Frozen zoo

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A frozen zoo is a storage facility in which genetic materials taken from animals (e.g. DNA, sperm, eggs, embryos and live tissue) are stored at very low temperatures (−196 °C) in tanks of liquid nitrogen. [1] Material preserved in this way can be stored indefinitely [2] and used for artificial insemination, in vitro fertilisation, embryo transfer, and cloning. Some facilities also collect and cryopreserve plant material (usually seeds).


The San Diego Zoo established the first "frozen zoo" program in 1972. San Diego Zoo Street Sign.jpg
The San Diego Zoo established the first "frozen zoo" program in 1972.

The first frozen zoo was established at the San Diego Zoo by pathologist Kurt Benirschke in 1972. [3] [4] [5] At the time there was no technology available to make use of the collection, but Benirschke believed such technology would be developed in the future. [6] The frozen zoo idea was later supported in Gregory Benford's 1992 paper proposing a Library of Life. [7] Zoos such as the San Diego Zoo and research programs such as the Audubon Center for Research of Endangered Species [8] [2] cryopreserve genetic material in order to protect the diversity of the gene pool of endangered species, or to provide for a prospective reintroduction of such extinct species as the Tasmanian tiger [9] and the mammoth. [10]

Gathering material for a frozen zoo is rendered simple by the abundance of sperm in males. Sperm can be taken from an animal following death. The production of eggs, which in females is usually low, can be increased through hormone treatment to obtain 10–20 oocytes, dependent on the species. Some frozen zoos prefer to fertilize eggs and freeze the resulting embryo, as embryos are more resilient under the cryopreservation process. [11] Some centers also collect skin cell samples of endangered animals or extinct species. The Scripps Research Institute has successfully made skin cells into cultures of special cells called induced pluripotent stem cells (IPS cells). It is theoretically possible to make sperm and egg cells from these IPS cells. [4]

Several animals whose cells were preserved in frozen zoos have been cloned to increase the genetic diversity of endangered species, as of 2021. One attempt to clone an extinct species was made in 2003; the newborn Pyrenean ibex died of a development disorder which may have been linked to the cloning, and there are not enough genetic samples in frozen zoos to re-create a breeding Pyrenean ibex population.

Existing frozen zoos

The Frozen Zoo at the San Diego Zoo's Institute for Conservation Research currently stores a collection of 8,400 samples from over 800 species and subspecies. [12] Frozen Zoo at San Diego Zoo Conservation Research has acted as a forbearer to similar projects at other zoos in the United States and Europe. [13] [14] However, there are still less than a dozen frozen zoos worldwide. [2]

At the United Arab Emirates' Breeding Centre for Endangered Arabian Wildlife (BCEAW) in Sharjah, the embryos stored include the extremely endangered Gordon’s wildcat (Felis silvestris gordoni) and the Arabian leopard (Panthera pardus nimr) (of which there are only 50 in the wild). [11]

The Audubon Center for Research of Endangered Species, affiliated with the University of New Orleans, is maintaining a frozen zoo. In 2000 the Center implanted a frozen-thawed embryo from the highly endangered African wildcat into the uterus of a domestic house cat, resulting in a healthy male wildcat. [15]

The Frozen Ark is a frozen zoo established in 2004 and jointly managed by the Zoological Society of London, the London Natural History Museum, and the University of Nottingham. [16] [17]

The University of Georgia's Regenerative Bioscience Center is building a frozen zoo. RBC Director Steven Stice and animal and dairy science assistant professor Franklin West created the facility with the thought of saving endangered cat species. The scientists have already extracted cells from a Sumatran tiger, which could be used for artificial insemination. Artificial insemination provides a remedy for animals who, due to anatomical or physiological reasons, are unable to reproduce in the natural way. Reproduction of stored genetic material also allows for the fostering of genetic improvements, and the prevention of inbreeding. Modern technology allows for genetic manipulation in animals without keeping them in captivity. However, the success of their restoration into the wild would require the application of new science and a sufficient amount of previously collected material. [11]



