REPRODUCTION BIO-TECHNOLOGIES IN THE
CONSERVATION OF WILD ANIMAL SPECIES
Are they really important?
1. Summary :
Key words.
2. Introduction
3. Assisted Reproduction Techniques (ART):
 Artificial Insemination
 In vitro Fertilization
 ICSI
 Embryo Transfer
 Fostering and Pouch Young Transfer.
 Cloning
 Spermatogonial Transplantation
 Ovarian Cryopreservation
4. Frozen Zoo (=Genetic Resource Banking) :
 Concept
 Institutions involved in Frozen Zoo Projects
1. Center for Reproduction of Endangered Species
(CRES)- San Diego Zoological Society
2. Center for Reproduction of Endangered Wildlife
(CREW)- Cincinnati Zoological Society
3. Audubon Center for Research of Endangered species
(ACRES)
4. Omaha´s Henry Doorly Zoo
5. Washington National Zoological Park (CRC)
6. Animal Gene Storage Resource Centre of Australia
7. Breeding Centre for Endangered Arabian Wildlife
8. WBRC – South Africa
9. Mohor Gazelle GRB
5. Final Considerations
6. References
Summary
Many captive breeding programmes have plans to manage small
captive populations which attempt to minimize inbreeding as well as
other processes that are believed to cause extinction. Considering
that genetic variability maintenance is dependent on reproduction,
assisted reproduction techniques are important tools for the
preservation of endangered species. Biodiversity is important for
sustaining a healthy Earth, but it also is immensely valuable to the
health and lifestyle of human society.
Science is key to conserving wildlife, whereas the wild animals
(and plants) comprise valuable genes and knowledge that can have a
positive impact on society. To conserve endangered species, an
understanding of their ecology and physiology is fundamental.
Zoological institutions can achieve this by maintaining captive
populations in conditions resembling those in the wild.
The reproductive biotechniques appear as alternative tools for
conservation programs of genetic material originating from
endangered animals.
Key words:
Cryopreservation, Biodiversity, Genetic Diversity,
Wildlife, Reproduction
Introduction
Biological diversity is the key to maintaining life as
we know it (Wilson 1992). However, rapidly growing
human populations place extraordinary pressures on
ecosystems,
such
as
large-scale
environmental
destruction, habitat conversion, habitat fragmentation, and
pollution. Virtually all conservation biologists agree that
habitat preservation is the best way to conserve
biodiversity. Captive breeding programs, genetic resource
banks (GRB) and artificial reproductive techniques (ART)
have been suggested as important tools for conservation
(Ballou, 1992, (Wildt, 1989; Wildt et al., 1993; Wildt et
al., 1997; Wildt and Wemmer, 1999)), including for
Neotropical carnivores (Morato and Barnabe, 2002).
Several problems arise when a species reaches a
very small population size: a. Loss of Genetic Variability:
When a population gets below about 500 breeding
individuals, the amount of genetic variation is reduced; b.
Inbreeding: Populations that fall below about 50 breeding
individuals are forced to breed with close relatives which
can lead to problems that includes decreased fertility, high
juvenile mortality and birth defects. Inbreeding also leads
to the expression of specific genetic defects, i.e.,a high
incidence of a genetic defect of the diaphragm in the
Golden lion tamarin, and kinky tail in the Florida panther;
c. Hibridization: seems to occur more often when species
are reduced to very small numbers; d. Selective breeding:
with long periods in captivity. Soulé (1991) has described
a “ biospatial hierarchy” to protect biodiversity, beginning
at the top with whole ecosystems (in situ protection) and
proceeding
down
through
communities,
species,
populations (ex situ zoo breeding programs), and
eventually to cryobanked biomaterials. The maintenance
of species in captivity in zoos, aquaria, and botanic
gardens is called ex situ conservation. Animal agriculture
has benefited from large-scale sperm and embryo
cryostorage for the purpose of improving meat and milk
production in domestic livestock.
In the early 1900s, waves of immigrants from
Europe settled in the American Midwest. As humans
transformed the land, they declared war on a perceived
pest: the prairie dog. An innocent victim of the eradication
campaign was the black-footed ferret, which preyed on
the prairie dogs. Using artificial insemination, National
Zoological Park’s team lead by Howard has brought the
species out of extinction red zone. This story offers three
important lessons. The first is that no species can be
managed in isolation. The second lesson is to expect
frustration when trying to get a little-known animal to
breed in captivity. The third lesson is: “high-tech”
approaches to getting a species to procreate can work. The
most important lesson learned during the past quartercentury has been that species vary remarkably- and
wondrously- in precisely how they reproduce. This
reproductive machinery varies significantly even within
families, species positioned in the same branches of the
evolutionary tree (Wildt et al., 1992, 1995).
The concept of genetic resource banking go back
more than fifty years to the discovery of method for
preservation of spermatozoa at very low temperatures.
There followed a revolution in animal breeding,
particularly in cattle. Development of methods for
freezing spermatozoa and embryos of wild animals relies
mainly on results obtained in domestic and laboratory
species. Practical, reliable methods for monitoring
gonadal status are essential for developing and using
assisted reproduction technology. Cryobiology is the
science integrating the cellular events that occur under
low temperature conditions. The key to cell survival is the
physiochemical relationship of heat and water transport
between the intra- and extracellular environment. The art
of cryobiology involves the addition of one or more
cryoprotectants (e.g., DMSO or glycerol), which generally
reduce both the eutectic and freezing points. Mammalian
embryos are composed of up to 80% water. As embryo
cryopreservation is developed and applied to new taxa,
some special technical modifications will be necessary
because
of
permeability
species
differences
characteristics,
and
in
embryo
size,
macro-molecular
cytoplasmic content. Even within species (e.g. laboratory
mice), there are reports of genotypic differences that
influence embryo survival following cryopreservation.
In the future, the constitution of embryo and gamete
banking could be an important strategy for restoring
genetic vigor and sustaining global biodiversity of
endangered species (Schiewe et al., 1995). In increasing
order of complexity and “sophistication”, some of the
reproductive biotechnologies seen as potential solutions to
the prevention of loss of biodiversity are AI, embryo
transfer, cryopreservation of gametes and embryos,
gamete intra- fallopian transfer (GIFT), IVF, oocyte
maturation, micromanipulation to produce twin embryos
or clones by nuclear transfer, intracytoplasmic sperm
injection, sperm sexing to separate X- and Y- bearing
spermatozoa, and in vitro development of ovarian
follicles.
1.
Assisted Reproduction Techniques (ART)
1.1
Artificial Insemination
Artificial Insemination is the introduction of
semen by means of a pipette into the reproductive tract of
the female at the correct stage of her cycle. The first
successful artificial insemination was performed in a dog
by Abbé Lazzaro Spallanzani in 1780. Three main sites of
insemination in mammals are: vaginal, intracervical and
intrauterine.
Laparoscopically
guided
intrauterine
insemination, initially developed in sheep, has now been
successfully applied to a wide range of non-domestic
species, including several wild feline species (Howard et
al. 1992; Wildt et al. 1992; Donoghue et al. 1993; Barone
et al. 1994; Swanson et al. 1996a), numerous deer species
(Jabbour et al., 1997; Pope and Loskutoff, 1997), non-
domestic bovids, Giraffes and Camels (Pope and
Loskutoff, 1997) as well as non-human primates (Gould
and Martin, 1986; Morrell, 1995). Insemination using
frozen semen has also been widely reported, but in
general, fertilization rates are lower than those achieved
with fresh semen. To date, AI with fresh or thawed sperm
has been used to produce offspring in ten felid species
(domestic Cat, Leopard, Puma, Leopard Cat, Cheetah,
Tiger, Clouded Leopard, Snow Leopard, Ocelot, Tigrina)
(Howard et al., 1992a; Dresser et al., 1982; Moore et al.,
1981; Barone et al., 1994; Wildt et al., 1992; Howard et
al., 1992b; Donougue et al., 1993; Howard et al., 1996;
Roth et al., 1997; Swanson et al., 1996b; Moraes et al.,
1997), with frozen-thawed spermatozoa used successfully
in three species (Leopard Cat, Ocelot, Cheetah) (Wildt et
al., 1992; Swanson et al., 1996b; Howard et al., 1997).
At Dallas Zoo an ocelot (Leopardus pardalis)
inseminated laparoscopically in utero gave birth to a
single kitten 78 days after insemination. The AI was
performed
using
frozen-thawed
sperm originally
obtained from a male at Colorado Springs (Swanson et
al. 1996; Howard et al., 1996). Banked germ plasm can
be used long into the future. For example, cattle sperm
that have been cryopreserved for 37 years retain
fertilization capacity (Leibo 1994).
Postmortem
collection
of spermatozoa has been described for injured or dead
domestic bulls and wild animal species. Studies
indicate
that
spermatozoa
must
be
recovered
immediately due to the deleterious effects of tissue
degeneration. Tissue degeneration can, however, be
retarded by cooling, and the spermatozoa within the
cauda of the epididymides are the most resistant to cold
shock damage and also have the highest fertility
potential (Watson, 1981).
