Smithsonian National Zoological Park l Friends of the National Zoo



Technical Details

by Caroline Treadway

Freezing sperm and eggs is a lot harder than it sounds. Why? They’re filled with water. And when water freezes, it expands, bursting fragile cell membranes. To avoid this, Zoo scientists use glycerol—an antifreeze similar to what you use in your car—as a cryoprotectant. It prevents ice crystal formation and minimizes cellular frost damage. When added to a sample, glycerol floods the cells and drives the water out. The glycerol-filled reproductive cells can then be safely frozen without damage.

“A grape is like a sperm head,” said JoGayle Howard, theriogenologist (animal reproduction specialist) at the National Zoo. “If you freeze a grape, the skin’s going to bust when it expands during freezing. But if you shrink it down like a raisin and then freeze it, you’re not going to have all that cryodamage. That’s exactly what we’re doing: sucking all that water out of the grape, making it a raisin, then freezing it.”

Before adding the cryoprotectant, however, scientists add a mixture of nutrient-rich “extenders” and antibiotics to fight contamination. Extenders are typically made from egg yolk or milk. They provide energy and protection for cryopreserved cells. Zoo scientists borrowed this idea from the commercial cattle-breeding industry. Scientists then cool the sperm or eggs to 4° Celsius.

Once the sample is cooled, scientists add the glycerol, which is toxic at room temperature. They then steadily cool the entire mixture to minus 80°C before freezing it in liquid nitrogen at minus 198°C. If kept at this constant temperature, cryopreserved material can be stored indefinitely, since such cold temperatures suspend all cellular activity. Upon thawing, the cryoprotectant is removed and replaced with water in the cell. After years of research, Zoo scientists have determined this method works best for cryopreserving wild animals’ sperm.

Scientists have another way to cryopreserve the genomes of endangered animals—vitrification. Faster than slow cooling, vitrification works better for freezing eggs and embryos. To vitrify a sample, scientists add large amounts of cryoprotectant to the cells and freeze them directly in liquid nitrogen. This cools the sample rapidly and creates a stable, glass-like state around the cell.

Smithsonian Zoogoer intern CAROLINE TREADWAY is a graduate journalism student at Boston University.


More: "Frozen Assets"

More: "Collecting Sperm Samples"


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Smithsonian Zoogoer 39(1) 2010. Copyright 2010 Friends of the National Zoo. All rights reserved.