A gaur that died of natural causes had some skin cells frozen and added to the San Diego Frozen Zoo. Eight years later, DNA from these cells was inserted into a domestic-cow egg to create an embryo (trans-species cloning), which was then implanted in a domestic cow (Bos taurus). On 8 January 2001, the gaur, named Noah, was born in Sioux Center, Iowa. Noah was initially healthy, but the next day, he came down with clostridial enteritis, and died of dysentery within 48 hours of birth. This is not uncommon in uncloned animals and the researchers did not think it was due to the cloning. [18]


The banteng was the second endangered species to be successfully cloned, and the first clone to survive beyond infancy. [19] [20] Scientists at Advanced Cell Technology in Worcester, Massachusetts extracted DNA from skin cells of a dead male banteng, that were preserved in San Diego 's Frozen Zoo facility, and transferred it into eggs from domestic banteng cows, a process called somatic cell nuclear transfer. Thirty embryos were created and implanted in domestic banteng cows. Two were carried to term and delivered by Caesarian section. The first was born on 1 April 2003, and the second two days later. The second was euthanised, apparently suffering from large offspring syndrome (an overgrowth disorder), but the first survived and lived for seven years at the San Diego Zoo, where it died in April 2010 after it broke a leg and was euthanised. [21] [22]

Przewalski's horse clone

A Przewalski's horse Wintercoat.jpg
A Przewalski's horse

In 2020, the first cloned Przewalski's horse was born, the result of a collaboration between San Diego Zoo Global, ViaGen Equine and Revive & Restore. [23] The cloning was carried out by somatic cell nuclear transfer (SCNT), whereby a viable embryo is created by transplanting the DNA-containing nucleus of a somatic cell into an immature egg cell (oocyte) that has had its own nucleus removed, producing offspring genetically identical to the somatic cell donor. [24] Since the oocyte used was from a domestic horse, this was an example of interspecies SCNT. [25]

The somatic cell donor was a Przewalski's horse stallion named Kuporovic, born in the UK in 1975, and relocated three years later to the US, where he died in 1998. Due to concerns over the loss of genetic variation in the captive Przewalski's horse population, and in anticipation of the development of new cloning techniques, tissue from the stallion was cryopreserved at the San Diego Zoo's Frozen Zoo. Breeding of this individual in the 1980s had already substantially increased the genetic diversity of the captive population, after he was discovered to have more unique alleles than any other horse living at the time, including otherwise-lost genetic material from two of the original captive founders. [23] To produce the clone, frozen skin fibroblasts were thawed, and grown in cell culture. [26] An oocyte was collected from a domestic horse, and its nucleus replaced by a nucleus collected from a cultured Przewalski's horse fibroblast. The resulting embryo was induced to begin division, and was cultured until it reached the blastocyst stage, then implanted into a domestic horse surrogate mare, [26] which carried the embryo to term and delivered a foal with the Przewalski's horse DNA of the long-deceased stallion.

The cloned horse was named Kurt, after Dr. Kurt Benirschke, a geneticist who developed the idea of cryopreserving genetic material from species considered to be endangered. His ideas led to the creation of the Frozen Zoo as a genetic library. [27] There is a breeding herd in the San Diego Zoo Safari Park. [28] Once the foal matures, he will be relocated to the breeding herd at the San Diego Zoo Safari Park, [29] so as to pass Kuporovic's genes into the larger captive Przewalski's horse population and increase the genetic variation of the species. [23]

Black-footed ferret

Elizabeth Ann, the first cloned black ferret, being weighed on the 18th of February 2021 (at 70 days old)

The cells of two 1980s wild-caught black-footed ferrets that never bred in captivity were preserved in the San Diego Wildlife Alliance Frozen Zoo. One of them was cloned to increase genetic diversity in this species in December 2020. More clones of both are planned. They will initially be bred separately from the non-cloned population. [30]

Pyrenean ibex

Diagram of trans-species cloning of the Pyrenean ibex Pyrenean ibex Cloning.svg
Diagram of trans-species cloning of the Pyrenean ibex

The Pyrenean ibex went extinct in 2003. In 2003 frozen cells from the last one (a male killed by a falling branch) were used to clone 208 embryos, of which 7 successfully implanted in goats, and one made it to term. That one ibex died of respiratory failure just after birth; quite possibly as a result of the cloning process, its lungs had not developed properly. There may not be enough individuals' cells preserved to create a breeding population. [31] [32]

See also

Related Research Articles

Cloning Process of producing genetically identical individuals of an organism

Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cloning is the process of creating cloned organisms (copies) of cells and of DNA fragments.

