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The Deep Freeze

by Dan Stone

Each time SECORE scientists have traveled to Rincon, a team of cryobiologists led by Mary Hagedorn has accompanied the group to study and freeze elkhorn sperm. Hagedorn is pioneering the cryopreservation—the freezing, storing, and thawing—of coral sperm and is considered the world’s foremost authority on aquatic cryopreservation. She heads up the only team doing this kind of research.

While in Puerto Rico, Hagedorn and her team worked with the coral’s reproductive material for six days—testing, freezing, and gathering thousands of samples. Once her team froze the sperm, they shipped it in containers that were negative 196 degrees Celsius—the temperature of liquid nitrogen—to genetic banks at Omaha’s Henry Doorly Zoo in Nebraska, the National Animal Germplasm Repository in Fort Collins, Colorado, and the Centre for Genetic Resources in Wageningen, Netherlands. The National Zoo has cryopreservation tanks that store frozen sperm samples from animals such as giant pandas and cheetahs.
Hagedorn hopes, with some expanded resources, to store coral samples at the Zoo in the next few years.

To guard against coral’s extinction, Hagedorn and her partners are creating the first genetic bank for coral reefs. This bank will hold frozen samples of coral sperm, embryos, stem cells, and DNA while habitats in the Caribbean are restored. Reproductive material could remain frozen but alive for hundreds of years in liquid nitrogen, and thawed samples could be used to seed shrinking coral populations.

The genetic bank initiative is part of the National Zoo’s Coral Conservation Program, a project designed and run by Hagedorn to prevent the extinction of coral and restore the species back to nature as needed.

Shenandoah Salamander

National Zoo's Mary Hagedorn (left) and Virginia Carter prepare coral for spawning on Coconut Island, Oahu. (Jim Danials/Jim Daniels Photography)

Until last year, no functioning genetic bank for coral existed. “Thanks to the efforts of Hagedorn’s group, the technology is now available to continue developing a bank for the world’s most endangered coral species,” says invertebrate keeper Mike Henley. “They have achieved important milestones over the past four years in coral physiology and cryopreservation—and they’re the only group that has successfully preserved coral sperm and produced viable coral larvae with it.”

The Zoo’s Coral Conservation Program is based at the Hawaii Institute of Marine Biology (HIMB) on Coconut Island, Hawaii— the same place “Gilligan’s Island” was filmed. Hagedorn was assigned this station for a number of reasons. First, most coral around the world (including elkhorn coral) spawn only once a year, but Hawaiian coral spawn many times a year—allowing Hagedorn’s team to collect data quickly. Second, HIMB has sophisticated equipment for understanding the coral’s complex processes. And third, the area has a variety of coral that are healthy and easy to collect—many are just offshore.

When freezing the coral’s reproductive material, Hagedorn uses the same techniques that are used in human fertility clinics. “No one thought we could do this,” she says. “But we use the same microscopes, equipment, procedures, and freezing material as human labs.”

To successfully freeze a cell, Hagedorn will remove some of its cellular water and add an antifreeze-like substance. This helps to prevent damaging crystals from forming. If you’ve ever seen freezer burn on ice cream, the harmful crystals look the same. Throughout the process, Hagedorn has to be patient. If sperm cells are frozen too quickly, the water will not have time to exit and ice crystals will puncture the membrane and kill the cells. On the other hand, if they are frozen too slowly, the cells will be damaged by dehydration.

For the past five years, Hagedorn has perfected her cryopreservation technique by partnering with a number of prestigious organizations, including Omaha’s Henry Doorly Zoo; Chicago’s John G. Shedd Aquarium; Rotterdam Zoo; the University of Houston’s Department of Biology and Biochemistry; Columbus Zoo and Aquarium; the University of California’s Department of Anatomy, Physiology, and Cell Biology; and Louisiana State University’s Aquaculture Research Station.

So far, Hagedorn has had no problem freezing coral sperm. But eggs and embryos have been more of a challenge. Embryos are particularly important because they contain both male and female DNA. At present, Hagedorn has not been successful at freezing these materials because they fall apart when chilled. With her partners, she is experimenting with extremely low temperatures that will likely prevent the formation of damaging crystals.

How do living cells survive such extreme temperatures? “When we freeze cells their metabolism stops and they live in suspended animation,” says Hagedorn. Since the 1950s, scientists have been cryopreserving animal cells. Many animals’ reproductive material has been frozen, including such marine life such as oysters, fish, and clams. But Hagedorn is the first to work with coral.