Sperm
cryopreservation procedures must be modified and
optimized for each species (Watson 1995). Thawed
sperm have high percentages of damaged acrosomal
membranes (Wood et al., 1993; Swanson et al. 1996;
Howard et al., 1996), however, the biological
competence of these sperm has been demonstrated in
the domestic cat (Platz et al., 1978), Leopard cat and
Ocelot (Swanson et al. 1996; Howard et al., 1996).
Mammalian spermatozoa, particularly those of
ungulate species, are sensitive to abrupt cooling from
physiological or room temperature to 0ºC, a phenomenon
referred to as “cold shock”. Manifestations of “cold
shock” are spermatozoa swimming in circular manner,
premature loss of sperm motility, decreased energy
production, increased membrane permeability, and loss of
intracellular molecules and ions. Moreover, addition of
egg yolk, which has been shown to protect spermatozoa
from domestic and laboratory animals against cold shock,
may prove to be beneficial for cryopreservation of
spermatozoa
from
wild
species.
While
semen
cryopreservation is successfully used for a few species,
application to other species can be problematic. The
species differences in female tract anatomy, subtle
differences in sperm transport mechanisms, ability to time
inseminations and deliver spermatozoa effectively are
powerful determinants of fertility with cryopreserved
spermatozoa.
The first birth after AI in antelopes occurred in the
Speke`s Gazelle (Gazelle spekei) using fresh spermatozoa
(Boever et al., 1980). Successful AI in Cervids has been
reported in the Wapiti (Cervus elaphus), Fallow Deer
(Dama dama), Axis Deer (Axis axis; Chapman et al.,
1999; Mylrea et al., 1992), Reindeer (Rangifer tarandus;
Dott & Usti, 1971; 1973) and Eld`s Deer (Cervus eldi;
Monfort et al., 1993).
The
first
successful
applications
of
artificial
insemination in cetacean occurred in the killer Whale and
then in the bottlenose Dolphin.
The
snow Leopard (Panthera uncia) differs among felid
species (even within the Panthera lineage) in that sperm
motility longevity in vitro is enhanced by a simple
(phosphate buffered saline; PBS) rather than a complex
(Ham`s F10) medium (Roth et al., 1995). This unique,
adverse response indicates a species- specific sensitivity
not observed in other felid taxa studied to date.
Artificial insemination is a feasible strategy for
perpetuating valuable genes from non-breeding giant
pandas with behavioral incompatibility problems or
compromised health. Assisted reproduction can be
utilized efficiently as a management tool to maintain
genetic diversity and avoid inbreeding depression in the
ex situ population. The artificial vagina has made possible
collect semen in camelids, bovids, cervids and even
cheetah (Acynonyx jubatus) (Watson, 1978). The first
documented birth of a Cheetah cub as the result of
assisted
reproductive
technology,
laparoscopic
intrauterine artificial insemination in this case, was in
1991. Laparoscopic intrauterine artificial insemination
consists in depositing sperm transabdominally into the
proximal aspect of each uterine horn. It may be useful for
overcoming barriers preventing sperm cells from reaching
the site of fertilization. Laparoscopic AI is finding utility
in other endangered taxa. Leopard cats (Felis bengalensis)
(Wildt et al.,1992), a Puma (Felis concolor) (Wildt et al.,
1994), and a Tiger (Panthera tigris) (Donoghue et al.,
1993) cub have been produced using this approach.
Cheetah
litters
have
been
born
from
artificially
inseminated females, including a cub born at the Rio
Grande Zoo in Albuquerque, New Mexico, with frozenthawed sperm that was collected from a wild, free-ranging
cheetah
from
Namibia
(D.E.Wildt,
T.L.Roth
&
J.G.Howard, unpubl. Data). Also, Eld´s Deer (Cervus
eldi) females produced offspring following laparoscopic
insemination with frozen-thawed sperm (Monfort et al.,
1993).
Semen
collection
and
cryopreservation
in
marsupials have received significant attention (Taggart et
al., 1996, 1997, 1998; Molinia & Rodger, 1996; Holt et
al., 1999; Johnston et al., 2000), and basic information is
now available for representatives from most Australian
marsupial families. In the Koala (Phascolarctus cinereus),
artificial insemination is a powerful tool for facilitating
genetic exchange between captive colonies, and the
maintenance
of
genetically
important
remnant
populations. Artificial Insemination has been successful in
the koala (Johnston et al., 2000a), brush-tailed Possum
(Molinia et al., 1998a; Jungnickel et al., 2000) and more
recently in the Tammar Wallaby (D. Paris, personal
communication). Unlike eutherian species, Trichosurus
vulpecula
and
Phascolarctos cinereus spermatozoa
require high levels of glycerol (up to 17,5%) for
cryopreservation (Rodger et al., 1991; Johnston et al.,
1993; Molinia and Rodger, 1996).
It may also be technically feasible to artificially
inseminate free-living females with sperm from males of
other wild, or even captive, populations. The latter
approach has already been made part of the conservation
management plan for Sumatran tigers living in highly
fragmented Indonesian habitats (Tilson and Brady 1992).
Moreover, a GRB could reduce or eliminate the need to
remove animals from the wild to support captive
populations. Concerning the use of cryoprotectants,
results from a preliminary study in the white rhinoceros
indicate that glycerol is toxic to spermatozoa (Williams et
al., 1995), but for black and Sumatran rhinoceroses the
glycerol can be used efficiently as a cryoprotectant (Roth
et al., 2000). Therefore, the potential toxicity of glycerol
may be species- or even individual-specific. It is possible
that spermatozoa from different types of sample
(epididymal
versus
ejaculated)
show
different
cryoprotectant sensitivities. The potential toxicity of
glycerol has been observed for rhesus monkey sperm
(Leverage et al., 1972) and marmoset sperm (Morrell,
1997a).
Dimethyl
sulphoxide
(DMSO)
is
the
cryoprotectant preferred for elephant spermatozoa (Jones,
1973).
For the scimitar-horned Oryx (Oryx dammah),
which thrives in large numbers in captivity, artificial
insemination has potential for eliminating the risks of
animal transport, and for optimizing the use of limited
enclosure space, while simultaneously preserving
extant gene diversity. Recent results demonstrate that
non-surgical AI in scimitar-horned Oryx using frozenthawed sperm results in pregnancy rates approaching
40% after a single fixed-time insemination. Evaluating
cryopreserved fringe-eared Oryx (Oryx gazella callotis)
sperm
function
using
a
heterologous
in
vitro
fertilization system previously developed to study
scmitar-horned Oryx (Oryx dammah) spermatozoa,
fringe-eared Oryx spermatozoa were incapable of
penetrating zona-free cow oocytes (Roth et al., 2001).
These results indicate that species- specific differences
in gamete interaction may exist even between very
closely related nondomestic bovids. Studies indicate
that
epididymal
needle
biopsy
under
ketamine
immobilization may be a useful method for laboratory
and field-based sperm collection in nonhuman primate
species
(Macaca
fascicularis)
for
posterior
cryopreservation. Assisted reproduction techniques can
contribute
to
jaguar
conservation
through
the
development of a resource genetic bank, in vitro, with
somatic cells, sperm, oocytes and embryos. Sperm
DNA-protein complex structure modifications can be
observed as a result of cryoinjury, affecting the oocytesperm interation (Morato et al., 2001). There is a
significant
loss
of
spermatic
quality
after
cryopreservation. The DNA-protein complex coloration
test is an in vitro test that helps cryopreservation
proceedings avaliation. Percoll and Swim-up are
efficient methods to perform the golden hamster zona
free oocyte penetration assay using frozen/thawed
jaguar (Panthera onca) spermatozoa (Morato et al.,
2001). The sperm penetration assay (SPA) is a
diagnostic test that measures the ability of sperm to
fertilize ova.
The low rate of penetration could be
related to the high rate of morphological abnormal
spermatozoa observed in the samples examined.
Samples from South China tigers males that produced
motile sperm were cryopreserved and stored in a
Genome Resource Bank located at Shanghai Zoo.
Acrosomal membranes of felid spermatozoa appear to
be especially sensitive to freezing injury: ~50% of
cryopreserved spermatozoa from both domestic and
non-domestic felid were damaged after freezing and
thawing (Tilson et al., 1987). A litter of red Wolf
(Canis rufus) pups has been born after artificial
insemination by surgical deposition of fresh semen into
the uterine horns (Waddell and Platz, personal
communication). Artificial breeding of wapiti (Cervus
elaphus) in North America is now a commercial
success. Several hundred inseminations have been
carried out in the last 5 years (Bringans, personal
communication).
Artificial insemination utilizing fresh extended
semen in elephants has recently been successful. The first
calf from AI was born in 1999 (Asian Elephant), and two
additional calves (African Elephant) were born in 2000.
Successful artificial insemination includes the
blackbuck (Antilope cervicapra) (Holt et al., 1988) and
Mohor Gazelle (Holt et al., 1996b), Giant Panda
(Ailuropoda melanoleuca) (Moore et al., 1984), Puma
(Felis concolor) (Moore et al., 1981) and scimitar-horned
Oryx (Oryx dammah) (Garland, 1989). The first offspring
from artificial insemination in Camelidae was reported in
a bactrian Camel inseminated with frozen semen collected
by electro-ejaculation in 1961 (Elliott, 1961).
