Test-tube conservation

When the last snow leopard has stalked among the crags, a spark of life will have gone, turning the mountain into stones of silence.

Each day, reality inches closer to naturalist George Schaller’s elegiac warning. Inhabiting the high mountains of Central Asia, snow leopards are the ghosts of the animal kingdom — solitary and secretive. There are an estimated 4,000 to 6,500 snow leopards left in the wild, but the breeding population is estimated at fewer than 2,500 animals. And those estimates may be optimistic — dating from 2003, they do not account for a further nine years of population decline. Threatened by poaching, lack of prey, conflicts with humans and military activity, the snow leopard’s fate remains imperiled.

But there is reason to hope. One patient pregnancy at a time, reproductive biologists are attempting to save endangered species like the snow leopard from extinction. Their tools include technologies developed and refined for human reproduction, like artificial insemination or in vitro fertilization. Recently, new advances in stem cell generation have revived the conversation about assisted reproductive technology (ART) and its role in wildlife conservation.

In November of 2011, researchers at Monash University, Australia, announced that they had generated induced pluripotent stem cells (iPS cells) from a snow leopard. Like embryonic stem cells, iPS cells are self-renewing and can develop into every cell type in the body. But iPS cells are not derived from fetuses — they are created from adult cells that are genetically reprogrammed into stem cells.

Only two months earlier, researchers from the Scripps Research Institute and the San Diego Zoo Institute for Conservation Research created the very first iPS cells from an endangered species, as they reported in Nature Methods. Headed by Jeanne Loring, the research team created iPS cells from the drill, a small primate, and the northern white rhinoceros.

“Obviously you cannot get embryos from endangered species,” says Loring, “so getting the same cell type another way was really very appealing.”

Loring’s research team is now setting out to generate and preserve iPS cells for many other endangered species. Since late 2011, they have generated iPS cells for the Somali wild ass and the Javan banteng, a species of wild cattle from Southeast Asia. Loring hopes that their success will encourage other stem cell researchers to take the next step — producing gametes (eggs and sperm) from iPS cells. This leap already has been successful in mice.

iPS cells are tantalizingly innovative, on par with the emergence of cloning. But the technology is decades away from saving species. “From a science perspective [iPS cells] are really interesting, but from a conservation perspective it has almost no impact whatsoever,” says Bill Swanson, a reproductive biologist at the Cincinnati Zoo.

Even proponents of iPS cells acknowledge that their use in wildlife conservation is in its infancy. “People are very excited in the conservation world about this,” Loring says, “but they’re also not sure what to do next.” In the meantime, something else will have to help the snow leopard.

Other forms of ART are more widely researched and have proven successful in certain species. But each successful pregnancy is backed by years of research and preceded by many failures. Even with a multitude of developing technologies, the ultimate question remains: Will they save species in time?

Assisted reproductive technology includes a diverse array of reproductive aids. The most widely used is cryopreservation, a technology akin to human sperm banks. Genetic material, often sperm, is frozen stored in genetic resource banks. Wildlife biologists refer to these repositories as “frozen zoos.”

Sperm from endangered species is collected from both wild and captive animals and frozen using liquid nitrogen. Before freezing, water is removed from the sperm cells and replaced with a protective agent, often glycerol. This prevents ice crystals from shattering the delicate cell membranes.

Cryopreservation allows precious genetic material from endangered species to be saved for the future — Noah’s ark at 200 degrees Fahrenheit. “Once a semen sample is cryopreserved, it theoretically is good forever,” says Adrienne Crosier, a cheetah research biologist at the Smithsonian’s Center for Species Survival. A majority of the genetic material in frozen zoos is semen, but facilities also save skin cells, eggs, and whole ovaries and testes.

Cryopreservation’s ability to promote genetic diversity is enormous. It allows researchers to “exchange genetics on a really large scale,” says Crosier. “You can collect sperm from a male and transfer the sperm sample, frozen, anywhere in the country,” she says. The ability to transport sperm, instead of the actual animal, makes managing the genetics of a population much easier. Semen collection also enables researchers to use genetic material from the wild, without taking a wild animal into captivity.

Cryopreserved sperm is just the first step, for the ultimate goal of assisted reproduction is pregnancy and offspring. Males and females are paired, often with the advice of the zoological version of genetic counselors to ensure the best match. If natural breeding will not work, due to logistical limitations or animal incompatibility, reproductive biologists have three options: artificial insemination, in vitro fertilization, or embryo transfer.

