The Frozen Zoo: High tech help

Plus: Good news for Borneo's orangutans. Palm oil nixed in Sri Lanka. Climate change migration in India. Sante Fe becomes pollinator friendly. Climbing health costs from climate change. And more.

From the Archive

This is the first in a series of “From the Archive,” pieces that I’ve written—some published, others unpublished—that stand out for their importance or quality. For this installment, we go back to September of 2006 and take a look at the San Diego Zoo’s Conservation and Research for Endangered Species (CRES) and other “frozen zoos.”

A mountain yellow-legged frog is small, only about as big as a prune. If you were to hold one in your hand, he would fit easily between your index finger and pinkie. Yellow legs rise up to dark green bodies, with splotches and speckles of yellow here and there. Their eyes don’t blink; rather, they seem to shut from time to time, Yoda-like, in a sad ponderous squint. Native to California, These amphibians used to be found in the San Bernardino Mountains and farther south to Mount Palomar, but since the 1970s are now extinct in these regions. They are severely threatened in their other habitat, the Sierras, with only about one percent of their original population remaining. Unless great strides are made to conserve this species, they will soon be gone.

At San Diego’s Wild Animal Park, in their Conservation and Research for Endangered Species (CRES) Center, a room holds dozens of steel cylinders that stand about waist high. Looking like every mad scientist’s laboratory, chilled water vapor pours down the cylinders sides and onto the floor. Inside one cylinder, frozen to –196 with liquid nitrogen, are sperm, fertilized eggs, and other tissue of the mountain yellow legged frog. Genetic materials of hundreds of other endangered species occupy this as well as the other cylinders.  This is CRES’ Frozen Zoo. Here and at other zoological institutions throughout the world, dedicated scientists are using the latest technologies of super cooled preservation, called cryogenics, to store the genes and DNA of endangered species.

Oliver Ryder heads the genetics division of the Frozen Zoo and has been at the institution from its inception in 1975. He is an adjunct professor at the University of California San Diego, where he received his PhD in biology. Ryder speaks clearly and maybe a little louder than most folks, like someone who has delivered a lot of lectures.  “This whole project started somewhat serendipitously as chromosome investigation,” he says. No one is quite certain who thought of preserving DNA from endangered species or what sort of application it might have, but by the early eighties CRES had dedicated a full facility to their Frozen Zoo with a mandate to store tissues and reproductive cells of species on the brink. “Choosing which species, at first, was opportunistic, species that we had easy access to or what we were studying at the time. It took a while before we developed some policies and actually have what we call a species survival plan.”

There are now hundreds of institutions that have a program for cryogenically preserving the tissues and reproductive samples of endangered species. “Just about every large zoo has a program similar to what we do here,” Ryder says. “We work with institutions in almost every state of the union and 38 other counties, but of all of those, we are the largest.”

The deep freeze

Working with liquid nitrogen is somewhat hazardous; it’s cold enough to cause frostbite instantly. The scientists nonetheless want to keep their frozen menagerie as cold as possible. Storing them in a normal freezer is not cold enough; even at the relatively bone chilling temperatures of –60 F, chemical reactions still occur, and that can lead to degradation of the tissues and cells. If the scientist could get things colder, using liquid helium for example, they would. Nitrogen is chosen because it cheap and plentiful. Ryder estimates that in the nitrogen filled cylinders the genetic material will be safe for 10,000 years.

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Once the cells are frozen, they’re safe. Getting them from room temperature to super freezing is the tricky part. “If you put an ice cube tray in the freezer and leave it there for a half hour, when you pull it out it won’t be frozen solid. But there will be ice crystals formed,” Ryder says. If an ice crystal forms in a tissue sample or cell, it can grow and break a cell wall or damage another cell structure. Just think of burst water pipes during a harsh winter. To avoid this, the cells are cooled slowly, over about six hours, so that they cool uniformly. Once they are at slightly lower temperature than 32F, right before they would start to freeze, the scientists place the samples in a bath of liquid nitrogen, freezing the cells almost instantly. Ice crystals don’t’ have a chance to grow.  Ryder estimates that this method works 98% of the time.          

For cloning purposes, the easiest tissues to gather are those of the animal’s skin. To take a sample, the researchers first shave the surface of the animal’s hide to remove dead and damaged cells. The site is then treated with alcohol, the same way you would swab someone’s skin before giving her a shot. The scientists then shave off another tiny piece of skin. They place this shaving into a flask filled with enzymes. The tissue floats around and little by little the enzymes go to work. The tissue breaks apart into individual cells called fibroblasts. Each fibroblast can be used to produce one clone.

The scientists at the Frozen Zoo have even successfully harvested tissue from animals that have been dead for two or three days. They also have found ways of getting tissue samples from feathers, an advance that allows the scientists to gather material from a nest rather than capturing an endangered bird. At the Frozen Zoo and elsewhere the emphasis has been on preserving mammals, and in the past few years the researchers have started adding more amphibians and birds to the list of species to store. Right now, worldwide, there are 52 or 53 mammalian species, mostly bovine, stored at the various cryogenic facilities.

The science of cloning is still in its early development, with only two endangered animals — a bantang and a gaur, both types of wild buffalo— being produced from this method. The science of preserving sperm, eggs, and zygotes for in vitro fertilization, however, goes back to the early fifties, when cattle ranchers started using new techniques to make cattle rearing more reliable.  Today all commercial cattle are produced from in vitro fertilization using frozen bull sperm. “All the milk, cheese, and beef that you eat is the result of the science that we use here on species preservation,” says Stanley Leibo, who spent most of his life working in the cattle industry in the United States and Canada. He now works in cryogenic preservation as a research associate with Audubon Center for Research of Endangered Species. He adds that soon after bull sperm was first successfully frozen, the first human sperm was frozen in 1951, with the first human from frozen sperm being born in 1953.