Female birds, unlike their mammalian counterparts,
have sperm storage tubules (SST) that allow viable
spermatozoa to be retained for extended periods in the
reproductive tract. Artificial Insemination with fresh or
frozen semen has also been used successfully to overcome
natural breeding failure in about 20 non-domestic species.
Non-domesticated avian species successfully breed with
cryopreserved spermatozoa: Sandhill crane; Canada
goose; American Kestrel; Peregrine Falcon; Houbara
Bustard and Edward´s pheasant. Especially important has
been the development of semen collection and AI that
have been essential to the effective management of many
species, but particularly to the Mississipi Sandhill Crane
(Gee et al., 1985; Ellis et al., 1996). In avian species,
dimethylacetamide
(DMA)
and
dimethylsulfoxide
(DMSO) have been used as alternative cryoprotectants to
glycerol because of its contraceptive effect.
The potential value of sperm cryopreservation and
AI to conserving and genetically managing amphibians is
significant. Anuran spermatozoa have an unusual
structure that may be essential for both sperm longevity
and post-thaw survival. This structure, first identified as
an accessory cell on Lepidobatrachus laevis spermatozoa
by Waggener & Carroll (1998a), isolated in the head
region of the spermatozoon.
AI with frozen-thawed sperm is relatively common
in the breeding and management of fish (Harvey 1993).
1.2
In vitro Fertilization
IVF is a process where sperm are mixed in a culture
dish with oocytes that are aspirated directly from a
female´s ovaries using a needle guided by fiber optic or
ultrasonic techniques. Amongst the felidae, IVF-derived
offspring have been produced in the Indian desert Cat
(Pope et al., 1989) and Tiger (Donoghue et al, 1990). IVF
has resulted in births in a variety of non-human primate
species (Loskutoff et al., 1991a), including a Western
Lowland Gorilla (Pope et al., 1997). Oocyte recovery, in
vitro maturation and in vitro fertilization have been
reported for the Gaur (Bos gaurus) (Johnston et al., 1994),
Klipspringer (Oreotragus oreotragus) (Raphael et al.,
1991), Bongo (Tragelaphus euryceros) (Pope et al., 1998)
and Addax (Addax nasomaculatus) (Asa et al., 1998).
In vitro fertilization (IVF) has been achieved in the
grey short tailed Opossum (Mo. Domestica) using mature
follicular eggs and epididymal spermatozoa (Moore and
Taggart, 1993). Studies suggest that hMG and hCG are
effective
for
stimulating
ovarian
activity
in
nonreproductive gorillas and that the recovered oocytes
are capable of being fertilized in vitro. Studies
demonstrate that ovaries of the caracal and aged cheetah
are responsive to pregnant mare´s serum gonadotropin
(PMSG) treatment and that mature oocytes can be
collected effectively from each species using laparoscopic
techniques. The ocelot has a low intrinsic sensitivity to
eCG/hCG and on a per body weight basis required at least
three to ten times the dose routinely administered to
cheetahs, pumas and clouded leopards (Swanson, Howard
et al., 1996). The superovulation and in vitro fertilization
protocols established for tigers need further evaluation for
jaguar since the actual rate of clivage is low (Morato,
2001). Between 0,5 to 3 hours after insemination, about
90% of oocytes are penetrated (Niwa et al., 1985). Most
of oocytes tend to clivage, in vitro, between 24 and 28
hours after insemination, and about 70% of the oocytes
can be at an embrionary stage of 2 to 4 cells (Pope, 2000).
Some authors cryopreserve embryos at the stage of 2 to 4
cells to, after thawing, make the transfer to oviduct
(Donoghue et al., 1990). It is possible that the several
feline species sperm cells have different preferences for
semen processing methods and fertilization environment
(Swanson et al., 1996). Studies of In vitro fertilization of
Bornean orangutan (Pongo pygmaeus pygmaeus) gametes
followed by embryo transfer into a surrogate hybrid
orangutan (Pongo pygmaeus) are developed at Woodland
Park Zoological Gardens in Seattle, Washington. This
type of program might enable zoos that house hybrid
orangutans to have infants born and raised in their
collections. In vitro fertilization has been reported in
numerous monkey species from several genera, including
Saimiri, Macaca, Cercophitecus, Callithrix and Papio.
1.3
ICSI
Intracytoplasmic sperm injection (ICSI) is a
process where an individual sperm cell is microinjected
into an oocyte to create an embryo. Methods for
Intracytoplasmic Sperm Injection (ICSI) have resulted in
live offspring in human, cattle and rabbits, although in
some species (e.g. felids) other approaches such as
subzonal insertion appear more successful (Pope et al.,
1995). The primary advantage to either approach is the
potential for generating embryos from a number of
previously untapped sperm sources such as oligo/teratospermic donors, testicular sperm and even from dead or
damaged sperm fragments. Sperm microinjection could be
especially important in cases where: 1-only a few males
remain; 2-overall semen quality is low or 3- there is a
high incidence of teratospermia (e.g. in wild felids; Wildt
et al., 1998). ICSI-fertilised embryos have been produced
in a number of primate species (Iritani et al., 1998).
Intracytoplasmic sperm injection (ICSI) with frozenthawed epididymal spermatozoa was performed in the
Cynomolgus Monkey (Macaca fascicularis) to produce
embryos in vitro. Overall 4 Monkeys became pregnant
resulting in the birth of 2 healthy infants. ICSI has
potential application in conservation of highly endangered
nonhuman primate species. A Rhesus Monkey offspring
have been produced following transfer of ICSI- generated
embryos (Chan et al., 2000a,b; Hewitson et al., 2000).
ICSI should be useful for producing embryos in vitro
when sperm quality is not suitable for doing standard IVF,
as has been demonstrated in Jaguarundi (Herpailurus
yaguarondi) (Pope, 2000).
Segregation of X- and Y- bearing sperm can
now be achieved with reasonable reliability using Flow
Cytometry to separate on the basis of DNA content (e.g.
Johnson, 1997). Birth of offspring of pre-determined sex
using flow cytometrically sorted fresh sperm was first
performed in rabbits (Johnson et al., 1989). Adaptation of
flow cytometry to sort sperm of wildlife species has been
achieved in non-human primates including the baboon and
marmoset (O`Brien et al., 2001). The combined approach
of sperm sexing and ART has great potential as a
population management strategy for species with singlesex dominated social structures such as the gorilla.
1.4
Embryo Transfer
In embryo transfer, fertilized eggs or early
embryos (usually about 8- cell stage) are removed from
the reproductive tract of a donor female and transferred
into the tract of a surrogate mother, who carries the
embryos to term and produces live young. Embryo
transfer procedures in non-domestic species are often
performed in conjunction with ovarian stimulation
protocols to enable the collection of larger number of
embryos either for immediate replacement or storage.
Embryo transfer increases the number of progeny per
female;
prevents
disease
transmission
between
populations; the offspring acquire passive immunity from
the surrogate mother via placental blood supply or milk;
animals can be transferred from one place to another as
embryos in test tubes and the embryos can be frozen
before transfer into the recipient female. In vitro embryo
production is the artificial generation of an embryo in
culture using gametes (oocytes and sperm) that have been
collected in a mature or immature state. All evidence
suggests that frozen mammalian embryos will remain
viable for generations.
Embryos are cryopreserved by slow freezing or by
vitrification (Martínez et al., 1998). Generally, the
survival of cryopreserved oocytes/embryos differs not
only among cryopreservation methods but also according
to the species, the maturational/developmental stage, and
even the quality of the oocyte/embryo. Unfertilized
oocytes are more sensitive to cryopreservation than
embryos. In vitro-derived embryos are more sensitive to
cryopreservation than in vivo fertilized embryos.In vitro
embryo production has been applied to a variety of
species
in
the
Bovidae
family:
addax
(Addax
nasomaculatus; Asa et al., 1998; Hall-Woods et al.,
1999a), African Buffalo (Syncerus caffer: Shaw et al.,
1995; Arlotto et al., 2000), Banteng (Bos javanicus;
Barnes et al., 1988; McHugh and Rutledge, 1998),
Blackbuck (Antilope cervicapra; Keller et al., 1999),
Blesbok (Damaliscus dorcas phillipsi; Winger et al.,
1997), Bongo(Tragelaphus euryceros; Pope et al., 1998),
Gaur (Bos gaurus; Eyestone et al., 1988; Loskutoff et al.,
2000),
Greater
Kudu
(Tragelaphus
strepsiceros;
Loskutoff et al., 1995), Impala (Aepyceros melampus;
Loskutoff et al., 1995), Klipspringer (Oreotragus
oreotragus; Raphael et al., 1991), Sable Antelope
(Hippotragus niger; Finnegan et al., 1999), Scimitarhorned Oryx (Oryx dammah; Roth et al., 1998) and water
Buffalo (Bubalus bubalis; Madan et al., 1994; Palta and
Chauban, 1998).
