srm ART and Animals

Rescuing endangered animals with

assisted reproductive technology
David Wildt, PhD
Senior Scientist, Center for
Species Survival
Conservation and Research Center
Smithsonian National Zoological Park
Front Royal, Virginia

W
hen it comes to reproduction, many of
the world’s endangered animals are not
unlike infertile couples struggling to con-
ceive. Before the problem of achieving
conception and full-term delivery can be solved, under-
lying fertility issues need to be studied and assessed.
Animal research parallels many of the same processes
used while investigating human infertility issues, such
as genetic diversity, sperm characteristics, estrogen
surges, using fresh or frozen sperm, and even diet and
mood swings in these animals. The case studies pre-
sented in this article describe how genetic management
and assisted reproductive technology (ART) have been
instrumental in helping scientists address reproductive
problems in several endangered animals—Florida pan-
thers, giant pandas, black-footed ferrets, ocelots, and
clouded leopards.
©2009 Carl Menz

Genetic diversity in small populations

A key factor in ensuring the continued survival of endan-
gered animals is genetic management. It is important
that an animal living in captivity has the same amount
of genetic diversity as its counterpart living in the wild.
This can be difficult because scientists almost always
Genetic management and assisted
work with small populations of animals that are geo- reproductive technology are saving
graphically dispersed across zoological collections or
fragmented habitats. The overriding concerns are sexual animals on the brink of extinction
incompatibility, inbreeding, loss of heterozygosity, and
expression of deleterious alleles. These species present
special challenges to managers ranging from being timid
to dangerous to self-destructive, even homicidal. The key
is ensuring that animals with the correct (outbred) geno-
type breed together to retain heterozygosity.
Although numerous ART techniques are available
to preserve and genetically manage giant pandas and
other wildlife, the ones used most often are “low-tech,”
To watch the complete video webcast
and read the accompanying transcript,
Disclosure: Dr Wildt has no conflicts of interest to disclose. log on to www.srm-ejournal.com.

Vol. 7, No.2 21
ART and Animals srm
Figure
Reproductive techniques and wildlife
Noninvasive monitoring of hormones
Artificial insemination
Embryo transfer
Genetic
In vitro fertilization
management
Cryopreservation of sperm
Cryopreservation of embryos and oocytes
Cryopreservation of gonadal tissue
Sperm sexing
Reprinted with permission from David Wildt.
for example, with fresh or frozen/thawed
sperm, combined with extensive noninva-
sive monitoring of urinary or fecal hormones
(Figure). There is less practical application of
embryo technologies, largely because of the
lack of basic knowledge about embryology in
these rare species. Also, a major issue is the
source of a surrogate species. For example, it
KeY Point is relatively easy to produce a Siberian tiger
embryo in vitro, but where is the embryo to
Low-tech ART gestate? Maintaining a large population of
generic tigers would be expensive and coun-
techniques used ter to traditional genetic management pro-
to preserve grams, and it is now well known that inter-
and genetically specific embryo transfer is not biologically
efficient.
manage wildlife
include fresh or Florida panthers: Low gene diversity
Consider the case of the Florida panther, a
frozen/thawed relic population (ie, isolated from traditional
sperm. populations of western pumas, or mountain
lions) of “big cat” living in a restricted area
of southern Florida. By the mid-1980s, there
were only about 40 animals remaining in
nature. Our colleague, Dr Stephen O’Brien
of the Laboratory of Genomic Diversity at the
National Cancer Institute discovered that this
subspecies had no gene diversity,1 being sim-
ilar to an inbred strain of laboratory mouse.
Because the second step in our studies
was to look at male reproductive fitness, we
focused on sperm characteristics, discov-
ering that 93% of the sperm had abnormal
morphology and 42% had a malformed acro-
some. These sperm were determined to be
functionally defective because they failed to
fertilize oocytes in our standardized in vitro
fertilization (IVF) system. Meanwhile, the
field team discovered additional problems,
22 May 2009
such as atrial septal defects, cryptorchidism
(55% of males), vaginal papillomas (93%),
and a very high seroprevalence to infectious
pathogens, which suggested a compromise
in the major histocompatibility complex.1
Together, these characteristics exemplify
what happens when gene diversity in a wild
population is lost—which also explains why
genetic management of rare populations
receives so much attention. This includes
rare populations maintained in zoos where
special programs are in place and techniques
used (including the movement of animals
between zoos) to ensure that only animals of
the appropriate genotype mate.2
Saving giant pandas
The giant panda is an excellent example of
an endangered species that has been helped
by genetic management and ART, both in
zoos in North America and at several breed-
ing and conservation centers in the People’s
Republic of China.
At the National Zoo, Mei Xiang, a female
panda, and Tian Tian, a male panda, have
never successfully mated naturally, largely
due to biology and incompetence (poor cop-
ulatory positioning). Biologically, the giant
panda is intriguing, as the female is mones-
trous, coming into estrus only once per year
for 24 to 72 hours. Therefore, when the single
estrus occurs, it is critically important to rec-
ognize it. This is usually achieved by a com-
bination of behavioral cues and an estrogen
surge detected in urine. The National Zoo’s
pandas have been unable to achieve a nor-
mal copulatory stance, with the female often
lying down and preventing the male from a
normal rear entry. As a result, transcervical
artificial insemination (AI) via endoscopy
has been important, and resulted in the birth
of Tai Shan, a male cub in 2005.
Studying giant pandas in captivity. On a much
larger scale, genetic management and ART
have boosted the population of giant pan-
das in China from 104 in the late 1990s to 271
in 2008. Our colleagues in China expect to
reach the targeted level of 300 pandas very
soon. This is the number needed to ensure
genetic diversity in the captive population;
at which time it would be possible to harvest
animals from captivity for reintroduction
into the wild, if deemed necessary by wild-
life managers.

