May-Final with Advt..Pmd

Feature Article MUKESH THAKUR & RUHINA JAVED Frozen Zoos!

DespiteDespite therethere beingbeing numerousnumerous genegene repositoriesrepositories inin thethe country,country, therethere isis nono initiativeinitiative asas yetyet toto developdevelop aa nationalnational genegene bankbank exclusivelyexclusively forfor thethe preservationpreservation andand utilizationutilization ofof wildwild animalanimal germplasm.germplasm. GenomeGenome ResourceResource BankBank (GRB)(GRB) oror FrozenFrozen ZooZoo wouldwould provideprovide significantsignificant benefitsbenefits forfor wildlifewildlife managersmanagers asas itit holdsholds particularparticular promisepromise forfor endangeredendangered species.species.

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 T’S not a zoo that you would particularly enjoy visiting. wildlife management. Semen can be collected at one site Unlike the conventional zoo where antics of animals in and shipped anywhere in the world. captivity keep you entertained, in a Frozen Zoo you Embryo Transfer (ET) is another captive breeding tool are more likely to come across tanks containing semen currently used. It requires two genetically related species. Iand embryos of wild animals frozen in liquid nitrogen. One species is super-ovulated, inseminated, and the Sometime in the future, when an animal in the wild is fertilized eggs are collected. The fertilized eggs are implanted threatened with extinction, it could be reproduced thanks into surrogate mothers. The offspring carry the genetic to the gene material safely stored in the Frozen Zoo. makeup of the two original parents, not the surrogates. Wildlife reproduction is a new and upcoming trend in This science provides many opportunities to captive conservation of wildlife. Practiced widely in several breeding programmes. Animals no longer have to be countries, it is not yet frequently applied in India. There are moved to accommodate natural mating. Conservation several new methods available today in wildlife biologists can work to counter the effects of genetic drift. reproduction including Cross-fostering, Artificial Even if an animal dies, gametes stay viable up to 24 hours, incubation, Artificial insemination, and Embryo transfer. allowing the possibility to re-introduce diversity back into All these methods go a long way in boosting genetic wild populations. variability, increasing population size, and improving reproductive success. Genome Resource Bank (GRB) or Frozen Cross fostering is a technique of inselective breeding, where Zoo would provide significant benefits for offspring are removed from their biological parents at birth and raised by surrogates. This can also occasionally occur wildlife managers. It has particular promise in nature. The surrogate can be a close relative of the species and does not have to be the same species. for endangered species. 44 SCIENCE REPORTER, May 2010 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: National Gene Bank Facility for conservation and preservation of domestic animal germplasm at the National Frozen Gene Bank Bureau of Animal Genetic Resources, Karnal, Haryana with The Frozen Zoo is a collection of animal genes in the form the responsibility of evaluating, certifying and conserving of frozen semen and embryos. In practical terms this is a the rich and varied germplasm resources available in the collection of sperm-holding straws stored in liquid nitrogen country and whose genetic base is shrinking fast. Thus, the tanks. The first so-called “frozen zoo” was created in 1975 establishment of the National Bureau of Animal Genetic by Kurt Benirschke, a visionary physician who switched Resources/National Institute of Animal Genetics in 1984 from his human practice to work with endangered species was the culmination of sustained efforts made by leading at the San Diego Zoo, studying gene pools and genetic geneticists in the country over the years. diseases made prevalent by inbreeding. National Genomic Resources Repository was established Modern technology allows the indefinite preservation in the premises of the National Bureau of Plant Genetic of genetic material through the freezing of semen and Resources, New Delhi for exchange, quarantine, collection, embryos (unfortunately, ova do not freeze very well). The conservation, evaluation and the systematic documentation implication of being able to freeze semen and embryos is of plant genetic resources. NBPGR act as an institutional that the species can be kept forever, even when they are framework for methodical and centralized efforts to collect, extinct, as their genes would be preserved. When the threat generate, conserve and distribute genomic resources for to the preserved species has been controlled, implantation agricultural research. 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 1. A researcher at the Cincinnati Zoo removes blood from a biopsy of a develop the technology required for related species not yet domestic cat’s follicles, the egg-nurturing structures in the ovary. endangered. Thus when the time comes to make 2. After the blood is extracted, the eggs in the sample are easier to conservation efforts for one of these species, the technology detect. would already be available. 3. A researcher opens up one of the chilled canisters containing sperm Today, in zoos around the world, these deep-freeze samples and frozen embryos. tanks act as a high-tech Ark, housing cells from the planet’s most endangered animals, including chimps, cheetahs, 4. Samples stored in liquid nitrogen cooled to -374 degrees Fahrenheit pandas, California condors and hundreds more. As a result, remain viable indefinitely. They are often used successfully after two long-dead animals are now becoming parents from the to five years in storage. 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.

