Simulated hibernation aids MSU toad work

MISSISSIPPI STATE ­ Mississippi State University researchers successfully
promoted egg laying in threatened Boreal toads when they moved the
amphibians out of the refrigerator and into the wine chiller.

Mississippi Agricultural and Forestry Experiment Station researchers at MSU
are working with a group of 52 threatened Boreal toads native to the
Colorado Rockies. The toads are housed in a special lab in the MSU
Department of Biochemistry, Molecular Biology, Entomology and Plant

Natalie Calatayud, a postdoctoral fellow, and Cecilia Langhorne, a graduate
student pursuing her doctorate in animal physiology, manage the lab and care
for the toads. Their work is part of a long-standing partnership with the
Memphis Zoo.

³Our goal is to increase the numbers of the wild populations,² Calatayud
said. ³We are establishing reproduction protocols to promote breeding in
captivity and produce animals for reintroduction to their natural habitat.²

In their native habitat, Boreal toads hibernate for six months before
mating. When the MSU project began, research focused on using hormones to
get the toads to breed without hibernation, but that approach was

³When that didn¹t work, we decided to hibernate the females,² Calatayud

To encourage hibernation, they use a refrigerator, a wine chiller and a
water cooler.

Four females are housed in plastic, lidded boxes and placed in the
refrigerator. The boxes have a layer of moist soil for the toads to sit on,
and temperatures are kept at a constant 39.2 degrees.

This lower temperature is used to simulate conditions found in the wild,
where Boreal toads spend the cold winters underground or in beaver dams.

³The females use their fat reserves to develop eggs while in hibernation,²
Langhorne said. ³They actually gain weight in the refrigerator as their
bodies hold moisture from the damp environment, and eggs develop.²

As a test, four females were kept in hibernation for one month in the
refrigerator, four others for three months and a last set of four for six
months. Toads in the six-month treatment came out of hibernation July 14.

³We have had success with females laying eggs a week later,² Calatayud said.

These studies determined the need for hibernation to get the animals to
reproduce successfully.

³We had a 50 percent success rate for both the one-month and three-month
hibernations,² Calatayud said. ³That is a great success because no female
had ever laid eggs in the two years before this test.²

Taking a toad from hibernation in a refrigerator is a two-step process.
Researchers first placed the female toads in a wine chiller with slightly
higher temperatures of 46.4-50 degrees. After a week in the wine chiller,
the toads were moved back into their regular laboratory habitat with 46- to
50-degree water supplied to their ponds from a water cooler.

³Temperature, humidity and altitude may be very important factors hindering
our efforts,² Calatayud said, but the researchers are learning how to work
around these challenges.

After collecting data on the initial hibernation study, the researchers will
adjust hormone treatment, hibernation length and other factors to help
optimize egg production. They are also working on timing the production of
eggs and sperm to create ideal conditions for in vitro fertilization.

Scott Willard, head of the Department of Biochemistry, Molecular Biology,
Entomology and Plant Pathology, said the MSU study is funded through the
Memphis Zoo as part of a larger Institute for Museum and Library Services

³This work is important because amphibians are sentinel species,² Willard
said. ³They are indicators of ecosystem health, which is important to all of
us. While this project is centered on the captive propagation of endangered
amphibians and working out the kinks of how to do this, there are larger
implications to the work in an ecological context regionally and