Browne, RK., J. Seratt, CK. Vance, and A. Kouba. 2004

Hormonal induction of spawning and spermiation in the Wyoming toad (Bufo baxteri) with in vitro fertilization. In prep.

A major program is to increase the efficiency and adaptability of captive breeding protocols for the endangered Wyoming toad (Bufo baxteri). The gregarious adult toads are housed in groups of up to five in modified aquariums. Their sponge matting substrate is washed every second day, and the toads love sunbasking on cork bark. They are ravenous feeders and consume large numbers of crickets of all sizes. They also love mealworms and waxworms that are fed sometimes as a treat.

Here a large female Wyoming toad is admired by two of the smaller males as she sunbasks on her bark. This female shows signs of oedema (excessive build up of fluid) such as the pouch beneath her throat. Present studies are addressing this common problem in Wyoming toads.

These were the first toads (or frogs) bred for release using in-vitro fertilization. In-vitro (in glass) fertilization needs the eggs and sperm to be sampled without the toads mating. We used hormones on both the females, in the larger boxes at the back, and on males in the boxes on the right hand side to encourage the production of eggs and sperm. We then mixed the sperm from different males with the eggs of each male and produced lots of genetic variation. This resulted in lots of samples of eggs in dishes to be checked for fertilization by sperm showed by the eggs dividing as the baby tadpoles grew, and for viability (the ability to live till hatch).

The eggs are examined under a microscope at 6-8 hours after fertilisation when they are at the 8-32 cell stage to check fertilisation rates. Only about 15% of the eggs are usually fertilized which is typical for Wyoming toads. We are now working on how to get more eggs to fertilise and grow. The eggs took about three days to hatch.

After checking the eggs for fertilization, obviously bad eggs were removed and the rest were left until neurulation (the peanut shaped embryo). A close watch must be kept for bacterial blooms from metabolites from decaying eggs as the viable eggs develop. Bacterial blooms are show as a milkiness in the water. Decaying eggs must be removed sequentially until neurulation when all eggs that fail to reach neurulation must be removed.

Once the larvae reach neurulation they were separated from the last unviable eggs, the surviving larvae were kept in Petri dishes or shallow trays with only 1.5cm (~1/2") of water. The water was changed daily and they were fed small amounts of spiralling. Regular inspection of the trays was necessary to remove any larvae that did not develop and died.

Because of artificially induced spawning, some eggs were smaller than normal and a lot of care was necessary to foster the compromised larvae. In this dish there are many small or deformed larvae some of which died before swim-up.

The floor of the trays when covered with spirulina were covered with tracks where the growing larvae had fed. The mechanism enabling this locomotion is unknown to me. I know of no information relating to microtubules or other structures on the surface of the skin that could cause the gliding motion.

The tadpoles were kept in tubs with a bubbler and water weed, and fed spirulina (a health food), along with plants and their roots. At different stages the tadpoles preferred different types of food.

Most of the tadpoles were shipped to Wyoming to be released in the wild at Lindzey's Lake, Wyoming. The Memphis Zoo retained some tadpoles to increase their captive breeding population. When the tadpoles had grown their front legs we put them in a tub with a slope and shallow water to enable them to get out of the water as they became little toads (metamorphosis). They took about three weeks from hatch to metamorphosis.

The fruit flies were put in a container that let them crawl out of the holes. This meant that there were not to many in the tub at one time and that all the juveniles had a chance at feeding.

After they had begun growing the metamorphs were first kept on moist paper towel which was changed daily. The reason for this is that there is always a compromise between juvenile toad density and feed density. The best way seems to use high densities of both with very frequent cleaning.

The toads quickly grew to over half an inch long in a few weeks and were well on their way to becoming adults.

Mortenson Lake: Picture taken by Michelle Geraud.

"After the capture of the last surviving Wyoming toads in 1998 for captive breeding programs tadpoles have been released yearly at their last known site at Mortenson Lake near Laramie, Wyoming. In 2004 a substantial number of toads were found to have bred at Mortenson Lake. Consequently, these 1900 tadpoles of 2004 were released at a new site, Lindzey's Lake, along with 8000 others. It will be very interesting to see if they establish and if they come down to breed in the spring of 2005." - Michelle Geraud

Picture taken by Michelle Geraud.

"Just back in from the field and thought I would pass along some great news! I found four Wyoming toad egg masses today on the far Northwest side of Mortenson Lake. Two appear to be recently laid, one was elongating, and the forth was at the "twitching" stage." - Michelle Geraud.