One technique for enhancing reproduction is
superovulation: treatment of the donor with fertility
hormones such as follicle-stimulating hormone causes the
release of large numbers of eggs (up to 31 in Eland), all of
which can potentially be fertilized, transferred and carried
to term in surrogate mothers. Another method of
increasing the reproductive rate is embryo bisection to
give identical twins or triplets; this has so far been
demonstrated only with laboratory and domestic animals.
There are a number of prerequisites essential to the
collection of embryos from any species: a. a sound
knowledge of the reproductive biology of the species; b.
good animal husbandry techniques and the availability of
facilities for animal restraint; c. laboratory facilities and
personnel trained in the collection and handling of
embryos and d.
the use of animals that are proven
breeders. Embryo collection is usually applied up to the
expanded blastocyst stage, that is during the first week of
development, and can be done using either surgical or
non-surgical techniques. Gaur females have been shown
to be responsive to superovulation procedures and embryo
transfer techniques developed for domestic cattle (Stover
and Evans, 1984; Pope et al., 1988).
Embryo transfer is considered a low risk method for
moving germ plasm of animals. The zona pellucida (ZP)
is a relatively thick barrier that protects transfer-stage
embryos of livestock. It is known to resist penetration by
a variety of bacterial and viral pathogens in domestic
cattle, goats, sheep, and swine. The embryos can be frozen
or immediately transferred to one or more surrogates that
gestate the embryos to term, with the offspring having the
original donor´s genotype. For example, a cow usually
produces only one calf per pregnancy. However, a
genetically valuable female that is given hormones can
produce many embryos, each of which can develop in the
reproductive tract of a less valuable cow. By avoiding
pregnancy, the original donor can be used to produce even
more embryos to rapidly proliferate her valuable genes.
The long-term storage of mammalian embryos by
cryopreservation was first accomplished in the mouse in
1972 (Whittingham et al., 1972; Wilmut, 1972). The
viability of embryos is reduced by freezing and thawing,
and it is therefore reasonable to expect a reduction of 1520% in the rate of overall survival to liveborn when
frozen-thawed embryos rather than fresh embryos are
transferred. The first live calf born following transfer of
embryos between two domestic cows was produced by
Willett and colleagues 50 years ago (Betteridge, 2000). In
most cases, methods have been developed in the species
under study; in some cases, a common “model” species is
studied first before the technology is applied to a wild
counterpart ( Wildt et al., 1986; Loskutoff & Betteridge,
1992). For example, the well-established protocols for
bovine embryo transfer are being applied to the
conservation of rare breeds of domesticated cattle, buffalo
(Solti et al., 2000) and other wild Bovidae (Pope &
Loskutoff, 1999). Correspondingly, the human has served
as an excellent model for applying IVF to the Great Apes
(Loskutoff et al., 1991a,b; Pope et al., 1997). Embryo
transfer has been most successfully in cervids in the Red
Deer, Wapiti, Fallow Deer and Axis Deer (Bringans,
1989; Chapman et al., 1999; Dradjat et al., 1996;
Fennessy et al., 1989; 1994; Haigh & Hudson, 1993;
Hunter, 1997; Wenkoff, 1987; Wenkoff & Bringans,
1991). Studies show that llama (Lama glama) embryos
can be successfully vitrified and recipient females can
become pregnant after transfer (Aller et al., 2002).
Embryo transfer of fresh in vivo- derived embryos has
been carried out both in the silver fox and the dog
resulting in live young, but with low success rates
(Jalkanen et al., 1998).
Successful embryo transfer in Macropods was
achieved several decades ago (Tyndale-Biscoe, 1963,170;
Renfree and Tyndale-Biscoe, 1978) and recently has been
carried out in the Fat-tailed Dunnart (Breed and Leigh,
1996).
Surrogacy (Embryo transfer interspecies): There are
several reports of successful interspecies transfer of
embryos from wildlife species to domestic heterospecific
species that resulted in living offspring. These include: a.
gaur and banteng embryos into domestic cattle; b. a bongo
embryo transferred to an eland (Dresser et al. 1985); c.
Mouflon and Armenian Red Sheep embryos into domestic
sheep; d. Grant´s Zebra and Przewalski´s horse embryos
transferred to domestic horses (reviewed by Loskutoff,
1998) and e. Indian desert Cat and African wild Cat
embryos into Domestic Cat recipients (Pope, 2000). The
first successful interspecies transfer of an embryo
collected from a
threatened species (Gaur) into a
domestic animal was reported by Stover et al (1981). The
placenta from the one live gaur calf born contained an
abnormally low number of placentomes and abnormal
histologic architecture.
In 1984 european researchers
successfully produced two Banteng (Bos javanicus) calves
through non-surgical embryo transfers to domestic red
and white heifers (Wiesner et al., 1984). There are a
number of important factors to consider when selecting a
surrogate mother of a different species: a.
the
reproductive physiology of the two species should be
similar; b.
if they hybridise readily; c.
if their
placentation is similar; d. if they are of comparable body
size. The biological compatibility between the trophoblast
of the embryo and the uterine endometrium will prevent
interspecies embryo transfer from becoming routine, at
least using current technology.
Intraspecies
embryo transfer has been used
successfully to produce calves in four species of antelope
– common Eland, Bongo, scimitar-horned Oryx and Suni.
The first successful non-surgical embryo transfer in an
exotic species was the birth of an Eland calf to a surrogate
eland mother in June 1983 (Kramer et al., 1982; Dresser,
1983).
1.5
Fostering and Pouch young transfer
Wallabies (M. eugenii) and Northern Bettongs
(Bettongia tropica) to Brsuh-tail Bettongs . Many egglaying animals (i.e. birds and reptiles) are capable of
producing many more eggs than they can rear. This raises
the possibility of collecting the extra eggs and hatching
and rearing the animals in captivity with a foster parent,
then using them to supplement wild populations. It has
worked very well with some birds, specially the peregrine
falcon. The problem with fostering programs is that the
young animals learn crucial behaviors in the wild but not
in captivity. The individual pouch young produced by one
species can be transferred to the pouch of a recipient
conspecific or congeneric female that is at a similar stage
of the donor´s reproductive cycle. Following removal of
her pouch young, the donor female will soon return to
oestrus and be mated again. Already used to transfer and
rear Black-footed Rock Wallabies (Petrogale lateralis) to
Wallabys Tammar ( M. eugenii) and Northern Bettongs (
Bettongia tropica) to Brush-tail Bettongs ( B. penicillata).
1.7
Cloning
True cloning, or the production of genetically
identical individuals, is a natural phenomenon. The birth
of monozygotic multiplets is a commom occurrence in
many taxa. This can be mimicked artificially by
mechanically dividing the embryo through blastomere
separation or embryo splitting. These clones produced by
blastomere separation are identical in nuclear as well as in
mitochondrial DNA constitutions. In contrast, clones
produced by nuclear transfer can have genetic differences
associated with cytoplasmic variation when cytoplasts are
used from different donors to produce the reconstructed
embryo (Plante et al., 1992). In such cases as Cheetah,
Asiatic Lion, Florida Panther, producing as many
individuals as possible from a single, split embryo would
ensure the perpetuation of genes from small, genetically
compromised populations (Watson, 2001). Nuclear
transfer (i.e. cloning) is the process whereby the nuclear
DNA of a single donor cell is transferred to an enucleated
oocyte that, in turn, is activated to produce an embryo
which is genetically identical to the original donor cell.
Following successful nuclear transfer in the sheep and the
mouse to produce surviving young, a calf from a wild
cattle species (Bos gaurus) has been produced using
nuclear transfer and interspecies embryo transfer (Lanza
et al., 2000). The same or a very closely related species
must serve as the surrogate for cloned embryos.
Calves produced from nuclear transfer tend to have
unusually high birth weights apparently due, in part, to
inadequate in vitro culture conditions that alter gene
expression (Blondin et al., 2000), resulting in excessive
growth in uterus (Farin & Farin, 1995).
Cloning techniques: a.
Somatic Cell Nuclear
Transfer: transfer of the nucleus from a somatic cell to an
egg cell; b. The Roslin Technique: somatic cells (with
nuclei intact) are allowed to grow and divide and are then
deprived of nutrients to induce the cells into a suspended
or dormant stage. An egg cell that has had its nucleus
removed is then placed in close proximity to a somatic
cell and both cells are shocked with an electrical pulse; c.
The Honolulu Technique: the nucleus from a somatic cell
is removed and injected into an egg that has had its
nucleus removed. The egg is bathed in a chemical solution
and cultured. The developing embryo is then implanted
into a surrogate and allowed to develop.