This accomplishment was backed by
more than a decade of basic and applied
research. When I first visited China in 1996
at the invitation of the Chinese Association
of Zoological Gardens, local scientists were
primarily concerned that the captive panda
population was not self-sustaining (eg, more
animals were dying every year than were
being born).
Together, Chinese colleagues and our
American team decided that the first step
in attempting to correct this situation was
to conduct a biomedical survey to deter-
mine the factors that were limiting repro-
ductive success. From 1998 through 2000,
more than 60 giant pandas were examined
in detail, especially at the Wolong National
Nature Reserve, the Chengdu Research Base
of Giant Panda Breeding, and the Beijing
Zoo. Vast amounts of data were generated—
enough to produce a book on giant panda
biology, veterinary medicine, and manage-
ment that was published by the Cambridge
University Press.3
Identifying significant problems. Among the
many problems identified were aggres-
sive behavior by males, inadequate fiber in
diets (due to the use of inappropriate artifi-
cial foodstuffs), and numerous health chal-
lenges, many of which could be corrected by
minor improvements in preventative health.3
More severe conditions were associated with
developmental abnormalities including a
stunted development syndrome observed in
more than 13% of examined pandas. These
individuals were only two-thirds the size of
“normal” individuals and had ascites, poor
teeth, miscellaneous health problems, and
consistent failure to reproduce. One by one,
each of these limiting factors was addressed
to gradually improve reproduction in adults
and survival of newborn cubs.
Often our applied studies were accom-
panied by systematic basic research stud-
ies, for example, understanding the quality
of giant panda sperm and its cryosensitivity
to eventually allow AI with thawed sperma-
tozoa. Additionally, substantial fundamental
research was directed at noninvasive hor-
monal monitoring, first with urine and later
in feces. This allowed managers to clearly
identify the single, annual estrogen surge and
the biphasic rise in progesterone—an initial
and very modest post-ovulatory rise followed
srm ART and Animals
by a later robust secondary rise (believed to
occur coincidentally with implantation).3
Furthermore, our more recent discovery that
hormone profiles can be measured in panda
feces (often comprised of bamboo remnants
of stems and leaves) has facilitated track-
ing of endocrine patterns in large facilities
that maintain too many pandas to effectively
collect daily urine samples. Fecal hormone
monitoring also likely has application to ani-
mals living in the wild where fecal samples
are routinely encountered.
All of this effort and information com-
bined has had a remarkable effect on giant
panda cub production. Generally, the Chinese
breeding facilities are producing an average
of 25 to 30 surviving cubs annually. This suc-
cess is a result of natural breeding supported
by AI using fresh as well as thawed spermato-
zoa. The giant panda is an excellent example
of a rare species that has benefited from a
scientific partnership and ART that has gen-
erated useful new knowledge to more than
double the numbers of individuals within the
captive population. KeY Point
Declared “extinct,”
The black-footed ferret returns to
nature thanks to ART
a remnant
The black-footed ferret is an example of an population of
endangered species that has been returned
to nature thanks to genetic management and
18 black-footed
ART. The black-footed ferret became nearly ferrets was
extinct with the demise of prairie dogs, its discovered,
primary prey. This chain of events started
in the early 1900s, when prairie dogs were brought into
subjected to poisoning as part of the effort to captivity and
convert grassy American plains to farmland
and rangeland. As the prairie dogs and their now number 6100
habitat disappeared, so did the black-footed descendants.
ferret. The US Fish and Wildlife Service put
the black-footed ferret on the endangered
species list in 1967, and by the late 1970s, the
species was declared extinct.4 However, in
the early 1980s, a remnant population of 18
black-footed ferrets was discovered in Wyo-
ming and brought into captivity.3 The first
step in developing an AI technique involved
working with a similar species as an ani-
mal model, the common European ferret. By
developing an understanding of the repro-
ductive biology of the European ferret, it was
then possible to develop a laparoscopic AI
technique that eventually resulted in consis-
