Even before the last surviving male northern white rhino died in Ol Pejeta Conservancy, Kenya, this year, the future of the species was more or less sealed. Down to three animals in the world, all from the same family, there wasn’t much hope for a progeny.
Until now. Earlier this month, a team of scientists from Europe, Australia and Japan used in vitro fertilisation (IVF) to create the first ever rhinoceros embryo using stored white rhino semen. “With a quality of this kind, there is a 50% chance that the embryo would yield a calf that is part northern white rhino,” Cesare Galli, a cloning expert involved in the work told The Wire.
Although IVF has been used with horses, cows and humans, it has never been extended to rhinoceroses. The feat was more daunting because only two northern white rhinos remain on earth, both females. Of them, “Najin suffers from Achilles heel and cannot carry a calf while her daughter, Fatu, has a damaged uterus, ruining any chances of implantation,” said Thomas Hildebrandt, head of reproduction management at the Leibniz Institute for Zoo and Wildlife Research, Berlin.
Hildebrandt, an author of the new study, had encountered a similar situation in an earlier assignment, when he assessed the reproductive health of northern white rhinos in the San Diego Safari Park, California. Authorities were concerned that no new calves were being born. Hildebrandt’s tests showed that this was because most animals were sterile. During this exercise, researchers also collected semen samples from the bull that were frozen on site for lab analysis. A few years later, Hildebrandt assessed seven northern rhinos of the Dvůr Králové Zoo, Czech Republic. This included three bulls: Saut, Suni and Sudan.
“At that time, we didn’t expect that in about a decade we would be using these [semen samples] for creating embryos,” Hildebrandt said.
Poaching had pushed these animals to extinction in the wild, their numbers plummeting from 2,000 in the 1960s to 15 by 1984. In 2009, only seven animals were alive: five at the Dvůr Králové and two in San Diego.
To help save them, Hildebrandt decided to use assisted reproductive techniques. He approached Galli, managing director at Avantea, an Italian biotechnology and animal reproduction laboratory, and together they decided to develop ways to create implantable white rhino embryos. From its start, the project was a bumpy ride: there were limited semen samples, two surviving females both infertile and no knowledge of how exactly to proceed.
Galli had improved upon artificial insemination of horse and cattle, but to try IVF in white rhinos, they would have to study several animals and rely on trial and error. To perfect the technique, Galli and his team turned to northern white rhinos’ closest relatives, the southern white rhino. This subspecies schism is thought to have happened about a million years ago, motivated by geographical isolation (although this explanation is contested). About 20,000 southern white rhinos exist in the world, and females of the subspecies were used as a source of eggs for IVF.
Also read: Was the death of the last male northern white rhino the end of a hoax?
The first challenge was to extract the eggs from their ovaries. The ovaries are located deeper in the bodies of rhinoceroses than in horses or cattle. So Hildebrandt and his colleagues constructed a device for egg pickup. The eggs collected in this fashion are immature so Galli’s team tinkered with culture techniques to mimic internal uterine environment, allowing the eggs to mature outside the body. The final challenge was to fertilise them with sperm.
Scientists selected semen samples from both the northern and the southern white rhinos and froze them. But after thawing, they found that the sperms had lost their mobility. When they checked if the sperm would fuse with a pig egg (as is the standard procedure), only a few samples were able to manage the feat. “We had limited sample and couldn’t afford to fail,” Galli said. So instead of adding sperm to the eggs and letting nature take its course, they resorted to intra-cellular sperm injection.
But for this technique to succeed, the sperm still has to ‘activate’ the egg, and there was no guarantee that this would happen. So the team used electro-activation to tip the odds. Within an hour after sperm injection, they struck the plate holding the egg with two mild electrical jolts of 1.5 KV/cm for 30 microseconds. This jumpstarted cell division in the embryo.
Nineteen eggs fertilised with southern white rhino sperm yielded three implantable embryos. Two of the partial northern white rhino embryos (hybrid) and one pure southern white rhino embryo will be implanted in a rhinoceros after Hildebrandt and team develop a reliable implantation technique. The remaining early embryos served as a source for embryonic stem cells. These cells have the potential to form any adult rhinoceros cell. So far, scientists have found ways to differentiate them into skin cells, brain cells and mesodermal cells (the kind that lines our gut). The end game is to create a line of germ cells that would mature and yield gametes.
Another goal for the team is to use induced pluripotent stem cells to generate gametes from adult rhinoceros cells. This is important because “embryos created from closely related members will not be viable for long,” Galli explained. Semen collected from a few northern white rhinos is not enough to ensure good genetic diversity. However, scientists have also collected tissues from about 12 different northern white rhinos, and gametes produced using these are expected to be diverse enough to sustain animals born through IVF.
This is tricky and so far humans have made it work only in mice. To expand it to rhinos would require more money and more time. Thus far, the project has been keep going by the scientists themselves, and if they are to successfully “conserve white rhinos through biotechnology”, Hildebrandt said both the government and private investors would have to cough up.
Of course, there is also doubt whether this approach will work – particularly in terms of delivering the best outcomes. For example, Jo Shaw, the African rhino lead for the World Wildlife Fund (WWF), told The Wire, “Innovative assistive reproductive techniques have limited value unless there are safe places for populations to thrive.” Amit Sharma, a senior wildlife conservationist with WWF India, pitched the additional concern that the embryos developed thus far are hybrids. “There is more value in conserving species in their natural habitat,” he said.
But where conventional conservation methods have failed to protect a species, Hildebrandt believes biotechnology is the only hope. “There has been a lot of scepticism on the use of biotechnology and cloning for conservation because [conservation scientists] say it is not natural,” Galli told The Wire, adding that he hopes that their new study will help change that view.
Next, the scientists plan to perfect implantation strategies in a surrogate. They also plan to retrieve oocytes from the remaining northern white rhinos to create embryos.
While Hildebrandt thinks that the story of northern white rhinos is not yet over, there is a big problem. Even if a hundred northern white rhino calves are born in this manner, the population will still be far too low to sustain the subspecies for long. Lochran Traill, an ecologist and conservation biologist based at John Moores University, the UK, has found that the minimum viable population size for mammals is over 1,000. “But the situation for northern white rhinos is desperate,” Traill said. “I think in such a situation any desperate measure is acceptable.”
Galli agrees that the conservation efforts would have made more impact if there were more individuals. “I mean, we are not pretending to save this species through biotechnology,” he said. ‘What we are saying is that biotechnology can be an effective tool if done right.”
Sarah Iqbal is a freelance science writer.