Probably
the
greatest
strength
of
cloning
technology is the relative ease of banking the genetic
material. The donor cells needed for nuclear transfer
cloning can be obtained from a variety of sources
including preimplantation embryo cells (Willadsen,
1986), differentiated embryo cells (Campbell et al., 1996),
fetal fibroblasts (Wilmut et al., 1997), oviduct cells (Kato
et al., 1998), mammary gland cells (Wilmut et al., 1997),
skin fibroblasts (Kato et al., 2000; Shin et al., 1999),
cumulus cells (Kato et al., 1998; Wakayama et al., 1998),
Sertoli cells (Ogura et al., 2000; Yanagimachi, 2002),
fetal testicular cells (Watagama and Yanagimachi, 2001),
mural granulose cells (Wells et al., 1999), embryo stem
cells (Wakayama et al., 1999) and leukocytes (Galli et al.,
1999). Exposure of early preimplantation embryos to an
in vitro or perturbed in vivo environment can induce a
range of feto-placental growth, birthweight and congenital
abnormalities. Animals suffering such symptoms are
described as being afflicted with the Large Offspring
Syndrome (LOS). The mechanism by which LOS is
induced may be peculiar to each specific embryotechnology or be of a single origin perturbed to varying
degrees. The symptoms of LOS are also dependent on
species. The only embryo-technology that appears to be
innocuous is sperm cryopreservation (Lansac and Royère,
2001). Nuclear transfer´s greatest asset to wildlife could
involve a situation in which a species is reduced to
critically low numbers, a population unlikely to recover
from natural mating or other assisted breeding strategies
(Dresser et al., 2000).
Genetic rescue of an endangered mammal by crossspecies nuclear transfer using post-mortem somatic cells:
A report of a successful cloning of a wild endangered
animal, Ovis orientalis musimon, using oocytes collected
from a closely related, domesticated species, Ovis aries.
They injected enucleated sheep oocytes with granulose
cells collected from two female Mouflons found dead in
the pasture. Blastocyst-stage cloned embryos transferred
into sheep foster mothers established two pregnancies,
one of which produced an apparently normal mouflon.
These findings support the use of cloning for the
expansion of critically endangered populations. The
cloned animal was phenotipically Mouflon , and
microsatellite analysis performed on blood samples
confirmed isogenicity with the somatic cell donor and not
the sheep foster mother.
Trans-species Somatic Cell Cloning is being
proposed for propagation of the Giant Panda (Nature, 30
July 1998). Scientists at China`s National Academy of
Sciences are hopimg to transfer nuclei from an adult giant
panda cell into the egg of another species, perhaps a black
Bear. A successful birth of a black Bear cub from an
embryo transferred from one pregnant black Bear to
another already occurred.
Similar nuclear transfer
methods are being proposed to help the recovery of the
Northern Hairy nosed Wombat (Australia`s rarest
mammal) using the Common Wombats. Some scientists
are even working on using cloning techniques to bring
extinct species back to life (p.e. wooly mammoths frozen
in Siberia and Thylacine in Australia) (Bryant, 2002). In
principle, somatic cell cloning methods could be used to
restore extinct species, subspecies, or genetic stocks from
cryopreserved cells.
The
Advanced
Worcester,
Mass.,
Cell
create
Technology
Noah,
the
(ACT)
first
in
cloned
endangered species (Gaur), in November of 2000. They
fused skin cells taken from a male Gaur with 692
enucleated cow eggs. Of those 692 cloned early embryos,
only 81 grew in the laboratory into blastocysts. They
ended up inserting 42 blastocysts into 32 cows, but only
eight became pregnant. The Mule, called Idaho Gem, is
the first equine animal to be cloned in USA in 2003. In
late 2003, researchers of Texas A and M University
announced the cloning of a white-tailed deer, that could
be instrumental in conserving endangered deer species.
1.8
Spermatogonial transplantation
Spermatogonial transplantation is a method in
which stem cell spermatogonia are transplanted into the
seminiferous tubules of sterile recipients (Brinster and
Zimmerman,
1994).
They
demonstrated
that
the
microinjection of a heterologous suspension of mouse
testis cells inside the seminiferous tubules of a receptor
mouse, which was securely sterile, could result in
spermatogenesis in that second animal. Spermatogenesis
is a complex and very efficient process that begins with
the division and differentiation of the spermatogonial
stem cells that are located in the basal membrane of the
seminiferous tubules, sustained by the Sertoli cells
(Schlatt,
2002).
This
technique
has
permitted
cryopreserved rat testes cells to continue spermatogenesis
when thawed and transplanted into immuno-incompetent
mice (Clouthier et al. 1996). The cryopreservation of
testis cells suspensions seems to be the largest promise for
the storage of germ cells destined for posterior
transplantation (Avarbock et al., 1996).
Single pieces of immature mouse or rabbit testis
were cryopreserved, thawed and transplanted into mouse
testes. Histological techniques were used to determine the
presence of spermatogenesis, which was restored in both
mouse and rabbit testicular pieces, and led to the
production of mature sperm after both cryopreservation
and syngeneic or xenogeneic transplantation into mouse
testes. Using sperm developed in the frozen-thawed
transplants, mouse offspring were born after in vitro
microinsemination. The testicular tissue banking is a
promised technique for the preservation of fertility in
prepubertal male oncology patients. Studies show that
testis- derived cells transplanted into the testis of an
infertile mouse will colonize seminiferous tubules and
initiate spermatogenesis in >70% of recipients.
1.9
Ovarian Cryopreservation
Whole ovarian sections have been found to
survive cryopreservation, with normal follicle growth and
ovulation occurring after transplantation, often to a host
whose immune responses are suppressed to keep her from
rejecting the foreign ovary tissue. Oocytes released from
such frozen-thawed material are viable; live births have
resulted in mice and sheep after autologous transplants of
ovarian grafts stored at –196 degrees Centigrades
(Gosden et al. 1994, Gunasena et al. 1997, Harp et al.
1994). Oocytes from pre-antral follicles may be less
sensitive to cryodamage than mature oocytes (Gosden et
al., 1994; Shaw et al., 2000) mainly because they are
smaller, have no zona pelucida and cortical granules and
have less intracytoplasmatic lipids that are sensitive to
low temperature. The reservoir of ovarian preantral
follicles represents a largely untapped source of
germplasm of great importance in the conservation of
endangered species. The preantral ovarian follicles (PAF)
represent 90% of the follicular population in mammals
(Figueiredo et al., 2002). So, small PAF recovered from
the ovaries of post mortem or convalescent animals are a
rich oocyte source, since they can be developed to
maturity in vitro. Cooling is a significant problem for
mammalian oocyte cryopreservation. All mature MII
oocytes are vulnerable to cryoinjury because the spindle
holding the chromosomes on the metaphase plate
depolymerizes when temperature is lowered.
Xenotransplantation is generally defined as the
transplantation of organs, tissues or cells across a species
barrier (i.e. a graft from one animal species to another
animal species) (Platt, 1998). Alternatively, allogeneic
transplantation involves the transplantation of a graft from
one individual to another individual within one species,
while
auto
or
syngeneic
transplantation
is
the
transplantation of a graft from one individual back into
the same individual. The development of ovarian tissue
banking may have significant implications for use in
animal conservation in years to come. Several specialized
reproductive tissue storage centers have been established
that cryobank ovarian tissue from valuable, rare, and
endangered
species
using
technologies
that
were
established in laboratory species. Ovarian tissue of nondomestic, and exotic species has been utilized and antral
follicle development has so far been reported in the
marmoset-monkey, the common wombat, the tammar
wallaby, and the African elephant (Choo et al., 1999,
Cleary et al., 2002, Mattiske et al., 2001, Gunasena et al.,
1998). The immune compromised nude mouse can
support the development of antral follicles in xenografted
cryopreserved- thawed elephant ovarian tissue.
Studies demonstrated that Wombat ovarian tissue
could
survive
and
function
when
grafted
into
immunocompromized rats and that Wombat ovarian
follicles can be recruited to growth and development in an
ovarian
xenograft. While
performing
xenografting,
revascularization of the transplanted tissue is another
important aspect to create a successful model. In the rat
model, revascularization of the tissue was evident in the
grafts recovered at week 2 as shown by the presence of
numerous blood vessels and erythrocytes in the grafts.
Such rapid restoration of blood supply is essential for the
graft to prevent post transplantation ischaemic damage
(Nugent et al., 1998).
Studies showed that cryopreserved Minke Whale (
Balaenoptera acutorostrata) follicular oocytes could
resume meiosis to M-II stage, but their postthaw survival
rate was lower than observed for other mammalian
oocytes.
2.
“Frozen Zoo”
2.1
Concept
“Frozen
Zoos”
(=Genome
Resource
Banks
(GRB)) are repositories of systematically collected germ
plasm (gametes), embryos, blood products, tissue, and
DNA for defined conservation programs. Advantages of
Genetic Resource Banks for managing small, isolated
populations of wild animals: Easy movement of genetic
material among living populations; Increased security
(Small populations are vulnerable to environmental
catastrophes, political upheavals, and disease outbreaks);
Unlimited
space;
Increased
gene
flow;
Minimize
introgression; Extend generation times; Maximize genetic
diversity;
Minimize
inbreeding;
Manage
effective
population size; Minimize selection; decrease mutation
and solve reproductive failure: increasing efficiency of
captive breeding (Like people, nonhuman animals have
sexual partner preferences). Genetic resource banking
(GRB) provides potentially useful support for managing
and conserving wildlife species. The GRB concept and
strategies have received increasing attention over the last
two decades (Holt et al., 1996b; Wildt et al., 1997; Holt &
Pickard., 1999; Watson & Holt, 2001). The important
point here is that stored materials for restoring
heterozygosity may be from animals that died generations
earlier.
