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Feature Article
MUKESH THAKUR & RUHINA JAVED
Frozen Zoos!
Despite
Despite there
there being
being numerous
numerous gene
gene repositories
repositories in
in the
the country,
country, there
there is
is
no
no initiative
initiative as
as yet
yet to
to develop
develop aa national
national gene
gene bank
bank exclusively
exclusively for
for the
the
preservation
preservation and
and utilization
utilization of
of wild
wild animal
animal germplasm.
germplasm. Genome
Genome
Resource
Resource Bank
Bank (GRB)
(GRB) or
or Frozen
Frozen Zoo
Zoo would
would provide
provide significant
significant benefits
benefits
for
for wildlife
wildlife managers
managers as
as itit holds
holds particular
particular promise
promise for
for endangered
endangered
species.
species.
I
T’S not a zoo that you would particularly enjoy visiting.
Unlike the conventional zoo where antics of animals in
captivity keep you entertained, in a Frozen Zoo you
are more likely to come across tanks containing semen
and embryos of wild animals frozen in liquid nitrogen.
Sometime in the future, when an animal in the wild is
threatened with extinction, it could be reproduced thanks
to the gene material safely stored in the Frozen Zoo.
Wildlife reproduction is a new and upcoming trend in
conservation of wildlife. Practiced widely in several
countries, it is not yet frequently applied in India. There are
several new methods available today in wildlife
reproduction including Cross-fostering, Artificial
incubation, Artificial insemination, and Embryo transfer.
All these methods go a long way in boosting genetic
variability, increasing population size, and improving
reproductive success.
Cross fostering is a technique of inselective breeding, where
offspring are removed from their biological parents at birth
and raised by surrogates. This can also occasionally occur
in nature. The surrogate can be a close relative of the species
and does not have to be the same species.
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SCIENCE REPORTER, May 2010
Artificial insemination (AI) is the process by which sperm
is placed into the reproductive tract of a female for the
purpose of impregnating the female by using means other
than sexual intercourse. It has been practiced in the livestock
industry for many years. It may be similarly practiced in
wildlife management. Semen can be collected at one site
and shipped anywhere in the world.
Embryo Transfer (ET) is another captive breeding tool
currently used. It requires two genetically related species.
One species is super-ovulated, inseminated, and the
fertilized eggs are collected. The fertilized eggs are implanted
into surrogate mothers. The offspring carry the genetic
makeup of the two original parents, not the surrogates.
This science provides many opportunities to captive
breeding programmes. Animals no longer have to be
moved to accommodate natural mating. Conservation
biologists can work to counter the effects of genetic drift.
Even if an animal dies, gametes stay viable up to 24 hours,
allowing the possibility to re-introduce diversity back into
wild populations.
Genome Resource Bank (GRB) or Frozen
Zoo would provide significant benefits for
wildlife managers. It has particular promise
for endangered species.
Feature Article
There are species that desperately need help from
captive situations, as we can’t save them in the wild. But
captive breeding is not always easy. Many animals are
disoriented by living in the wrong climate or social system,
or being squeezed into small “habitats” in the presence of
humans. Scientists and keepers try to help animals breed
naturally. But often, researchers do not even know how
normal mating takes place. Therefore, AI and IVF might be
helpful in such cases.
Actually, Gene Bank is not a new concept for India.
These have been earlier only used for domestic animals,
cultivated agriculture crops and for microbes. Some of the
prominent examples are:
Frozen Gene Bank
The Frozen Zoo is a collection of animal genes in the form
of frozen semen and embryos. In practical terms this is a
collection of sperm-holding straws stored in liquid nitrogen
tanks. 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.
Modern technology allows the indefinite preservation
of genetic material through the freezing of semen and
embryos (unfortunately, ova do not freeze very well). The
implication of being able to freeze semen and embryos is
that the species can be kept forever, even when they are
extinct, as their genes would be preserved. When the threat
to the preserved species has been controlled, implantation
and development of an embryo can then be carried out.
On the research front it allows much to be learned about
species-specific reproductive physiology and will help to
develop the technology required for related species not yet
endangered. Thus when the time comes to make
conservation efforts for one of these species, the technology
would already be available.
