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Terri Roth: saving endangered species through cryogenics.

Conservationists may have to measure their success in hundreds of years, but their vision for it begins in a single moment.

For Dr. Terri Roth, director of a cryogenic station located within the grounds of the Cincinnati Zoo and Botanical Garden, that moment came when she discovered that Sumatran rhinos ovulate only after mating. In response to a hormonal change that occurs within the reproductive tract of the rhino, its ovary releases one or more eggs available for fertilization.

When Roth and her associates at the Center for Conservation and Research of Endangered Wildlife (CREW) made this discovery, they began a new era in the use of science as a tool to preserve a species that dwells on the edge of extinction.

Using ultrasound, blood work, and hormonal therapy, CREW has been responsible for the viable delivery of two Sumatran rhinos since 2001. With fewer than three hundred Sumatrans remaining in the wilds of Indonesia and Malaysia, Roth set out on a mission in 1996 to assist in the collection of their reproductive materials for cryogenic storage, as well to offer services to an existing captive-breeding program in the Cincinnati area.

Procuring a grant from the International Rhino Foundation, Roth began an investigation to determine when Sumatran rhinos ovulate, thus indicating when they would be most receptive to male advances. Since Sumatrans are loners by nature, they can be aggressive and hostile when paired for mating.

Selecting an appropriate breeding time is crucial. For eight months, she studied blood samples and ovarian ultrasounds before determining that the rhino (Emi) showed no signs of estrous or receptivity. She was frustrated, but not beaten.

Roth, a recipient of the 2004 ChevronTexaco Conservation Award, began her stint at the Cincinnati station in 1996 after leaving her post as a gamete biologist at the National Zoo's Conservation and Research Center, an associate organization of the Smithsonian Institution in Washington, D.C. Roth, who grew up in Pleasanton, California, recalled her childhood stuffed-animal collection, her involvement with the 4-H Club, and a fascination with animal reproduction as precursors for a career set to the rhythm of nature's cycle. As a young college student, she considered a major in veterinary medicine or genetics, but felt bogged down with too many formulas and not enough fur.

After earning a master's degree in animal science from the University of California, Roth obtained a doctorate in animal reproductive physiology before attending the Smithsonian for postdoctoral training, where she gravitated toward the study of hoof-stock animals, and charted a course for rhino conservation.

The ChevronTexaco board awarded Roth $10,000 for her commitment to the management and protection of natural resources. She donated the money to support rhino-protection units in state and national forests in Malaysia and Indonesia that support anti-poaching programs. The units, funded largely by global collaborators, including a division of the United Nations, look for snares, arrest offenders, and offer educational opportunities to neighboring communities.

Protection programs are in place for the five remaining species of rhinos on the planet. The Indonesian government protects the Javan Rhino in a local reserve dedicated to propagation; while Species Survival Plans cover the African black, the African white, the Indian, and Sumatran rhinos, which are hunted for their horns. Roth's forte, the Sumatrans, are represented by fifteen individuals in captivity to date--two of which, a mother and its calf, live in Cincinnati.

Since her acquisition, Emi has given birth to two calves after five miscarriages. The first calf, born in the shadow of the 2001 terrorist attacks, was the product of a pregnancy aided by the use of progesterone-soaked bread. Regarding hormonal intervention as an unnatural process, Roth hoped for Emi to conceive again and to maintain the pregnancy without the use of medication--a feat the team accomplished in July 2004. These two rhino deliveries were the first successful captive-breeding births in more than a hundred years.

Following the footprints

Calibrating a balance between natural selection and scientific intervention requires conservationists to examine the human impact on animal populations.

Because destruction of habitats is a main cause of extinction, researchers are forced to create captive-breeding grounds, as well as use technology, such as in vitro fertilization, embryonic transfers, and cryogenics in order to preserve reproductive material, including components that allow for genetic diversity within species.

CREW has little hope of releasing their successes into the wild, but maintains an interest in sharing their information acquired through research and observation with global wildlife reserves. One such example includes the use of rhino footprints, which Roth keeps behind her desk as artwork, to determine the age of wild calves, unseen. When compared with footprints in the forests of Asia and Africa, these data allow researchers to track individual calves, as well as keep tabs on their numbers, which serve as indicators of poaching activity.

"It's all about the science," Roth said.

Indeed, her lab, which is often referred to as a genome research bank, has numerous liquid-nitrogen tanks containing gametes from more than sixty animals and one hundred fifty plant species from around the world, including sperm from the Indian rhino. After breeding Emi, researchers collected sperm from her mate's semen as it drained from her, and then froze it.

Anesthetizing large endangered animals for sperm extraction is risky, although a time may come when it's necessary. The use of electrodes to induce ejaculation is another option.

Other frozen samples include embryos, sperm, and unfertilized eggs, as well as shoot tips, seeds, and spores that will allow for ex situ propagation. When a black rhino died unexpectedly a few years ago, CREW was able to remove and freeze its sperm thirty hours after its death. In a test thaw, half of the sperm remained motile.

Thawing sperm is relatively simple, according to Roth. She removes the straw from the tank (-196 degrees Centigrade) for ten seconds before plunging it into a 37 degree Centigrade bath. In contrast, techniques for embryos vary from one species to another and generally do better than unfertilized eggs, which the freezing process often damages. Since the egg is a single cell, ice crystals easily can break its membrane.

