enucleated oocytes, while Lee et al. (2002) cloned zebra-
fish by transferring nuclei from long-term cultured fibro-
blasts into enucleated eggs. Both studies had low success
rates (#2%), consistent with the results in mammals.
Cloning might logically be successfully applied to threa-
tened species of fish, and it might even be relatively
straightforward to mass-produce the offspring. The eggs
are often produced in large quantities, sometimes thou-
sands or millions at a time, and are physically large in
comparison to mammalian oocytes. It is possible to cul-
ture fish cells in vitro, so making an extensive collection
of endangered fish cell lines should perhaps be a current
conservation priority. However, methods for obtaining the
eggs may be the most challenging aspect of such a
programme.
Fish species are extremely diverse, as are their repro-
ductive systems. One species of molly (Poecilia formosa),
for example, reproduces entirely by parthenogenesis, and
occurs naturally as all-female populations. Although this
process differs biologically from cloning, it is similar in
some respects, being a variant of asexual reproduction.
Females mate with another sympatric Poecilia species, but
spermatozoa play no role in fertilisation and the female
Figure 3 This example shows how the problem of mitochondrial inheritance, shown in Fig. 2, could be avoided, while still using oocytes
derived from a common species such as a cow. The original donor nucleus and accompanying cytoplasm are derived from a male of the
endangered species of interest. Although the reconstructed embryo and therefore the first-generation offspring would exhibit mitochondrial het-
eroplasmy, this could be eliminated if a cloned male offspring were to breed naturally with a female of the endangered species of interest
because paternal mitochondria are not transmitted to the next generation.
322 W V Holt and others
Reproduction (2004) 127 317–324 www.reproduction-online.org
genome is copied in its entirety from one generation to
the next. Mating simply stimulates egg development, the
male does not contribute to the genome of the offspring,
and all-female clones are produced (Paxton & Eschmeyer
1998).
This extreme example underlines an important, relevant
principle. While the detrimental effects of inbreeding are
widely recognised as something to be avoided, the
specific consequences of inbreeding differ between
species. The reasons are unclear and could be a matter of
chance, but they could also reflect the environment within
which species have evolved. Thus a species that lives in
small isolated groups may be less affected by inbreeding
depression than one that is normally found in large
groups, where it would be unusual to have restricted mate
choice. This is relevant when considering the desirability
of a cloning programme; survival and fitness of offspring
may differ between species for the same reasons.
Conclusions
Biological problems associated with the cloning of mam-
mals have stimulated considerable debate about ethical
aspects of the procedure, both among scientists and the
general public. Developmental biologists at the forefront
of cloning research emphasise the need for caution, while
encouraging continued research. Unfortunately, those few
who abandon caution tend to be both highly vociferous
and good at capturing media attention. Hence, a highly
publicised plan to clone a Tasmanian tiger using DNA
recovered from a single alcohol-fixed museum specimen
has generated considerable publicity worldwide, attracted
funding for the project and raised public expectations of
success (Anon 2002, Meek 2002). Examples of this sort
manage, however, to create the impression among the
conservation community that reproductive and develop-
mental biologists are unthinking zealots who only want to
perform the latest hi-tech procedures. In an era where
rapid advances in cloning technology are being made,
perhaps it is more appropriate to focus on developing rea-
listic strategies for using these methods in wildlife conser-
vation and ensuring that scarce resources are deployed
where they will be most effective.
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