Today, in zoos around the world, these deep-freeze
tanks act as a high-tech Ark, housing cells from the planet’s
most endangered animals, including chimps, cheetahs,
pandas, California condors and hundreds more. As a result,
long-dead animals are now becoming parents from the
grave, with their precious DNA still circulating in the gene
pool. The real goal is the management of the eroding gene
pools of endangered species.
National Gene Bank Facility for conservation and
preservation of domestic animal germplasm at the National
Bureau of Animal Genetic Resources, Karnal, Haryana with
the responsibility of evaluating, certifying and conserving
the rich and varied germplasm resources available in the
country and whose genetic base is shrinking fast. Thus, the
establishment of the National Bureau of Animal Genetic
Resources/National Institute of Animal Genetics in 1984
was the culmination of sustained efforts made by leading
geneticists in the country over the years.
National Genomic Resources Repository was established
in the premises of the National Bureau of Plant Genetic
Resources, New Delhi for exchange, quarantine, collection,
conservation, evaluation and the systematic documentation
of plant genetic resources. NBPGR act as an institutional
framework for methodical and centralized efforts to collect,
generate, conserve and distribute genomic resources for
agricultural research.
1. A researcher at the Cincinnati Zoo removes blood from a biopsy of a
domestic cat’s follicles, the egg-nurturing structures in the ovary.
2. After the blood is extracted, the eggs in the sample are easier to
detect.
3. A researcher opens up one of the chilled canisters containing sperm
samples and frozen embryos.
4. Samples stored in liquid nitrogen cooled to -374 degrees Fahrenheit
remain viable indefinitely. They are often used successfully after two
to five years in storage.
left:
This ocelot
kitten was
conceived in
the lab using
sperm and a
stored egg
SCIENCE REPORTER, May 2010
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Feature Article
The San Diego Zoo where the first frozen zoo was created (above), Animals at LaCones (above right); and Samples preserved in a Frozen Zoo (below).
Microbial Type Culture Collection and Gene Bank
(MTCC), a national facility, was established in 1986, funded
jointly by the Department of Biotechnology (DBT) and the
Council of Scientific and Industrial Research (CSIR),
Government of India. The MTCC is a modern facility
housed at the Institute of Microbial Technology (IMTECH),
Chandigarh. The main objectives of this national facility
are to act as a depository, to supply authentic microbial
cultures and to provide related services to scientists
working in research institutions, universities and
industries. Presently, the MTCC has five sections, the
Actinomycetes, Bacteria, Fungi, Yeasts and Plasmids
collectively holding over nine thousand cultures. The
MTCC scientists are actively involved in research
programmes relating to microbial diversity, ecology and
taxonomy using both classical and molecular approaches.
Laboratory for the Conservation of Endangered Species
(LaCONES) at CCMB, Hyderabad aims to conserve
endangered animals of India by using modern techniques
of DNA fingerprinting, genetic polymorphism analysis and
assisted reproductive techniques such as artificial
insemination, in vitro fertilization, cryo-banking of DNA,
cells and tissues and eventually somatic cell cloning.
Scientists at Centre for Cellular and Molecular Biology
(CCMB) have been successful in the first artificial
insemination of a spotted deer at the Nehru Zoological Park
in Hyderabad.
A group of scientists at LaCONES under the leadership
of Dr. S. Shivaji, has also been working towards developing
assisted reproductive technologies to successfully induce
pregnancy in endangered animals by artificial
insemination. Scientists were delighted with their success
when the Indian Black Buck, a Schedule 1 under Wildlife
(Protection) Act 1972, delivered a live fawn (later named as
Blacky) on 23 August 2007. This was the first successful
artificial insemination in the world in estrus-induced Black
Buck, by non-surgical intra-vaginal insemination, leading
to the birth of a live fawn. Scientists are also planning to
take genes from live cheetah cells from Iran, where a close
relative of the extinct Indian cheetah is found, and fuse it
with empty leopard eggs. Any resulting embryos would
then be carried in leopard surrogates.
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SCIENCE REPORTER, May 2010
Only good
quality
sperm
samples
fulfilling all
the
essential
parameters
are banked.