Dolly (sheep) First cloned mammal (1996–2003)

Dolly was a female Finnish Dorset sheep and the first mammal cloned from an adult somatic cell. She was cloned by associates of the Roslin Institute in Scotland, using the process of nuclear transfer from a cell taken from a mammary gland. Her cloning proved that a cloned organism could be produced from a mature cell from a specific body part. Contrary to popular belief, she was not the first animal to be cloned.

Embryo Multicellular diploid eukaryote in its earliest stage of development

An embryo is the early stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sperm cell. The resulting fusion of these two cells produces a single-celled zygote that undergoes many cell divisions that produce cells known as blastomeres. The blastomeres are arranged as a solid ball that when reaching a certain size, called a morula, takes in fluid to create a cavity called a blastocoel. The structure is then termed a blastula, or a blastocyst in mammals.

Przewalskis horse Subspecies of mammal

Przewalski's horse, also called the takhi, Mongolian wild horse or Dzungarian horse, is a rare and endangered horse originally native to the steppes of Central Asia. It is named after the Russian geographer and explorer Nikołaj Przewalski. Once extinct in the wild, it has been reintroduced to its native habitat since the 1990s in Mongolia at the Khustain Nuruu National Park, Takhin Tal Nature Reserve, and Khomiin Tal, as well as several other locales in Central Asia and Eastern Europe.

Somatic cell nuclear transfer Method of creating a cloned embryo by replacing the egg nucleus with a body cell nucleus

In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte and implanting a donor nucleus from a somatic (body) cell. It is used in both therapeutic and reproductive cloning. In 1996, Dolly the sheep became famous for being the first successful case of the reproductive cloning of a mammal. In January 2018, a team of scientists in Shanghai announced the successful cloning of two female crab-eating macaques from foetal nuclei.

<i>Ex situ</i> conservation Preservation of plants or animals outside their natural habitats

Ex situ conservation literally means, "off-site conservation". It is the process of protecting an endangered species, variety or breed, of plant or animal outside its natural habitat; for example, by removing part of the population from a threatened habitat and placing it in a new location, an artificial environment which is similar to the natural habitat of the respective animal and within the care of humans, example are zoological parks and wildlife safaris. The degree to which humans control or modify the natural dynamics of the managed population varies widely, and this may include alteration of living environments, reproductive patterns, access to resources, and protection from predation and mortality. Ex situ management can occur within or outside a species' natural geographic range. Individuals maintained ex situ exist outside an ecological niche. This means that they are not under the same selection pressures as wild populations, and they may undergo artificial selection if maintained ex situ for multiple generations.

Banteng Species of wild cattle found in Southeast Asia

The banteng, also known as tembadau, is a species of cattle found in Southeast Asia. The head-and-body length is between 1.9 and 3.68 m. Wild banteng are typically larger and heavier than their domesticated counterparts, but are otherwise similar in appearance. The banteng shows extensive sexual dimorphism; adult bulls are generally dark brown to black, larger and more sturdily built than adult cows, which are thinner and usually pale brown or chestnut red. There is a big white patch on the rump. Horns are present on both sexes, and are typically 60 to 95 cm long. Three subspecies are generally recognised.

Commercial animal cloning is the cloning of animals for commercial purposes, currently, including livestock, competition camels and horses, pets, medical uses, endangered and extinct animals, as first demonstrated in 1996 for Dolly the sheep.

Captive breeding Intentional breeding of wild plants or animals in captivity

Captive breeding, also known as captive propagation, is the process of maintaining plants or animals in controlled environments, such as wildlife reserves, zoos, botanic gardens, and other conservation facilities. It is sometimes employed to help species that are being threatened by the effects of human activities such as climate change, habitat loss, fragmentation, over hunting or fishing, pollution, predation, disease, and parasitism.