Gametic rescue from tissues has considerable value
for spermatozoa, where epididymal spermatozoa are
readily obtainable post mortem. Unfertilized oocytes are
much harder to cryopreserve than embryos because the
haploid female gamete is highly susceptible to chilling
injury and cryoprotectant toxicity (Parks and Ruffing
1996). This susceptibility is thought to be associated with
the unique characteristics of mature oocytes, namely their
large size and the presence of a meiotic spindle and
cortical granules in the ooplasm (Candy et al. 1994).
Because cryopreserving mammalian oocytes is so
difficult, there has been considerable interest in the freezestorage of slices of ovarian tissue as a feasible alternative
(Sztein et al., 1998; Candy et al., 2000). Although
recovery and maturation of primordial follicles from
cryopreserved
ovarian
tissue
remains
a
technical
challenge, some success has been achieved (Carroll et al.,
1990). Studies conducted in a variety of non-domestic
species suggest that the pellet method is particularly
suitable for freezing carnivore and ungulate semen.
Preserving sperm, eggs, and other tissue in what some call
a “Frozen Zoo” (and scientists call “genome resource
bank”) will provide an insurance policy against a
catastrophic decline in Giant Panda numbers. Accidental
introduction of infectious agents can have catastrophic
consequences on population viability and on the welfare
of individual animals, so great care must be taken to
ensure that cryopreserved germplasm does not harbour
infections. For successful low-temperature storage, cells
need to be cooled below – 100ºC, and under these
conditions they may remain viable for many years (Mazun
et al., 1981).
With recent progress in understanding mechanisms
of injury to cells during cryopreservation, physical
modeling using mathematical formulations has been
developed to stimulate a cell’s response to environmental
change during the cryopreservation process and to predict
optimal cryopreservation conditions. When cells are
frozen they are subjected to stresses resulting from the
water-solute
interactions
that
arise
through
ice
crystallization. Exposure of cells to the hyperosmotic, yet
unfrozen, solution causes withdrawal of intracellular
water, consequent cell shrinkage and possible influx of
ions (Mazur, 1984). Thawing involves a reversal of these
effects, and the consequent inward flux may cause cell
membrane disruption. Mammalian spermatozoa show a
variable
degree
of
adverse
response
to
reduced
temperature depending upon species.
In the process of vitrification, high levels
of cryoprotectant are introduced and cooling is carried out
relatively rapidly. Such methods have been established for
embryos (Rall and Fahy, 1985; Trounson, 1990) and,
recently, for oocytes (Wood et al., 1993; Kuleshova et al.,
1999). In some species there is a subpopulation of
vulnerable
spermatozoa
that
do
not
survive
cryopreservation (Curry and Watson, 1994). Since the
first application of vitrification in domestic animal
embryology, this technology was regarded as a possible
alternative for traditional freezing (slow cooling) by
offering the advantages of considerable reduction in time,
equipment and labor costs to cryopreserve.
General components and specific factors of an
Action Plan for a Specific Taxon/ Species:
a. Summary: Synopsis providing brief description of
justification, goals and overall conservation plan in the
context of a GRB.
b. Justification: Provide specific short- and long-term goals
for the GRB.
c. Current
knowledge
reproduction.
of
life
history
and
natural
d. Current knowledge of assisted reproduction.
e. ISIS, studbook and regional collection plan status.
f. Status in the wild.
g. Accessibility of existing animals for banking.
h. Type and amount of germplasm (and other biological
materials) to preserve.
i. Technical
germplasm collection, storage, use and
ownership.
A mobile laboratory developed at Texas A&M
University has proven to be successful in allowing
performing some ART for the preservation of desert
bighorn sheep in the Davis Mountains of west Texas. A
coordinated international approach to collecting gametes
from the reproductive tracts of dead, sick or injured
animals has been advocated to avoid loss of potentially
valuable genetic material (Graham et al., 1978). Studies
from the Institute of Zoology of London Zoo showed that
common marmoset monkey (Callithrix jacchus) as all
mammalian oocytes can be induced to resume meiosis
without
fertilization,
using
ethanol
and
electrical
stimulation, and the resulting parthenogenetic embryos
carry only maternal chromosomes. Clouded leopards
might represent an ideal species for GRB application. In
USA, the clouded leopard SSP population suffers from
extremely low levels of genetic variation (<75%),
primarily due to the incompatibility and aggression of
males when paired with unfamiliar females. Artificial
insemination with frozen spermatozoa could offer an
alternative to population managers who wish to avoid the
inherent risks association with pairing and natural
breeding of this species.
GRBs also can offer a mechanism to moves genes
between wild and captive populations. As one example,
this approach has been used successfully to obtain new
founders for the Cheetah SSP, using frozen spermatozoa
collected from wild Namibian cheetahs to inseminate and
produce offspring in captive cheetahs in U.S. zoos
(Howard et al., 1997). Another potential benefit of GRBs
is the ability to extend the generation interval for
reproduction, an invaluable attribute when managing
small populations and species with naturally short
generation intervals (Ballou, 1992). Developing GRBs in
range countries: a.
A Cheetah sperm GRB is being
implemented with a NGO partner in Namibia; b.
the
Giant Panda is another species with a sperm GRB being
developed with Chinese partners; c. there are plans to
develop a GRB for the Asian Elephant, Clouded Leopard
and fishing Cat in Thailand. The Genome Resource
Banking Advisory Group, a scientific advisory group
under the auspices of the American Zoo and Aquarium
Association,
serves
to
guide
and
facilitate
the
development of GRBs. Recent refinements based on
systematic research studies have included altering the
initial cooling rates and dilution rate with cryoprotectants
which substantially improves acrosomal and plasma
membrane status, especially for samples with high
percentages of pleiomorphic spermatozoa (Pukazhenthia
et al., 1999; 2002).
The concept of Biological Resource Bank (BRB):
As stated by Holt (2001) BRBs can be organized for two
objectives: a. to develop a collection of preserved somatic
tissues, cell lines, DNA and serum samples from a variety
of species, primarily for taxonomic, demographic and
medical research and b.
to develop a collection of
gametes and embryos that will clearly be aimed towards
animal breeding. BRBs can help to accumulate biological
samples that can be used for genetic analysis. It is
particularly important if we develop an integrated network
that involves all countries of America sharing common
species in collaborative work. Nuclear DNA markers
called microsatellites are ideal tools to develop data for
pedigree
analysis,
migration,
phylogeography
and
hybridization detection.
2.2
Institutions involved in Frozen Zoo Projects:
2.2.1
Center
for
Endangered
Diego Zoo :
reproduction
of
Species(CRES)-
San
The first so-called “frozen zoo” was created
in 1975 by Kurt Benirschke, a visionary physician who
switched from his human practice to work with
endangered species at the San Diego Zoo, studying gene
pools and genetic diseases made prevalent by inbreeding.
Housed in an array of large cryogenic freezers, the Frozen
Zoo contains carefully accessioned and cryopreserved
cells, representing a wide range of mammals and birds. It
is composed of two parts: somatic cells (diploid fibroblast
skin strains) and gametes (haploid sperm and eggs). Cells
immersed in a special cryoprotectant medium are frozen
at a rate of 1ºC per minute and then placed in liquid
nitrogen holding tanks for storage at – 196ºC. Samples are
split between freezers in two different locations to ensure
safekeeping of the cells.
The
San
Diego
Zoo’s
Center
for
Reproduction of Endangered Species has created a
collection of frozen fibroblast cell cultures as well as
tissue samples collected from various rare and threatened
animal species, such as Przewalski’s horse and the
Sumatran rhinoceros. In the “Frozen Zoo” you can find
the genetic material of over 500 Przewalski’s Horses, 150
Western
lowland
Gorillas,
80
black
Rhinos,
22
Queensland Koalas, 19 Bornean bearded Pigs, Pandas,
Condors and even a California gray Whale among others.
The “Frozen Zoo” consists of specimens from over 4300
individual mammals, birds, and reptiles, representing 353
species. CRES is one a few laboratories that undertake
chromosomal studies of wildlife species. Chromosomal
analysis is a powerful indicator of reproductive isolation.
Numerical or structural variation in karyotipe is an
indicator
of
potential
reduction
in
reproductive
performance, including sterility (Robinson and Elder,
1993; Benirschke and Kumamoto, 1991). Chromosomal
analysis can also be used for sexing in birds (e.g. condorsChemnick et al., 2000) and mammals (e.g., hyenasWurster et al., 1970). Chromosomal errors, like Down’s
syndrome, also occur in wildlife species.
The Bird Department of San Diego CRES develops
artificial insemination with fresh semen, resulting in hatch
rates of 100% for Temminck’s Tragopans, 100% for
Himalayan Monals, and 57% for Chinese Monals.
