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Frozen Sperm Could Someday Save Salmon by Mike O'Bryant Columbia Basin Bulletin - January 12, 2001

Freezing endangered salmon sperm could be the most unique effort in the Northwest to help save wild runs of salmon. However, researchers hope the sperm will never have to be used to resuscitate any of the endangered salmon stocks of the lower Snake River basin since its use would occur only after all else fails and one of the stocks nears extinction.

Biologists have collected sperm from Snake River chinook salmon and steelhead every year since 1992 and added it to sperm banks at the University of Idaho and Washington State University where the specimen's are being cryopreserved. The effort is only in case recovery efforts fail for the two stocks listed as endangered under the federal Endangered Species Act.

"We were concerned enough about the possibility of specific stocks disappearing, that we began to collect sperm," said Joe Cloud, professor of zoology at the University of Idaho. "We look at this as a genetic insurance policy, but hopefully we'll never have to use it."

Cloud and another scientist at Washington State University, along with Nez Perce biologists, initially collected sperm samples with volunteer labor and money, but today the effort is funded through the Bonneville Power Administration's fish budget at about $160,000 per year. That grant is administered through the Nez Perce tribes.

Cloud said samples of both wild and hatchery fish are collected and carefully brought to university laboratories for freezing and preservation.

"At this point, we're trying to save the genetic background of the stocks, hoping that all the genetic material will be available in both," Cloud said of the practice of collecting sperm from both hatchery and wild fish. He's not sure the assumption is completely accurate, but he and others are looking at it as an insurance policy against extinction.

So far the sperm banks at the two universities have preserved sperm from 1,286 individual chinook salmon and 540 steelhead. Samples come from lower Snake River fish and include those from the Clearwater and Salmon rivers in Idaho, and the Grande Ronde and Imnaha rivers in Oregon. Although he's not sure because it hasn't been tested, Cloud believes frozen sperm should last more than 200 years.

Biologists collect samples every spring and fall from fish that have already spawned, milking every last bit of left over sperm from the fish. The samples are kept separate, marked as to type and date, and flown immediately to the universities where they must be frozen within 24 hours.

"When we collect the semen or milt, it must remain in such a state that the cells are not activated," Cloud said. "Few people realize that sperm from salmon are not immediately motile, like mammals. Once motility is induced, a salmon sperm only lasts about a half a minute and after that it's not fertile."

Samples are kept at both universities as a redundancy measure. If something happens to sperm in one laboratory from a fire or other disaster, it will still be preserved at the other. Electricity is not required to keep the specimens frozen, only a consistent fix of liquid nitrogen, which can be added even without electricity.

In the laboratory, researchers add a cryoprotectant to keep the water inside the cell from freezing, along with a freezing solution of DMSO, a substance that goes through the cell membrane very quickly. The samples are labeled and sealed in semen straws -- long straws like a swizzle stick -- and placed in goblets, two to a tray, and frozen in liquid nitrogen to minus 196 degrees centigrade. The reverse process is similar.

"Just as we have to freeze the sperm at a certain rate, we also thaw it at a specific rate to maximize its fertility," Cloud said. "It's like baking a cake. All you have to do is follow a recipe."

Still, the process results in a loss in fertility of about half the sperm due to the severe stress it has endured, which Cloud said is the cost of cryopreservation.

Salmon eggs or embryos cannot survive the cryopreservation process because of the egg's yolk, but the process of building a new salmon from frozen sperm still uses eggs from a female, he said. "We can generate an animal from a just the sperm," Cloud said.

The first step is to remove the nuclear DNA from an egg of a closely-related stock. The easiest way to do that is to irradiate it with gamma radiation, something that breaks up the egg's DNA. The egg is sterilized and the yolk is removed. It is fertilized with sperm, which makes a haploid zygote, which is an egg with just one chromosomal set, according to Cloud.

But they need to have an egg with two chromosomal sets, or a diploid zygote, so researchers then inhibit the division of the egg and force the two eggs back together, now providing the egg with its second chromosomal set. Cloud said this is a process that can be done with salmon, but cannot be duplicated in humans or any mammals.

However, the zygote is androgenetic, meaning that it is based wholly on the male and this worries Cloud.

"Is that important and should we be trying to preserve eggs?" Cloud asked. "That's something we think and worry about in fisheries science."

He hypothesized that one way to circumvent the problems encountered when freezing eggs is to freeze a cell that will become an egg, since the problem of freezing has only to do with the yolk. Freezing the cell or an organ of a female would be like freezing the salmon's female genome.

Funding from BPA is an annual exercise and Cloud doesn't know what the future outlook is for his project. However, he has approached officials at the U.S. Department of Agriculture's animal germ plasm bank in Colorado about storing salmon sperm. The plasm bank is now storing sperm from domestic livestock and they agree that ESA listed species are an important resource, according to Cloud. While they haven't agreed yet, it is still a possibility, he said.

Link Information:
University of Idaho: www.uidaho.edu