left: This ocelot kitten was conceived in the lab using sperm and a stored egg

SCIENCE REPORTER, May 2010 45 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 Only good jointly by the Department of Biotechnology (DBT) and the quality Council of Scientific and Industrial Research (CSIR), Government of India. The MTCC is a modern facility sperm housed at the Institute of Microbial Technology (IMTECH), Chandigarh. The main objectives of this national facility samples are to act as a depository, to supply authentic microbial cultures and to provide related services to scientists fulfilling all working in research institutions, universities and the industries. Presently, the MTCC has five sections, the Actinomycetes, Bacteria, Fungi, Yeasts and Plasmids essential collectively holding over nine thousand cultures. The MTCC scientists are actively involved in research parameters programmes relating to microbial diversity, ecology and taxonomy using both classical and molecular approaches. are banked. Laboratory for the Conservation of Endangered Species (LaCONES) at CCMB, Hyderabad aims to conserve endangered animals of India by using modern techniques Frozen Zoo—Great Opportunity of DNA fingerprinting, genetic polymorphism analysis and Genes from wild-caught endangered animals are valuable, assisted reproductive techniques such as artificial as they are needed to increase the founder population of insemination, in vitro fertilization, cryo-banking of DNA, breeding groups. Domestic cattle are constantly being cells and tissues and eventually somatic cell cloning. improved genetically by means of artificial breeding. The Scientists at Centre for Cellular and Molecular Biology advantage of speeding up the genetic progress in a breeding (CCMB) have been successful in the first artificial programme can similarly be used for exotic species. The insemination of a spotted deer at the Nehru Zoological Park frozen zoo could provide the valuable material to produce in Hyderabad. genetically superior stock so that natural breeding in one A group of scientists at LaCONES under the leadership herd or colony could be reproduced. Furthermore, the of Dr. S. Shivaji, has also been working towards developing production of genetically healthier animals will assisted reproductive technologies to successfully induce automatically reduce inbreeding problems caused by loss pregnancy in endangered animals by artificial of heterozygosity. insemination. Scientists were delighted with their success There are several ways in which this type of assisted when the Indian Black Buck, a Schedule 1 under Wildlife breeding can be done. Semen from a sperm bank can be (Protection) Act 1972, delivered a live fawn (later named as used for Artificial Insemination. Other techniques include Blacky) on 23 August 2007. This was the first successful Embryo Transfer and In-Vitro Fertilisation, both of which artificial insemination in the world in estrus-induced Black are skillful methods that are species-specific. Buck, by non-surgical intra-vaginal insemination, leading The success rate of assisted breeding is low compared to the birth of a live fawn. Scientists are also planning to to natural breeding, but it is very useful for animals that are take genes from live cheetah cells from Iran, where a close anatomically or physiologically unable to breed naturally. relative of the extinct Indian cheetah is found, and fuse it It bypasses the hurdles of infertile animals that fail to with empty leopard eggs. Any resulting embryos would produce oocytes or ovulate, have blocked fallopian tubes then be carried in leopard surrogates. or are unable to sustain a pregnancy. Also, it allows a female 46 SCIENCE REPORTER, May 2010 Feature Article CREW’s CryoBioBank™ contains Creating a Frozen Zoo one of the world’s most Males hugely overproduce sperms, which facilitates the valuable rhino sperm banks at establishment of a sperm bank. There are various methods of collecting semen including training to mount as done in Cincinnati Zoo. Frozen semen horses, camels, primates and certain wild animals and samples from the most manual stimulation as done in dogs. But by far the most genetically valuable Indian common method in wild animals is electro ejaculation. This rhinos in North America and the method uses an electrical stimulus over the accessory sex glands to elicit ejaculation. It is performed under heavy only cryopreserved Sumatran sedation or general anaesthetic. Semen can also be collected rhino semen in the world are post mortem if it is retrieved soon enough. just a few examples of the But there are several factors that play an important role. important samples stored in For instance, such species have a different semen composition and this changes the freezing technique and quality of post- CREW’s CryoBioBank. thawing. 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 to donate oocytes and carry a natural pregnancy, potentially use of female genetic material. However, using a specific doubling her breeding rate. hormone it is possible to stimulate the ovary to produce

Gene Repositories in India

S.No Repository Type Place of Collection Type Services Establishment

1. Blue Green Algae IARI, New Delhi Collection of more than 700 uni-algal Cultures supplied to different research Collection (BGAC) strains of blue-green algae and six /private organizations, BGA biofertilizer species of Azolla – developed and distributed to farmers and entrepreneurs