A partnership from CRES and Advanced Cell
Technology, Inc. produced the first cloned endangered
animal born on Monday, January 8, 2001. The birth of the
baby bull Gaur, “Noah”, is the first successful birth of a
cloned endangered animal. Another partnership from
CRES, Advanced Cell Technology and Sioux Center in
Iowa produced a clone of two calves of a more
endangered wild bovid species, the Banteng. They were
born on April 2003 and are the identical copy of a male
Banteng that died at San Diego Wild Animal Park in
1980. The male Banteng clone, named “Jahava” is in his
new home at the San Diego Zoo, marking the first time a
cloned endangered species is viewable to the public. He
was born to an Angus cow in Iowa and joins s mall herd
of females at the Zoo. One of the goals of the cloning
project is that a cloned animal will be able to reproduce
healthy offspring.
2.2.2
Cincinnati Zoo (CREW)
At the heart of the Center for Conservation and
Research of Endangered Wildlife (CREW), are the Frozen
Zoo and Garden, that consists of numerous tanks filled
with liquid nitrogen. Submerged within the –196ºC are
thousands of samples representing over 60 animal and 150
plant species. Plant samples range from seeds to spores to
tiny shoot tips and embryos. Animal samples include
sperm, oocytes and embryos. Species currently stored
within CREW’s Frozen Zoo and Garden represent a
variety of taxa. For example, Sumatran Rhino, Wyoming
Toad, Cheetah and Penguin sperm samples occupy one
tank. In another, there are Ocelot, Eland and Gorilla
embryos. In 1984 Dr. Betsy Dresser performed the first
interspecies embryo transfer. He took embryos from a rare
Bongo antelope at the Los Angeles Zoo and transferred
them to a surrogate mother- another species of antelope
called an Eland – at the Cincinnati Zoo. “Timu”, the
world’s first “Test-tube” Gorilla, was born as a result of in
vitro fertilization with frozen thawed sperm and embryo
transfer at CREW. Recently, CREW scientists produced
the first endangered cat, an Ocelot (named “Sihil”), by the
transfer of frozen-thawed embryos. In collaboration with
Brazilian colleagues, CREW has produced more than 75
embryos from wild caught Ocelots by in vitro fertilization
and has them safely stored in liquid nitrogen in Brasil.
Develop studies on Post-coital sperm recovery and
cryopreservation
(Dicerorhinus
in
the
sumatrensis):
Sumatran
Rhinoceros
Sumatran
Rhinoceros
spermatozoa of moderate quality can be collected from
post-copulatory females. Rhinoceros sperm samples show
only slight reductions in quality after cryopreservation
and thawing and have potential for use in artificial
insemination. One advantage to collecting this type of
sample is that it represents a sample of a natural ejaculate,
whereas the small volumes of fluid emitted during manual
stimulation or electroejaculation may not consist of the
appropriate mixture of seminal fluids.
Cincinnati Zoo reported a birth of a Western
Lowland Gorilla (Gorilla gorilla gorilla) following in
vitro fertilization and embryo transfer:
A 21-year-old
multiparous female was given hFSH and hCG. At 35 h
after hCG, follicles were aspirated by controlled suction
under trans-vaginal ultrasound guidance. At 21 h postinsemination (p.i.) , eight oocytes were at two cell stage,
five were cryopreserved, and three were cultured to the
six- to eight- cell stage before transcervical uterine
transfer at 47 h p.i. Ultrasound examination revealed a
single fetus at 15 weeks post-transfer, and unassisted
delivery of a live 1,37 kg female infant occurred at 29
weeks.
2.2.3
Audubon Center for Research of
Endangered Species (ACRES)
ACRES opened in 1999 in New Orleans on
the 1,200 acre grounds of the Freeport-McMoRan
Audubon Species Survival Center, a facility of 36,000
square-foot. The Research Center’s frozen zoo currently
holds cryopreserved semen from a number of animals
such as: Gorilla, Sumatran Tiger, Jaguar, Jabiru Stork,
Mississipi sandhill Crane, mountain Bongo Antelope,
Eland, Gaur, African Wildcat, Caracal, as well as
domestic Cattle, Dog and Cats. In vitro-produced Tiger,
African Wildcat, Serval, Caracal and other endangered cat
embryos are also stored at the Frozen Zoo. A variety of
antelope (Bongo, Eland), and Cattle embryos are also
present. African and Asian Elephants, Baird´s Tapir,
Colobus Monkey, Lion, Jaguar, Gaur, Roan Antelope,
Bongo, black Bear, Tompson Gazelle, giant Eland, Nyala,
Serval, fishing Cat, Cape Buffalo are just a few examples
of the gametes and tissue samples in their expanding
collection.
The world’s first cloned endangered African
wildcat has been born in New Orleans. The kitten is the
first cloned wild carnivore. Born to a common domestic
housecat on August 6, 2003, the kitten, named “
Ditteaux”, was created using frozen/thawed genetic
material from the African wildcat Jazz, who was also born
to a domestic cat, but as a result of a different kind of
procedure, the first successful in vitro
fertilized
frozen/thawed embryo transfer.
To create the cloned
embryo, scientists took tissue samples from the male
African wildcat, Jazz. These cells were grown in tissue
culture to provide a supply of thousands of cells (with the
Wildcat’s DNA). The cells were frozen in the Frozen Zoo.
Then, DNA was removed from an egg of a domestic cat.
Frozen-thawed cells from “Jazz” were inserted into the
domestic cat egg cells. The egg was exposed to an electric
current, causing the new DNA to fuse with the egg, which
divided to become an embryo. The embryo was implanted
into the uterus of a domestic cat surrogate, who had a
normal pregnancy before delivering the cloned kitten. In
addition, researchers at the Audubon research center
announced the birth of the world’s first Caracal cat
created from a frozen/thawed embryo. The kitten, created
from an IVF procedure where the frozen/thawed embryo
was transferred to a surrogate mother Caracal, was born
September 6, 2003. On May 24, 2000, two young
Caracals were born resulting of in-vitro fertilization
utilizing frozen sperm.
Audubon Research Center also announced the birth
of an African Serval cat, born October 1, 2003 as a result
of in vitro fertilization followed by embryo transfer.
In a innovative project, Dr. Betsy Dresser, director
of ACRES, bring to Mt. Kenya Game Ranch about 50
Bongo frozen embryos from new Orleans to implant into
about 25 surrogate Eland mothers that have received three
weeks of hormone injections.
At ACRES, Sandhill Cranes are produced through
artificial
insemination,
incubated,
and
raised
by
researchers costumed as Cranes to prevent human
imprinting. They are being reintroduced to the Mississipi
Sand Hill Crane Refuge in Pascagoula.
2.2.4
Omaha Zoo
The Center for Conservation and Research (CCR)
at the Henry Doorly Zoo encompasses the entire medical
and the majority of the research facilities at the Zoo. The
Center for Conservation and Research includes research
laboratories for: reproductive physiology; genetics;
support laboratories; Genome Resource Bank and
horticulture laboratory.
Programs
in
reproductive
physiology at the Henry Doorly Zoo: a. Gorilla Assisted
Reproduction Program, where oocytes will be collected
from SSP managed females for embryo production by
intracytoplasmic injection of sex selected sperm to
produce female offspring; b. In- vitro embryo production
and cryopreservation in Siberian Tigers; c.
In- vitro
embryo production and cryopreservation in Brazilian
Jaguars; d.
Development of optimal methods for
cryopreserving sperm in a variety of species: African and
Asian Elephants (K. Suedmeyer and D. Schimitt), Jaguars
(R. Morato) and Tasmanian Devils (W. Pryor) ; e.
Developing optimal methods for cryopreserving in-vivoderived embryos and semen collected from Dromedary
camels (J. Skidmore and N. Loskutoff); f. Developing
techniques for sperm microinjection in exotic felids and
wild cattle (S. Hoffaman, R. Godke and N. Loskutoff); g.
Henry Doorly Zoo CCR Genome Resource Bank:
Currently, over 18.000 individual samples of sperm and
embryos from approximately 45 species are in liquid
nitrogen storage and monitored by a state-of-the-art
computerized system. Ultrasound-guided transvaginal
follicle aspiration is an effective method for recovering
oocytes from superestimulated gaur cows, and the
embryos derived from these oocytes appear to be
developmentally
competent.
Coupled
with
semen
cryopreservation, oocyte retrieval and in-vitro embryo
production techniques are particularly valuable for the
conservation and genetic management of endangered
species such as the Gaur.
2.2.5
Washington Zoo
The National Zoological Park’s Conservation &
Research Center was invited to take a lead role in
studying
ferret
reproductive
biology
as
well
as
participating in the ex situ breeding programme.
Extensive studies were conducted in common
ferrets to develop a reliable approach for collecting,
processing
and
analyzing
fresh
or
cryopreserved
spermatozoa (Curry et al., 1989; Wildt et al., 1989;
Howard et al., 1991; Van der Horst et al., 1991).
The domestic ferret strategy was subsequently applied
to the Siberian Polecat and finally the Black-footed Ferret.
The laparoscopic intrauterine AI technique, developed in
the domestic ferret, proved to be effective in its close
relatives. Four of six (66,7%) Black-footed Ferrets
inseminated with fresh or frozen-thawed semen became
pregnant and delivered live young (Howard 1999;
Howard et al., 1996).