tent births of baby ferrets, or kits.
Vol. 7, No.2 23
ART and Animals srm
This success gave us the confidence to
start detailed studies on the black-footed
ferret, which included breeding animals
naturally and conducting basic research
studies. The end result of more than 20 years
of work that involved the help of many zoos,
state and federal agencies is more than 6100
descendants from the original 18 ferrets. At
the Conservation & Research Center, our
work has resulted in more than 500 black-
footed ferrets, including 135 ferrets by AI,
mostly with fresh sperm but also with fro-
zen/thawed spermatozoa. Due to sexual
incompatibility problems, these 135 ferrets
would never have been born without AI.
Today, there are more than 1000 free-living
black-footed ferrets in 8 states in the Ameri-
can West and Mexico.
The ocelot: Embryo technologies
Despite the success of IVF and embryo
transfer in humans and livestock species, no
wildlife species is currently being genetically
KeY Point managed using such technology. However,
these methods have produced some signifi-
ART is not always cant births, often “one-time wonders,” that,
while attracting substantial media attention
needed to enhance have not translated into practical application.
reproduction. The challenges associated with using
Often, it is simply embryo transfer and IVF relate largely to
the lack of data on embryology in virtually
a matter of all wildlife species as well as logistical issues
changing related to finding an appropriate surrogate.
Zoos do not have sufficient space to maintain
environmental large numbers of potential embryo recipients.
factors. And not only is interspecies embryo transfer
poorly effective, but there is the concern that,
for example, a tiger cub produced in a lion, or
an addax calf born to a scimitar-horned oryx
would be behaviorally confused.
Despite the complexities of adapting
embryo technologies to wildlife, I am encour-
aged by studies being done with ocelots at
the Center for Conservation and Research
of Endangered Animals at the Cincinnati
Zoo and Botanical Garden by Dr William
Swanson. His group is collaborating with
colleagues in Brazil to use IVF to genetically
manage this species with the eventual goal
of allowing international transport of ocelot
diploid genetic material.5 Extensive basic
studies in Brazil have resulted in substantial
new information on the basic biology of this
species. That, in turn, resulted in the ability
24 May 2009
to successfully treat ocelots (many wild born
and, thus, extremely valuable genetically)
with hormones to allow laparoscopic aspi-
ration of oocytes followed by successful IVF
and embryo freezing. Following the initial
studies in 2000, frozen embryos were thawed
in 2007, transferred to ocelot recipients and,
to date, have resulted in 3 pregnancies and 3
living offspring (from 24 total embryos trans-
ferred to 8 surrogates). Although such per-
centages may be considered comparatively
low by human IVF/transfer standards, this
is one of the first examples of consistency in
the wildlife world, demonstrating the poten-
tial of the technology after persistence in
documenting the basic reproductive biology
of the target species.
Sperm sexing
Although not routinely used in man-
aging wildlife species, there is enormous
potential in developing sperm sexing to be
used largely with AI. Unbalanced sex ratios
are common in captive animal collections.
For example, 70% of all calves produced by
2 rhinoceros species in North American zoos
are males.6 This places unusual stress on
space resources and forces the management
of many large, singleton males.
The potential of sorting sperm (Figure) on
the basis of DNA content in X-bearing sperm
compared with Y-bearing sperm deserves
significant attention in addressing this issue.
The most progress has been reported by Jus-
tine O’Brien and Todd Robeck of SeaWorld
Incorporated, who have used flow cytometry
to produce a preponderance of X-bearing
sperm and, thus, higher ratios of calves in
various marine mammals, including artifi-
cially inseminated killer whales.
The clouded leopard: Environmental
influences on reproductive success
Finally, it is important to realize that
not all enhancement of reproduction needs
to occur as a result of ART. In many cases, it
is simply a matter of changing environmen-
tal factors that also are well known to affect
human and livestock reproductive efficiency.
A prime example has been our interest in
the conservation and genetic management
of the clouded leopard, a mid-sized cat
species indigenous to southeastern Asia.