2. Marine Cyanobacterial Bharathidasan Collection of 290 strains of cyano- Cultures supplied to different Germ Plasm Collection University, Trichy bacteria including 70 species of marine research/private (MCGPC) cyanobacteria belonging to 25 genera organizations of 10 families of North Andaman Island and adjoining smaller islands

3. National Facility for Pusa, New Delhi Holding germplasm of approx. 1000 Cultures supplied to different Plant Tissue Repository accessions of various including over 100 research/private organizations (NFPTCR) crops accessions of suborthodox seed species as base collection under seed cryopreservation

4. Repository for Filarial Filaria Research Collection of 300 filarial sera belonging Bulk filarial kits were provided to Institute parasites and Group in CBT, Anna to different groups i.e. Microfilaraemia, for Vector Control and Zoonoses, Hosur, reagents University, Chennai Acute, Chronic and Occult clinical cases Regional Centre, Calicut, RMRC, and CDRI, Lucknow from different endemic zones of India Bhubaneshwar and ISPAT General Hospital, Rourkela.

5. Repository for Medicinal CIMAP, Lucknow About 200 accessions of Aromatic and Documentation, identification and and Aromatic Plant Medicinal Plants and a phytochemical confirmation of structure of active Materials repositoryof around 100 pure compounds compound through spectral analysis for various medicinal and aromatic plants

SCIENCE REPORTER, May 2010 47 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 embryo sexing, sperm sexing and cloning. This human made being able to reproduction has the advantage of greater funding but is, freeze semen and fortunately and unfortunately, curtailed by ethics. Nonetheless, such techniques are carried through and are embryos is that being applied to animal breeding, though they are the species can expensive. The number of institutions actively involved in be kept forever, assisted reproduction of exotic animals is still small. Modern technology allows assisted breeding in wild even when they animals without the need to keep them in captivity, but are extinct, as requires close collaboration between wildlife managers and their genes would scientists. Much research is called for, so it remains imperative that zoos, wildlife centres and field stations be preserved. cooperate on assisted reproduction projects to harvest opportunistic data on large-scale wild animal manipulations more. This process, called superovulation, enables collection to facilitate the growth of developing frozen zoos. of 10-20 oocytes at one time. Each species does vary though Despite there being numerous gene repositories, there in its ability to be stimulated in this way. is no initiative as yet to develop a national gene bank/ The oocytes are then inseminated in an incubated frozen zoo exclusively for the preservation and utilization petridish to produce embryos by IVF. Embryos are more of wild animal germplasm. Genome Resource Bank (GRB) resistant to being frozen than oocytes and will develop into or Frozen Zoo would provide significant benefits for normal offspring after implantation into a female. If there wildlife managers. It has particular promise for endangered are too few females the embryos can be used for interspecies species. With the help of this technology, it is possible to transfer into a related species. bring back species from extinction. Currently in the Arabian Peninsula work has been Wildlife Institute of India, an internationally acclaimed carried out on several endangered species. An Arabian Institution in wildlife research and management, may also leopard, sandgazelle, Asian cheetah and Gordon’s wildcat put emphasis on conserving wild animals, develop policy sperm bank has been set up at the Breeding Centre for to preserve germplasm and work on creating a Frozen Zoo. Endangered Arabian Wildlife (BCEAW) in Sharjah, UAE. They have been successful in cryopreservation of embryos Mr. Mukesh Thakur is a Senior Research Fellow at the Wildlife Institute of by IVF for the Arabian leopard and Gordon’s wildcat. These India, Dehradun. Address: C/o Dr. S. Sathyakumar, Scientist F & Head, will be kept frozen until suitable recipient mothers become Department of Endangered Species Management, Wildlife Institute of India, available. This is a fast growing area of research. However, Post Box 18, Chandrabani, Dehradun–248001, Uttarakhand; Email: there is still a lot of work to be done on endangered species [email protected], [email protected] to enable a significant amount of material to be Ms Ruhina Javed is a Senior Research Fellow at the National Bureau of cryopreserved before it is too late. Animal Genetic Resources, Karnal, Haryana. Address: C/o Dr. Ramesh Kumar Human assisted reproduction research has developed Vijh, Principal Scientist, DNA Sequencing lab, National Bureau of Animal a great deal in the last decade, with improved preservation Genetic Resources, PB 129, GT Road Bypass, Karnal-132001, Haryana; of genetic material, improved sperm injection techniques, Email: [email protected] 48 SCIENCE REPORTER, May 2010