At the National Zoological Park’s Conservation &
Research Center, the natural breeding programme for
Black-footed Ferrets was adapted to include AI. The goals
were to: a.
Produce offspring from behaviourally
incompatible animals, especially non-breeding males, to
meet reintroduction demands, and b. Increase founder
representation in the under-represented lineage.
This has allowed reintroduction of captive ferrets to the
wild at a number of sites, initially in Wyoming then in
South Dakota, Montana, and Arizona.
The laboratories of the National Zoological Park
and its Conservation & Research Center have developed
protocols for routinely producing offspring in the Cheetah
(Acinonyx jubatus), Eld`s Deer (Cervus eldi), and
scimitar-horned
Oryx
(Oryx
dammah),
all
using
cryopreserved sperm.
Among
the
milestones
achieved
using
assisted
reproduction are: a. Successful pregnancies in eight cat
species; b. Helping save the Florida panther using fertility
assessments; c.
Producing cheetah cubs by artificial
insemination with frozen sperm (imported from Africa);
d. Tiger cubs born from in vitro fertilization and embryo
transfer.
The National Zoo’s Conservation & Research
Center maintains the world’s leading Endocrine Research
Laboratory for wildlife species.
2.2.6
Animal
Gene
Storage
Centre of Australia
Resource
A consortium involving Monash University and
Taronga Zoo has formed the Animal Gene Storage and
Resource Centre of Australia (AGSRA).
To guarantee the survival of endangered species
and to complement breeding programs in the wild or
captivity (zoos), the AGSRA has established a living
frozen zoo, which preserves reproductive cells (semen,
embryos, ovaries) and genetic material in a frozen state, at
– 196ºC in liquid nitrogen. At the Gene Bank, there are
Rhinos, Elephants, Hairy-nosed Wombats, Bilbies and
100 other species.
The AGSRA has designed and developed
GeneSearch, a computerized database where critical
information is stored and has the capacity to form a
network linking gene banks around the world for
international conservation of wildlife. The AGSRA
contributes to animal conservation of endangered species
by
using
new
reproductive
techniques
made
in
embryology, cryobiology, and molecular cellular biology.
The AGSRA concentrates on conservation and recovery
programs of native species, which are classified as
endangered or threatened.
The highlights of AGSRA are: a.
The
establishment of the Animal Gene Bank; b. Design and
production of the computer database GeneSearch; c. The
black rhinoceros recovery program; d. Preservation of the
Northern Hairy-nosed wombat; e. Interspecies surrogates.
In the majority of marsupials, a short period of
embryonic differentiation in utero, less than the duration
of an oestrous cycle, is followed by the birth of an altricial
fetus so that their primary reproductive investment is
placed in lactation rather than in gestation and
placentation.
The
Northern
Hairy-nosed
Wombat
(Lasiorhinus krefftii) is a highly endangered marsupial
species and every possible option for sustaining the
species needs to be explored. So at Monash Institute they
developed assisted reproductive technologies in the nonendangered Common Wombat (Vombatus ursinus) for
application in breeding the Northern Hairy-nosed
Wombat,
specially
the
follicular
development
in
cryopreserved
Common
Wombat
ovarian
tissue
xenografted to Nude rats.
2.2.7
Breeding Centre for Endangered
Arabian Wildlife
Over the last hundred years Arabian species
such as the Ostrich, Cheetah and Caspian Turtle have
become extinct and many more Arabian species are
destined for a similar fate if loss of natural habitat and
poaching continues.
Currently in the Arabian Peninsula, work has been
carried out on several endangered species. An Arabian
Leopard, sand Gazelle, Asian Cheetah and Gordon´s
Wildcat sperm bank has been set up in the Breeding
Centre for Endangered Arabian Wildlife in Sharjah, UAE.
The Breeding Centre for Endangered Arabian
Wildlife at Sharjah in collaboration with Henry Doorly
Zoo are applying techniques of assisted reproduction (Invitro embryo production and cryopreservation) to the
conservation of the Arabian leopard in the range country
(A. Aziz, C. Gross, N. Loskutoff and D. Armstrong).
Already, IVF for the Arabian leopard and Gordon´s
wildcat has resulted in cryopreservation of embryos.
2.2.8
WBRC
The Wildlife Biological Resource Centre
(WRBC) is a working group of the Endangered Wildlife
Trust (EWT), a not-for-profit wildlife NGO, dedicated to
the conservation of biodiversity in southern Africa. The
WRBC has established a Biological Resource Bank
(BRB) of wildlife biomaterials for use in conservation,
research and management of rare and endangered wildlife
species. Biomaterials, such as sperm, blood, serum, eggcells, skin, hair, embryos, muscle are collected, processed,
banked and made available to regional and international
conservation, research and management institutions.
Biomaterials
are
utilized
by
a
multidisciplinary
conservation and research community, integrating wildlife
management, genetics, reproduction, animal (and human)
disease, nutrition and general physiology to the benefit of
rare and endangered wildlife species.
Animals that have been bred using assisted
reproduction include eland, sable antelope, tigers,
cheetahs and elephants.
A very special birth took place at the
Johannesburg Zoo in 2003, where an eland heifer was
artificially inseminated with frozen sperm collected a year
ago from a dead bull.
They developed an alternative to the
conservation of endangered equid species: In vitro
fertilization of in vitro matured oocytes recovered from
free ranging zebra species (Burchell and Hartmann) in
South Africa. Developing the In-vitro production of
African buffalo (Syncerus caffer) embryos derived from
follicular oocytes and epididymal sperm.
2.2.9 Mohor Gazelle GRB
The Institute of Zoology in London
collaborated with the Estacion Experimental de Zonas
Aridas (EEZA) in Almeria, Spain. The aim has been to
support
conservation
breeding
programmes
and
reintroduction projects for three endangered Gazelles
(Mohor, Cuvier and Dorcas Gazelles).
Conclusions
In vitro techniques could facilitate genetic management
by offering:

More offspring from a female than she could
produce in a natural reproductive lifespan.

The ability to produce viable embryos from
prepubertal (Earl et al., 1998) and pregnant (Meintjes et
al., 1995) females, from ovarian tissue excised from
individuals undergoing damaging therapies (Shaw et al.,
2000) or from males (Hopkins et al., 1998) and females
(Johnston et al., 1993) after death.

The possibility of eliminating certain pathogens
from diseased animals by utilizing established processing
and treatment methods for semen (Wrathall & Sutmoller,
1998) or embryos (Bielanski, 1998; Stringfellow, 1998).

The ability to determine or select the sex of embryos
directly (Seidel, 1999) or by the flow cytometric- sorting
of X- and Y- chromosome- bearing sperm before IVF
(Johnson & Welch, 1999).

The ability to produce multiple, genetically identical
offspring by embryo division (i.e. blastomere separation
or embryo splitting; Loskutoff et al., 1993).
There is now less need to preserve viability in
sperm cells since it has been shown that spermatozoa can
be freeze dried and then subsequently used for
intracytoplasmic sperm injection producing live births
(Wakayama and Yanagimachi, 1998). Nowadays, the
possibility
of
using
stored
spermatogonia
(undifferentiated male germ cells) for repopulating the
testis of a common “host” species in order to obtain
spermatozoa for IVF is a reality (Avarbock et al., 1996;
Clouthier et al., 1996). In the female, the recovery of
oocytes from primordial follicles in stored ovarian tissue
came a step nearer with studies published in the 1990s
(Newton et al., 1990).
GRBs could be developed under the care of the
intensive species management strategies of zoos. There
are more than 80 species survival plans in North America,
in which zoos work together to maintain maximum
genetic heterogeneity in a given species (Hutchins and
Wiese 1991). These efforts are occasionally linked to the
range countries of species origin, with support provided to
local zoos or field researchers. For wild populations, GRB
activities could be coordinated through IUCN- World
Conservation Union taxon specialist groups that have a
mandate to develop species or taxon action plans for both
wild and captive populations. At least 19 species have
been reintroduced into the wild after captive propagation.
In at least seven species (Pere David`s Deer, European
Bison, Arabian Oryx, Guam Kingfisher, Red Wolf, Guam
Rail and California Condor) they were extinct in the wild
at the time of reintroduction.
Usually the concept of frozen repositories of
biomaterials is associated with germ plasm and embryos.
But collecting and storing blood and tissues is also
important because these materials can be processed into
serum, plasma, blood cells, DNA, and tissue and cell
cultures.
These
biomaterials
have
wide-ranging
applications for studying genetic variation, phylogeny,
paternity, and the processes underlying diversity, such as
gene flow, selection, and mating. Nowadays, there are
several methods to recover gametes from convalescent or
post mortem animals. Those gametes would be a valuable
arsenal in the formation of germoplasma banks,
contributing to the preservation of genetic patrimony,
essential for the maintenance of the species. Man needs to
be conscious of his actions and try to have a peaceful
coexistence with the ecosystems and all the animals inside
it, as well as providing methods to restore the losses
caused by him.
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