Olaf grasps Diane Barber’s gloved hands together with his sticky, 4 fingered legs. His skin is bumpy & moist, the color of pebbles at bottom of a river when dappled sun hits them. Olaf eyes are deep amber. His body lifts & falls with each breath. “The males get really pretty,” says Diane Barber, ectotherms curator at Fort Worth Zoo in Texas. “Sometimes they will turn a solid yellow when they’re in breeding form.”
In some ways, this toad shouldn’t exist. He’s the progeny of an egg from a captive mother & sperm from a wild father, simply, a hybrid from parents who were both dead. Olaf isn’t the first amphibian to be born via IVF that has been happening for years, but he’s the first of his species to be born from sperm that was frozen & thawed.
“We were able to recover a genetic lineage that had disappeared, so we were able to produce an offspring from dead-parents,” says Andy Kouba, an ecologist at Mississippi State University, who assisted with the project. “So that was an exciting first, to re-introduce genetic lines back to the population.”
Scientists have tons of tools to conserve species, says Kouba, but they still got-to hedge against extinction in the wild. First way could be to bank the genetic lineages of species by freezing sperm & egg deposits then later thawing & mixing them during a dish to make offspring.
Amphibians are at the forefront of an uphill-battle against extinction, losing a greater proportion of species than the other vertebrate group. The IUCN estimates that a minimum of 41% of amphibians are at imminent risk of extinction. Habitat loss, climate crisis & a fungal disease, all play a role in their demise.
But amphibians aren’t the sole species which get benefit new technologies. In vitro fertilization, hormone therapy & cryopreservation are increasingly getting used as tools for conservation for amphibians & beyond. In a fast-changing world, frozen zoos could also be the places where tissues are kept in the hope of boosting numbers or resurrecting species in the future.
Frozen zoos
How did Olaf come to exist at all? A year ago, in Puerto Rico, researchers captured 6 male toads, injected them with hormones then collected the sperm they ejected once they urinated. The toads usually pee when picked-up by humans, but the researchers also barked at them, Kouba explains, as toads urinate when frightened, and dogs barking are a sure-fire thanks to scare them. The males were then released back to the wild.
The team of international scientists preserved the semen in nitrogen & transported it to Fort Worth Zoo, where female toads were injected with hormones to release eggs. Olaf & his roommates were created during a dish, eliciting cheers from the scientists once they hatched from their eggs.
The idea-of making a frozen zoo first emerged in the 1970s, when a medical pathologist named Kurt Benirschke started banking animal sperm & eggs at San Diego zoo in the same way that human gametes were starting to be stored: in giant vials of nitrogen that dropped the temperature of the materials to -196 Fo. When Benisrschke started banking genetic material, no technology existed to form use of it, but he believed it had been important to carry on anyway.
Now, biobanks for animal species, from fish to reptiles to birds & even snails & molluscs are shooting up around the globe. The Frozen Zoo repository at San Diego zoo holds more than 10000 cell cultures from nearly 1000 different species. They’re mostly from mammals, but also from birds, reptiles & amphibians.
Scientists had been performing on the method of bringing together sperm & egg outside the bodies of animals like rabbits & guinea pigs since the 1870s. But making babies in a test tube is not a simple task because every species is different. IVF success rates for mammals are much higher than with reptiles & with some species, it’s incredibly low. Even within species, some cells are harder than others. When it involves amphibians, Kouba says, their sperm is pretty simple to freeze, but eggs aren’t, because they’re such a lot larger than the sperm. “Inside that cell, you’ve water content and once you start to freeze anything with water, they form ice crystals that rupture the cell.”
Because fish like salmon & cod are widely available commercially, they have been studied far more than amphibians and amphibian researchers like Barber & Kouba are expecting breakthroughs that they can adopt. “There is a huge interest in freezing sperm to move genetics around the world, for commercially viable species,” Kouba says.
One reason that conservationists want to increase the numbers of amphibian populations is because diseases are devastating them in the wild. Kouba says cryopreservation features a place as a last-ditch effort to bank the remaining genetic material from a dying species, but that it’s really better to take action before a species numbers have crashed. “We can produce tens of thousands for re-introduction, which can allow a species-to return to a healthy population,” he says.
That’s because amphibians have a naturally high mutation rate, so sending thousands of amphibians into the wild to mate & repopulate could mean they create a disease-resistant strain on their own. “You release animals with the hope that disease resistant lines will develop, and people animals will reproduce,” says Kouba.
Healthy herds
In Colorado, Jennifer Barfield is using IVF to make bison with healthy genes to introduce-into existing wild herds. Left to their own devices, bison breed well but what they can’t do is move around the globe, so to transplant new genes to different areas requires the utilization of IVF.
Using assisted reproductive technologies, she produced healthy Yellowstone bison from ones that had a bacterial disease called brucellosis and established a new conservation herd in Colorado called the Laramie Foothills Bison Conservation Herd.
There is another-advantage to using IVF in bison says Barfield. “Bio-banking the genetic preservation is simply a good idea, like a sort of genetic insurance. You never know how science will advance-to use that material in the future.”
She says that one among the challenges in endangered species is that researchers don’t know enough about the reproductive physiology of rare animals to use solutions before the animals become extinct. “You can’t experiment on species,” says Barfield.
In some species, assisted reproduction is the last-hope. There are only 2 remaining northern white rhinos left in the world, both female, both past reproductive age. Last year, researchers were able to harvest eggs from one among the females and have created 3 embryos with frozen sperm from dead males. The embryos are now frozen, with the hope of implanting them into surrogate southern white rhino females later this year. The eventual goal is to make 5 animals that would live in the wild in Africa, an idea that would take decades to come to fruition.
Back at Fort Worth Zoo, Barber carefully puts Olaf back in his cage the isolation room. In the wild, Puerto Rican crested toads like Olaf spend 95% of their time in small holes between crushed rocks, coming-out to feed at night & when it rains. In his home at the zoo, the staff have constructed small pyramids made up of sections of white PVC pipes for Olaf & the other toads to hang-out in. Of the toads that used to live in this room, 193 were recently shipped via FedEx back to Puerto Rico for re-introduction; now more are being raised here. Other rooms house frogs raised from other parts of North America, beat need of a population boost.
Barber says, in the 1980s, zoos thought of themselves as arks. “That was sort of the buzz word for zoos: we’re becoming arks rather than just having animals for exhibit. We’d like to start doing more about thinking about conservation & maintaining species future.”
But those arks are full & more species need assistance, there’s no space to keep animals even as an insurance policy. “We’re not arks, we’re life rafts,” Barber says. “And we have very limited seating.”
As she speaks-about her passion for overlooked species, Barber eyes fill with emotion. “Especially with the things that I work with the creepy crawlies, most of the people wonder: why should we care? And it’s like, well, we should always care about everything because we’re all connected. Sometimes it’s just really hard to communicate why we should always care and why it is our responsibility to try to mitigate things that we humans have caused.”
So far, IVF conservation work is merely being done with about 10 species, but Kouba predicts it’ll become far more widespread in the next decade. “I think it’s getting to become mainstream,” he says, adding that it could become a primary resource for conservation. “We left those species in the world and that we were ready to collect their material, bring it back & put new founder lines back in the population, so we don’t got to remove animals from the wild any more.
“That will revolutionize how we manage wildlife.”