Frozen Zoo—Great Opportunity
Genes from wild-caught endangered animals are valuable,
as they are needed to increase the founder population of
breeding groups. Domestic cattle are constantly being
improved genetically by means of artificial breeding. The
advantage of speeding up the genetic progress in a breeding
programme can similarly be used for exotic species. The
frozen zoo could provide the valuable material to produce
genetically superior stock so that natural breeding in one
herd or colony could be reproduced. Furthermore, the
production of genetically healthier animals will
automatically reduce inbreeding problems caused by loss
of heterozygosity.
There are several ways in which this type of assisted
breeding can be done. Semen from a sperm bank can be
used for Artificial Insemination. Other techniques include
Embryo Transfer and In-Vitro Fertilisation, both of which
are skillful methods that are species-specific.
The success rate of assisted breeding is low compared
to natural breeding, but it is very useful for animals that are
anatomically or physiologically unable to breed naturally.
It bypasses the hurdles of infertile animals that fail to
produce oocytes or ovulate, have blocked fallopian tubes
or are unable to sustain a pregnancy. Also, it allows a female
Feature Article
CREW’s CryoBioBank™ contains
one of the world’s most
valuable rhino sperm banks at
Cincinnati Zoo. Frozen semen
samples from the most
genetically valuable Indian
rhinos in North America and the
only cryopreserved Sumatran
rhino semen in the world are
just a few examples of the
important samples stored in
CREW’s CryoBioBank.
to donate oocytes and carry a natural pregnancy, potentially
doubling her breeding rate.
Creating a Frozen Zoo
Males hugely overproduce sperms, which facilitates the
establishment of a sperm bank. There are various methods
of collecting semen including training to mount as done in
horses, camels, primates and certain wild animals and
manual stimulation as done in dogs. But by far the most
common method in wild animals is electro ejaculation. This
method uses an electrical stimulus over the accessory sex
glands to elicit ejaculation. It is performed under heavy
sedation or general anaesthetic. Semen can also be collected
post mortem if it is retrieved soon enough.
But there are several factors that play an important role.
For instance, such species have a different semen composition
and this changes the freezing technique and quality of postthawing. Traditional wildlife research needs to be geared to
establish semen cryopreservation protocols for endangered
species. During preservation, the semen quality also needs to
be fully evaluated for its normal parameters. The movement
of the sperm has to be assessed as to its capability to reach the
ova. The viability of the sperm and acrosome integrity are
also assessed using special stains. Only good quality sperm
samples fulfilling all the essential parameters are banked.
Washed and concentrated semen is drawn up in straws,
frozen in liquid nitrogen and stored in tanks. Females
produce very few oocytes each cycle, greatly limiting the
use of female genetic material. However, using a specific
hormone it is possible to stimulate the ovary to produce
Gene Repositories in India
S.No Repository TType
ype
Place of
Establishment
Collection TType
ype
Services
1.
Blue Green Algae
Collection (BGAC)
IARI, New Delhi
Collection of more than 700 uni-algal
strains of blue-green algae and six
species of Azolla
Cultures supplied to different research
/private organizations, BGA biofertilizer
– developed and distributed to farmers
and entrepreneurs
2.
Marine Cyanobacterial Bharathidasan
Germ Plasm Collection University, Trichy
(MCGPC)
Collection of 290 strains of cyanoCultures supplied to different
bacteria including 70 species of marine research/private
cyanobacteria belonging to 25 genera organizations
of 10 families of North Andaman Island
and adjoining smaller islands
3.
National Facility for
Pusa, New Delhi
Plant Tissue Repository
(NFPTCR)
Holding germplasm of approx. 1000
Cultures supplied to different
accessions of various including over 100 research/private organizations
crops accessions of suborthodox seed
species as base collection under seed
cryopreservation
4.
Repository for Filarial
parasites and
reagents
Collection of 300 filarial sera belonging
to different groups i.e. Microfilaraemia,
Acute, Chronic and Occult clinical cases
from different endemic zones of India
5.