Each of these technologies has proven successful in some species, but biologists do not simply choose a method and begin. Every successful birth is preceded by extensive research, on both models and the endangered species.

“Oftentimes we need to do basic science on either a domestic animal model or a common family member,” says Alexander Travis, the director for the Cornell Center for Wildlife Conservation in Ithaca, New York. “You don’t want to go and practice on your endangered species.”

Even when researchers know enough about a species’ reproductive biology to apply ART, the success rates are low. At the National Zoo, Crosier says that the success rate for artificial insemination of cheetahs is less than 30 percent.

Such low success rates are not unusual. “Artificial insemination is not some sort of magic bullet,” says Swanson. “If I can do three artificial insemination procedures and get one pregnancy,” he says, “that’s good enough to use to manage the population.”

Crosier hopes that upcoming research on embryo transfer in cheetahs will yield higher success rates, citing the successes of embryo transfer in the cattle industry. Like in vitro fertilization, embryo transfer begins in the lab, but the embryos are implanted into a different female animal, not the biological mother.

Embryo transfer allows reproductive biologists to breed females who are unable to reproduce or who have behavioral problems and will not breed naturally. Their embryos can be brought to term and raised by more suitable mothers, keeping their valuable genetic material in the cheetah population.

This procedure can also help found captive populations of endangered subspecies. Swanson used this technique to establish a population of Brazilian ocelots in the United States. The Brazilian ocelot, Leopardus pardalis mitis, is a subspecies of ocelot found in southern Brazil, Paraguay, and northern Argentina. While ocelots are not endangered, the Brazilian ocelot subspecies is considered vulnerable. Swanson estimates that there are between 1,000 and 5,000 individuals left in the wild.

In 1999 and 2000, Swanson traveled to Brazil and used in vitro fertilization to create 86 ocelot embryos. The intention was to transport the frozen embryos to the US and transfer them into captive ocelots. Artificial insemination was not an option, because captive ocelots are generic mixtures of many subspecies. Therefore the resulting kittens would not be true Brazilian ocelots.

After years of trying to obtain export permits, Swanson performed the embryo transfers in Brazil in 2007. Twenty-four embryos were transferred into eight ocelots, resulting in three pregnancies and three healthy kittens. Swanson later transported some of these founder kittens back to the US.

There are now 28 Brazilian ocelots in US zoos, and Swanson estimates that 50 percent are either ocelots that he imported or are offspring produced by his founders through natural breeding or artificial insemination.

The continued improvement of all forms of ART will allow biologists to tailor the technology to the animals in their care. The choice of which technology to use “comes down to the particular animal, species and situation that you are dealing with,” says Swanson.

Though each technology still needs extensive development, embryo transfer, in vitro fertilization and artificial insemination are having concrete effects on current species conservation. In contrast, the value of iPS cells and their sister technology, cloning, remains unknown.

In response article to Loring’s paper, reproductive biologists David E. Wildt, Vimal Selvaraj and Budhan S. Pukazhenthi state that though the tantalizing possibility of embryo creation or cloning from iPS cells exists, “there will need to be decades of basic research investment before it is possible to reap the dividends of viable offspring production.”

ART is having a real-time, albeit limited, effect on managing captive wildlife populations in the United States. But its role in future conservation efforts depends on continued technological advances. These advances depend, unsurprisingly, on funding.

ART’s potential will also be constrained by the fate of wild populations. Captive breeding programs are fruitless unless they are combined with legal protection and habitation conservation to preserve animals in the wild.

“Wild populations are becoming more fragmented and more restricted,” says Swanson. As a result “it’s becoming more and more difficult to… develop these global connections between wild and captive populations.”

Loring’s iPS cell version of a frozen zoo will lay the groundwork for any future advances in technology. Her research team’s work could very well have a significant effect on the livelihood of those species in 50 years.  But in the intermediate decades, many animals could become endangered or go extinct without continued improvements in technologies that help produce offspring.

Loring’s team chose the northern white rhinoceros because the species was virtually extinct. There were only eight northern white rhinos left on earth when they started their research. By the time they finished, there were only seven.

It is too late for the northern white rhino. The snow leopard population is plummeting, and the Brazilian ocelot is creeping toward endangerment. In the end, assisted reproductive technology falls prey to the same maxim as all other conservation strategies: For imperiled species, it is a race against time.