Northern white rhinoceros Subspecies of white rhinoceros

The northern white rhinoceros, or northern square-lipped rhinoceros, is one of two subspecies of the white rhinoceros. Formerly found in several countries in East and Central Africa south of the Sahara, this subspecies is a grazer in grasslands and savanna woodlands. Since 19 March 2018, there are only two known rhinos of this subspecies left, called Najin and Fatu, both of which are female; barring the existence of unknown or misclassified male northern white rhinos elsewhere in Africa, this makes the subspecies functionally extinct. The two female rhinos belong to the Dvůr Králové Zoo in the Czech Republic but live in the Ol Pejeta Conservancy in Kenya and are protected round-the-clock by armed guards.

Kurt Benirschke was a German-American pathologist, geneticist and expert on the placenta and reproduction in humans and myriad mammalian species. At the San Diego Zoo, he created the world's first frozen zoo for the cryopreservation of genetic material from endangered species.

Genetic erosion is a process where the limited gene pool of an endangered species diminishes even more when reproductive individuals die off before reproducing with others in their endangered low population. The term is sometimes used in a narrow sense, such as when describing the loss of particular alleles or genes, as well as being used more broadly, as when referring to the loss of a phenotype or whole species.

San Diego Zoo Wildlife Alliance Non-profit organisation in the U.S.

San Diego Zoo Wildlife Alliance is a not-for-profit organization headquartered in San Diego that operates the San Diego Zoo and the San Diego Zoo Safari Park. Founded in 1916 as the Zoological Society of San Diego under the leadership of Harry M. Wegeforth, the organization claims the largest zoological society membership in the world, with more than 250,000 member households and 130,000 child memberships, representing more than half a million people. The organization's mission is to save species worldwide by uniting their expertise in animal care and conservation science with their dedication to inspiring passion for nature.

Revival of the woolly mammoth Efforts to revive the woolly mammoth

The existence of frozen soft-tissue remains and DNA of woolly mammoths has led to the idea that the species could be recreated by scientific means. In 2003 the pyrenean ibex was briefly revived, giving credence to the idea that the mammoth could be successfully revived. As of today, several methods have been proposed to achieve this goal, including cloning, artificial insemination, and genome editing. The ethics of reviving the animal have been disputed.

De-extinction Process of re-creating an extinct species

De-extinction is the process of generating an organism that either resembles or is an extinct species. There are several ways to carry out the process of de-extinction. Cloning is the most widely proposed method, although genome editing and selective breeding have also been considered. Similar techniques have been applied to certain endangered species, in hopes to boost their genetic diversity. The only method of the three that would provide an animal with the same genetic identity is cloning. There are both pros and cons to the process of de-extinction ranging from technological advancements to ethical issues.

Pyrenean ibex Extinct subspecies of Iberian ibex

The Pyrenean ibex, Aragonese and Spanish common name bucardo, Basque common name bukardo, Catalan common name herc and French common name bouquetin, was one of the four subspecies of the Iberian ibex or Iberian wild goat, a species endemic to the Pyrenees. Pyrenean ibex were most common in the Cantabrian Mountains, Southern France, and the northern Pyrenees. This species was common during the Holocene and Upper Pleistocene, during which their morphology, primarily some skulls, of the Pyrenean ibex was found to be larger than other Capra subspecies in southwestern Europe from the same time.

Morné de la Rey South African veterinarian

Morné de la Rey is a South African veterinary surgeon and embryo transfer specialist. In 2003, he was one of a team of scientists and veterinarians from his company Embryo Plus and the Danish Agriculture Institute to clone a cow, the first animal to be cloned in Africa. In 2016, he was one of a team to use in vitro fertilisation successfully for the first time in the Cape buffalo.

Elizabeth Ann (ferret) First cloned ferret

Elizabeth Ann is a black-footed ferret, the first U.S. endangered species to be cloned.

Revive & Restore is a California-based nonprofit that works to bring biotechnologies to conservation biology, with the mission to enhance biodiversity through the genetic rescue of endangered and extinct animals.


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