CREW, one of ten "frozen zoos" in the U.S., also lays claim to the successful reproduction of an ocelot, a member of the cat family, using a frozen-thawed embryo in 2000. The Cincinnati Zoo houses the only program that is focused on the conservation and research of five often-ignored cat species.

Under the supervision of Dr. William Swanson (Roth's husband) for two years, the team collected unfertilized eggs and produced ocelot embryos using in vitro techniques, then stored them in their freezers. Using laparoscopy, they transferred three embryos into a female, which gave birth to one kitten eighty-three days later.

In addition to the ocelot and sand, fishing, and Pallas cats, CREW feline projects include the expansion of its research to study the reproductive biology, as it pertains to the feasibility of artificial techniques, of the elusive black-footed cat found only in Botswana, Namibia, and South Africa. So far, scientists, in collaboration with the San Diego Wild Animal Park and the McGregor Museum in South America, have obtained blood samples and fifteen semen straws for their freezers without permanently removing the donor from its habitat. CREW has established conservation efforts with each of the five cat species in their indigenous lands in Thailand, Mongolia, South Africa, and Brazil.

In the tank

Cryogenics as a tool of conservation is helpful because it allows for scientists to save reproductive materials from a species without the expense and danger of animal transportation. Equally as important to the long-term survival of a species is the procurement of the diversity within it.

"It's a way to move genetics around without moving the animals," said Roth, who just attended a meeting at Emi's barn to discuss an expansion for her offspring. Producing diversity is an issue CREW struggles with while breeding Emi, because she has only one mate. Since cryopreservation is lagging behind when it comes to rhinos, artificial insemination or embryo transfer is not yet an option.

Plans for a third calf are already under way, but Ipuh and Emi will mate naturally.

Aside from rhino and cat reproduction, CREW supports a study of the red howling monkey in Trinidad in addition to numerous other investigations involving the prenatal gender identification of Sumatrans, and a study on the immune system of black rhinos, which characteristically suffer from depressed biological defenses.

Under its greenhouses, CREW, which was established in 1981, also has one of the world's largest repositories of endangered plant samples. Founded in 1988, the plant division initiated the Endangered Species Propagation Program by beginning with rare Trillium specimens.

Since that time, CREW's plant contingent has been involved in the assisted proliferation of plants from around the world. Under the auspices of the Ohio Seed Bank, Wilson Popenow-Award recipient Dr. Valerie Pence and her associates targeted seeds acquired from the 10 percent of Ohio's remaining wetlands and learned that they are adaptable to dry conditions and are suitable for long-term storage in the tanks.

Although they can obtain many rare plant specimens from simple seed collection or by cuttings, researchers must turn toward alternative methods for some species, such as the use of nutrient mediums in test tubes. CREW froze tissue samples from each culture line for future use.

Once they reproduce the plants, they return its products to its collaborators for added research, display, or for reintroduction into the wilderness.

A moment in time

Figuring out Emi's time of estrous was Roth's most daunting challenge, but one that eventually would provide her with international attention. Noticing no behavioral changes or receptivity toward Ipuh, Roth's team depended upon science to determine an appropriate introduction. But with no signs of ovulation showing up in the blood work or on an ultrasound, Roth began to wonder if Emi's ovaries were defective.

One day, Emi responded positively to the advances of Ipuh. Within forty-eight hours, an ultrasound revealed breakdown on the cellular level of the ovarian follicle, which allowed for the escape of an egg.

"Wow! I'll bet she [and her species] is an induced ovulator," said Roth, who also serves as vice president of animal science at the Cincinnati location.

Black and white rhinos that travel in herds, which will increase the odds of conception, ovulate every twenty-five days and forty-five days respectively; the Sumatrans, which are solitary animals, ovulate only after mating. Ovulation-upon-demand might be beneficial in nature, because it conserves a limited supply of eggs.

Emi's pregnancy lasted forty-two days before her first miscarriage. She became pregnant four more times before Roth prescribed progesterone to maintain a full-term gestation, which typically runs between fourteen to sixteen months. After a successful medication-free delivery of a calf in 2004, Roth began planning for yet another rhino delivery.

Determining whether or not conservationists are making progress requires more than a scientific notion. They may look at the numbers of births, the knowledge gained, or the duplicability of accomplishments to measure success, but the real value of their work might go unnoticed for decades or even centuries. While the reintroduction of the Sumatran calves, still unborn, into the wild is not unachievable, Roth questions their safety. Aside from poaching, habitat destruction is unabated.

"People are struggling to survive. There has to be a mutual benefit," she said.

Deforestation in the Philippians has ruined the watershed, for example. Once they've lost the forest, the water runs off and the cycle of destruction, human and otherwise, continues.

Roth looks to her immediate accomplishments as a means for appraising a vision that in practicality exceeds countless lifetimes. She sees progress in conservation as a continuum throughout the years, beginning with a change of human heart. Then, as if she understood the calf footprints near her desk to be an icon for progress, she added, "One step at a time."

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Kym Kuenning is a freelance science writer.
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Title Annotation:NATURAL SCIENCE
Author:Kuenning, Kym
Publication:World and I
Date:May 1, 2005
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