There are two things to remember when freezing cells for in vitro fertilization: Size matters, and there is strength in numbers. Leibo explains that the smaller the cell, the easier it is to freeze. Just as a taller stack, of blocks is easier to knock over than a smaller stack, the larger the cell, which has more surface area, the more easily it is damaged. Sperm have the advantage of being extremely small cells. Even the largest Bull Moose produces sperm that is about twenty times smaller than the eggs of his mate. Additionally, because males produce so many sperm, the mortality of the samples does not present a problem. Leibo says, “With a sperm sample of 100,000 sperm, what do we care if 25% of them die? We still have 75,000. And you only need one sperm to fertilize an egg.”

We all check the carton of eggs at the grocery store, removing the cracked ones and replacing them with good ones. All eggs, from snake eggs to whale eggs, are similarly delicate when it comes to cryogenic preservation. They have what is called a spindle on which chromosomes reside. Freezing tends to damage this spindle and hence the chromosomes themselves. Also, whereas males produce millions of sperm, females may only produce dozens of eggs. Embryos, zygotes, and other products of fertilization are thus preferred for cryogenic preservation. Once eggs are fertilized they loose their spindle, and the chromosomes are less likely to be damaged when they are frozen. The advantage of strength in numbers applies here as well. When the fertilized egg becomes a zygote of 32 cells, if a quarter of those cells are destroyed upon freezing, a healthy individual can still be produced from the surviving cells. And as the zygote progresses, it becomes a hollow ball of cells, a blastocyst. At this stage the cells become permeable to water; the scientists take advantage of this development and dehydrate the cells to avoid damage from ice crystals. “We’ve worked out ways to squeeze water out of the cell,” Leibo says. “We also remove the intercellular water as well.”

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The first attempt at cryogenic species preservation was the work of T.C. Hsu (pronounced shoe) at the University of Texas' M.D. Anderson Hospital and Tumor Institute, now known as the M.D. Anderson Cancer Institute. In 1971, the year after the first Earth Day, Hsu froze in dry ice tissue samples of species he feared were on the brink of extinction. Much of the technology used for cryogenic species preservation had not been developed, but Hsu believed that these samples of 60 to 100 million cells he collected from each endangered species would one day become the genetic “seeds” that could regenerate the species if they were to go extinct.

No, this isn’t Jurassic Park

Hsu may have imagined his efforts as being able to revive extinct species. Scientists now believe that is unrealistic. Instead the materials they preserve are used to help increase the numbers of species that are presently endangered. As in the case of the mountain yellow-legged frog, its dwindling numbers threaten its survival not only because there are fewer individuals, but also because there is less and less genetic variation of that species. In a captive breeding program CRES reintroduces the frozen genes to the population of mountain yellow-legged frogs to help maintain the species’ genetic diversity. In other cases frozen sperm and zygotes can be used to produce young in zoo animals that have difficulty breeding in captivity.

Bernadette Plair, a Research Associate at the Cincinnati Zoo’s Center for Conservation and Research of Endangered Wildlife (CREW), became interested in conservation when the blue and yellow macaw went extinct on her native island of Trinidad. “It left an indelible impression on me when I found out that the Macaw was gone from Trinidad. I remembered the bird from my childhood.” she says. The last bird was seen in 1961.  In 2001 Plair spearheaded a successful reintroduction of the bird to the island. Presently there are 52 macaws on Trinidad. 

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Plair leads a research team that specializes in plant preservation. At a collection site her associates at CREW do not remove a plant from its environment. They only need to snip the terminal buds from branches or shoots. The plants hence have a chance to regenerate. Once collected, they put the buds, which are about one millimeter in size, in a plant growth medium. Also, to keep out contamination from the field, the samples are treated with an antibiotic and a fungicide. To ensure genetic variation, the CREW team tries to collect from as many individuals as possible, and in one case they have 17 different genetic lines preserved for one species. The total inventory of the frozen zoo is about 1500 frozen buds.

Each bud is encapsulated in a gel made of sodium fluoride, resulting in beads of uniform size that look like frogs eggs. Plair then dries the beads by placing them in a medium of calcium chloride for up to a day. She says, “You want the tissue dry but not to be totally without water. The tissue won’t regenerate if all the water is removed. We leave about 20% of the water encapsulated in the beads.” For buds that require more water, she immerses the beads in a solution of glycerol and ethyl glycol that creates a matrix. About 1.8 milliliters of the matrix can hold 10 beads. These are CREW’s general protocols to freeze the terminal buds, but each plant requires a slightly different approach; the scientists at CREW have had to find through trial and error what works best to freeze each species.

In San Diego Ryder places another sample of tissue, this time from a desert horned toad, in a flask. Almost a speck, it’s difficult to see it float in the solution. Ryder says that cryogenic preservation is only a part of the effort to keep our planet a livable place for all species. “This is not an ark,” he says, emphasizing that cryogenic preservation should not give anyone false hope that scientist can save samples of all the endangered species and bring them back someday to a more snake, snail, and ocelot friendly world. The greater effort is in habitat preservation and big picture issues such as global warming. “We are here to save what we can now. Just look at mammals, which is what we’ve worked mostly with. We’ve stored almost 60 species, and it’s estimated that there are 4000 mammal species. We’ve only started. We’ve only scratched the surface.”

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