Repository for Medicinal CIMAP, Lucknow
and Aromatic Plant
Materials
Filaria Research
Group in CBT, Anna
University, Chennai
and CDRI, Lucknow
Bulk filarial kits were provided to Institute
for Vector Control and Zoonoses, Hosur,
Regional Centre, Calicut, RMRC,
Bhubaneshwar and ISPAT General
Hospital, Rourkela.
About 200 accessions of Aromatic and Documentation, identification and
Medicinal Plants and a phytochemical confirmation of structure of active
repositoryof around 100 pure compounds compound through spectral analysis
for various medicinal and aromatic plants
SCIENCE REPORTER, May 2010
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Feature Article
The animal holding area at LaCONES
When an animal in
the wild is
threatened with
extinction, it could be
reproduced thanks
to the gene material
safely stored in the
Frozen Zoo.
Wildlife reproduction
is a new and
upcoming trend in
conservation of
wildlife.
The implication of
being able to
freeze semen and
embryos is that
the species can
be kept forever,
even when they
are extinct, as
their genes would
be preserved.
more. This process, called superovulation, enables collection
of 10-20 oocytes at one time. Each species does vary though
in its ability to be stimulated in this way.
The oocytes are then inseminated in an incubated
petridish to produce embryos by IVF. Embryos are more
resistant to being frozen than oocytes and will develop into
normal offspring after implantation into a female. If there
are too few females the embryos can be used for interspecies
transfer into a related species.
Currently in the Arabian Peninsula work has been
carried out on several endangered species. An Arabian
leopard, sandgazelle, Asian cheetah and Gordon’s wildcat
sperm bank has been set up at the Breeding Centre for
Endangered Arabian Wildlife (BCEAW) in Sharjah, UAE.
They have been successful in cryopreservation of embryos
by IVF for the Arabian leopard and Gordon’s wildcat. These
will be kept frozen until suitable recipient mothers become
available. This is a fast growing area of research. However,
there is still a lot of work to be done on endangered species
to enable a significant amount of material to be
cryopreserved before it is too late.
Human assisted reproduction research has developed
a great deal in the last decade, with improved preservation
of genetic material, improved sperm injection techniques,
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SCIENCE REPORTER, May 2010
embryo sexing, sperm sexing and cloning. This human made
reproduction has the advantage of greater funding but is,
fortunately and unfortunately, curtailed by ethics.
Nonetheless, such techniques are carried through and are
being applied to animal breeding, though they are
expensive. The number of institutions actively involved in
assisted reproduction of exotic animals is still small.
Modern technology allows assisted breeding in wild
animals without the need to keep them in captivity, but
requires close collaboration between wildlife managers and
scientists. Much research is called for, so it remains
imperative that zoos, wildlife centres and field stations
cooperate on assisted reproduction projects to harvest
opportunistic data on large-scale wild animal manipulations
to facilitate the growth of developing frozen zoos.
Despite there being numerous gene repositories, there
is no initiative as yet to develop a national gene bank/
frozen zoo exclusively for the preservation and utilization
of wild animal germplasm. Genome Resource Bank (GRB)
or Frozen Zoo would provide significant benefits for
wildlife managers. It has particular promise for endangered
species. With the help of this technology, it is possible to
bring back species from extinction.
Wildlife Institute of India, an internationally acclaimed
Institution in wildlife research and management, may also
put emphasis on conserving wild animals, develop policy
to preserve germplasm and work on creating a Frozen Zoo.
Mr. Mukesh Thakur is a Senior Research Fellow at the Wildlife Institute of
India, Dehradun. Address: C/o Dr. S. Sathyakumar, Scientist F & Head,
Department of Endangered Species Management, Wildlife Institute of India,
Post Box 18, Chandrabani, Dehradun–248001, Uttarakhand; Email:
[email protected], [email protected]
Ms Ruhina Javed is a Senior Research Fellow at the National Bureau of
Animal Genetic Resources, Karnal, Haryana. Address: C/o Dr. Ramesh Kumar
Vijh, Principal Scientist, DNA Sequencing lab, National Bureau of Animal
Genetic Resources, PB 129, GT Road Bypass, Karnal-132001, Haryana;
Email: [email protected]