Our country of interest has been Thailand
because zoos there have a high incidence
of wild-born clouded leopards, often con-
fiscated from illegal holders. Until our col-
laborative studies, however, these animal
had never reproduced. A survey of Thai zoos
revealed suboptimal housing, for example,
inadequate diets and conditions where
clouded leopards were housed adjacent to
larger, predatory tigers or leopards. In fact,
fecal hormone monitoring determined that
clouded leopards housed in such conditions
produced higher fecal corticoid concentra-
tions than conspecifics maintained off-exhibit
and away from larger carnivores. Clearly
stress was playing a major role in suppress-
ing successful reproduction.
Likewise, obesity was recognized as
another issue, with animals receiving high
fat, low protein diets without vitamin or min-
eral supplementation.This dietary imbalance
also caused metabolic bone disease, charac-
terized by extreme joint laxity, eye lesions,
cardiac defects, poor lactation, and poor cub
survival. Not surprisingly, a lack of exer-
cise also contributed to obesity, since many
leopards were living in small enclosures.
When the environment, including housing
and diet, was improved, reproduction com-
menced with more than 30 surviving cubs
produced in the program.
References
1. Roelke ME, Martenson JS, O’Brien SJ. The conse-
quences of demographic reduction and genetic
depletion in the endangered Florida panther. Curr
Biol. 2003;3:340-350.
2. Ballou J, Gilpin M, Foose TJ, eds. Population Man-
agement for Survival and Recovery. New York, NY:
Columbia University Press; 1995:375.
3. Wildt D, Zhang A, Zhang H, et al, eds. Giant Pandas:
Biology, Veterinary Medicine and Management. New
srm ART and Animals
Conclusion
ART will continue to play a significant role
in both understanding the biology and sav-
ing endangered animal species. However,
progress will take many years, because the
successful use of ART in one animal species
cannot easily be transferred to another—the
way that animals reproduce is as different as
their genetic diversity and physical appear-
ances. Nonetheless, as outlined here, there
are exciting examples of successes demon-
strating how advances that have occurred
in human reproductive health and livestock
production can have consistent application
once the basic biology of a given species is
well established. n
Author’s acknowledgment
This work was possible due to the dedicated
efforts of the following colleagues: JoGayle
Howard, Budhan Pukazhenthi, Ken Lang, and
Mitch Bush of the Smithsonian National Zoo-
logical Park; Stephen O’Brien and Melody
Roelke, National Cancer Institute; Susie Ellis, KeY Point
International Rhino Foundation; Don Jans-
sen, Zoological Society of San Diego; Zhang The way that
Zhihe, Chengdu Research Base of Giant Panda
Breeding; Zhang Hemin, Wolong National
animals reproduce
Nature Reserve; and William Swanson of the is as different as
Cincinnati Zoo and Botanical Garden.
their genetic
diversity and
physical
York, NY: Cambridge University Press; 2006. linkage of in situ—ex situ populations. In: Roldan E,
4. Howard JG, Marinari PE, Wildt DE. Black-footed
ferret: Model for assisted reproductive technologies
Gomenido M, eds. Spermatology. Nottingham, Eng-
land: Nottingham University Press; 2007:417-432.
appearances.
contributing to in situ conservation. In: Holt WV, 6. Pukazhenthi BS, Wildt DE. Which reproductive
Pickard A, Rodger JC, et al, eds. Reproductive Sci- technologies are most relevant to studying, man-
ences and Integrated Conservation. Cambridge, MA: aging and conserving wildlife? Reprod Fertil Dev.
Cambridge University Press; 2003:249-266. 2004;16:1-14.
5. Swanson WF, Stoops MA, Magarey GM, et al. Sperm
cryopreservation in endangered felids: Developing
Vol. 7, No.2 25