Among rhinos and giraffes, at the far end of San Diego's Safari Park, lies a research institute that safeguards a precious treasure in liquid nitrogen: The Frozen Zoo—an extensive cell biobank from animal species on the brink of extinction. Armed with today’s state-of-the-art cloning, in vitro fertilization, and genomics technologies, it gives us another tool in the battle to conserve and restore these rare creatures, before they are gone forever.

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*This article was inspired by a family visit to the San Diego Safari Park and a “behind-the-scenes” tour of the Frozen Zoo laboratories.

Zoos are an inseparable part of the effort to restore ecosystems that have been damaged by human activity and other factors, through the restoration and conservation of endangered animal species. The effort mostly focuses on collecting animals whose habitats are shrinking and breeding them in captivity or in nature reserves to boost their numbers. When possible, they are even re-released into the wild [1]. The problem is that it is not always enough to put a male and female rhinoceros together and “let nature take its course”. For some species we still do not know how to provide suitable breeding conditions in captivity, and technological measures, such as in vitro fertilization, are sometimes required, much like for a human couple who have difficulty conceiving. In addition, when only a handful of individuals remain in the world, genetic diversity suffers, meaning that the population effectively consists of “first cousins”. As we also know from humans, inbreeding can preserve or spread genetic diseases and traits that constitute an evolutionary disadvantage in the wild.

This is where our story begins: In 1975, at the San Diego Zoo, a fertility expert named Dr. Kurt Benirschke decided to establish a biobank for endangered animals and preserve their cells in liquid nitrogen—a “Frozen Zoo” [2–4]. These were practically prehistoric times in terms of cloning (Dolly the cloned sheep would not arrive for more than twenty years), molecular biology, and genome mapping, but Dr. Benirschke believed that at some point in the future, science would figure out what to do with the frozen cells to save the species stored there. And indeed, nowadays we can clone animals [5], sequence entire genomes within hours [6], and “reprogram” any type of body cell into a stem cell [7]. Today, fifty years after it was founded, the Frozen Zoo holds cells (mainly skin-derived fibroblasts) from more than a thousand animal species—mammals, birds, reptiles, amphibians, and fish. It also stores reproductive cells (sperm cells and eggs) from various animals, in the hope of gaining a better understanding of each species’ reproductive processes and using them to create healthy individuals that will enrich the zoo population—and, hopefully, wild populations as well.

Beyond cloning and in vitro fertilization, the cells are also used to assess the existing genetic diversity of each species in the zoo and beyond, to determine whether there really is an “inbreeding problem”. Assessing genetic variation is fairly straightforward for species that are more familiar to us: Humans, pets, laboratory animals, and livestock. In humans, for example, we can relatively easily estimate the genetic heterogeneity of the population because the entire human genome has been mapped for more than twenty years. In other words, we know how to break down the long sequence of DNA “letters” into genes—segments that contain instructions for producing proteins and are therefore transcribed [8] into RNA.

For species that are not as well-studies, and especially when very few individuals remain, genome mapping must start almost from scratch. This year, for instance, the zoo published a paper reporting that about 50 percent of the genome of the northern white rhinoceros (Ceratotherium simum cottoni)—a subspecies on the verge of extinction with only two females left—has been mapped [9]. This achievement is considered extraordinary [10]. The mapping involved the painstaking comparison of numerous RNA transcripts from cells of the closely related southern white rhinoceros to the DNA sequence of the northern subspecies stored in the bank, until researchers figured out how to “divide” it into genes. Without a critical mass of mapped genome for the specific species, it is hard to assess the genetic diversity of the population. How do we know when there is “enough” genetic diversity in a bank or in a population? That is the million-dollar question, and there is no definitive answer (we even asked the scientists working there 😉).

Northern white rhinoceros, the last male that lived at the San Diego Safari Park until his death in 2014. Source: Wikipedia, public domain

Suppose we want to enrich a population that began with a very small number of individuals, as happened with the Przewalski’s horse (Equus przewalskii) in the mid-20th century. The global population of these wild horses dwindled to just twelve animals(!). Efforts were gradually made to increase their numbers, but because the founding population was so small, genetic diversity remained extremely low.

Przewalski’s horse. Photo: Claudia Feh, Wikipedia

The San Diego Safari Park houses a small group of such mares, all are more or less “cousins” of one another. Fortunately, a stallion that was relatively genetically distinct from the rest of the population had once existed—but he died in 1998. This is where the Frozen Zoo came into play: Cells from that stallion had been preserved, and in the meantime a cloning industry for domestic animals, like domesticated horses, had developed. In a collaboration between the zoo and a cloning company, two clones were created from that Przewalski’s stallion who has been gone since 1998. The method used was slightly different from classic cloning, but proved effective in this case: Only the genetic material from the Przewalski’s horse was taken and was then inserted into an egg from a domestic mare; then, the resulting embryo was implanted in another mare’s uterus [11]. Thus, an ordinary domestic mare gave birth to a rare Przewalski’s foal! Today the two clones live at the San Diego Safari Park, where staff are monitoring their fertility in the hope that they will add genetic diversity to the family of mares [12].

Additional “Frozen Zoos” now operate in a similar format, sharing protocols and cells to broaden the genetic diversity they store and to make sure that “not all the eggs are in one basket” [13]. The Frozen Zoo has even made its way to the big screen: after the creators of the film “Jurassic Park” visited it in the early 1990s, they were inspired to write the scene in which dinosaur embryos are stolen from racks of vials arranged in liquid nitrogen. Unlike the film, however, we have no dinosaur genetic material, nor could we, because of DNA’s limited survival time. We will therefore have to abandon the fantasy of a real-life Jurassic Park and focus on saving the species we still have a chance to rescue—here and now.

Hebrew editing: Smadar Raban
English editing: Elee Shimshoni

References:

1. Why preserving and restoring animal species matters
2. Explanation of the Frozen Zoo on the San Diego Zoo Wildlife Alliance website
3. Fifty years of the Frozen Zoo
4. Dr. Kurt Benirschke, Wikipedia
5. On cloning pets
6. Guinness World Record for fastest DNA sequencing, Broad Institute
7. UCLA Center for Stem Cell Biology: Induced pluripotent stem cells
8. Explanation of RNA and how DNA gives protein-building instructions
9. Article on the last male northern white rhinoceros
10. Paper on mapping the northern white rhinoceros genome
11. Paper describing the cloning of a Przewalski’s horse
12. Announcement naming the second cloned Przewalski’s horse—Ollie
13. The biobank of the European Association of Zoos and Aquaria