Better than sex? For hard-to-breed rhinos, technology strives for a solution

In March 2018, Sudan, the last known northern white rhino (Ceratotherium simum cottoni) male, was euthanized at the Ol Pejeta Conservancy in Kenya. With only two known female northern white rhinos remaining, Sudan’s death rendered his sub-species functionally extinct.

However, not all scientists have given up hope.

A team of researchers at San Diego Zoo has embarked on an ambitious project to use assisted reproductive technology (ART) to bring the northern white rhino back from the brink. They aim to use northern white rhino skin cells held in the zoo’s cryo-lab to produce stem cells they hope could ultimately grow into viable eggs and sperm. It’s one of four similarly audacious projects currently underway around the world.

ARTs, which aim to tackle fertility problems using techniques such as artificial insemination (AI) and in vitro fertilization (IVF), have been used successfully in humans and domestic livestock since the 1970s. The potential for ART to improve breeding success and diversify the gene pool has obvious appeal for wildlife managers caring for endangered species like rhinos that don’t breed well in captivity.

However, a recent article published in Mammal Review  summarizing more than two decades of ART use on captive rhinos illustrates that the technology’s full potential has yet to be achieved when it comes to these animals. The knowledge researchers have gained has already proved invaluable, largely in assisting natural breeding, but the vision of creating a viable rhino embryo outside of the womb is still some way off.

The potential benefits of ART

Due to poaching and habitat loss, the status of the world’s five rhino species is dire. The Sumatran rhino (Dicerorhinus sumatrensis), Javan rhino (Rhinoceros sondaicus) and the black rhino (Diceros bicornis) are all listed as critically endangered by the IUCN, and the greater one-horned or Indian rhino (Rhinoceros unicornis) is classified as vulnerable. Even the white rhino, the only species not considered threatened, thanks to the success of the southern sub-species, isn’t safe. An explosion in rhino poaching over the past decade has seen 7,912 white and black rhinos killed across Africa between 2008 and 2018.

“The risk of losing wild populations is great,” says Terri Roth, a reproductive physiologist and vice president of conservation and science at Cincinnati Zoo, who isn’t affiliated with the recent article. “We should make [captive populations] self-sustaining so we have a backup population.”

Populations of white, black, Sumatran and Indian rhinos are already kept in captivity, but they’ve struggled to breed in a zoo setting. Captive-breeding programs are often hampered by problems such as behavioral incompatibilities or low fertility — exacerbated by the limited number of rhinos available.

“With ARTs, like Artificial Insemination (AI), we can overcome some of that,” says Parker Pennington, a reproductive physiologist working on San Diego Zoo’s Northern White Rhino Initiative and lead author of the article.

ART practitioners are concerned about more than just numbers. “We need to have babies that are not only a contribution to the population but are genetically healthy,” Pennington says. If researchers can perfect AI, where collected sperm is introduced into the uterus, then they can better manage genetic diversity and could even add favorable genes back into wild populations.

With each captive rhino now so valuable, another appeal of ART for animal managers is that it avoids the risk of aggression between animals. Aggression is a natural part of the rhino courtship process and there’s always a risk of injury when two animals weighing well over a ton each come together. For example, a female greater one-horned rhino was recently killed by a male while mating in Nepal’s Bardia National Park. The same can also happen in zoos. In 2018, India’s Assam State Zoo lost a young female rhino when her back was broken after a much larger male attempted to mount her.

Roth says she’s seen zoos become more averse to risk in their animal management over the years, a trend she says could be having negative consequences. “We don’t want to see any aggression from our animals towards each other and we don’t want to put them at risk of any kind of injury and because of that we’re getting less and less effective at breeding animals naturally.”

What’s worked so far

The basis for the successful application of ART is a thorough understanding of the underlying reproductive biology. The problem for reproductive scientists is that “each species is so very, very different,” Roth says.

As a starting point, researchers used one of the rhinos’ closest genetic cousins, the horse (Equus ferus), on which ART has been used extensively. “There is a lot of similarities between the horse and the rhino,” Roth says. In particular, the parallels in embryo development between horses and rhinos have proved useful for scientists monitoring early rhino pregnancies.

The “horse model” could only take researchers so far, though, and it’s taken years to understand the finer details of rhinos’ reproductive system. Even among the different rhino species there’s great variation in key factors such as ovulation cycle, hormone levels, and the size of pre-ovulatory follicles. So researchers have had to conduct the same detailed studies on all of the different species.

“We have a good foundation of information to build on,” Pennington says. Her recent review collated published and unpublished data to provide a clear picture of the status of ART research in rhinos. “Unfortunately, unpublished data is everywhere,” she says. “In science we are taught that unless the experiment went well and we got positive results multiple times, it shouldn’t or can’t be published.”

In a field made up of many small projects working independently, failures that go unpublished may well be repeated, wasting precious resources. However, both Pennington and Roth agree that researchers in the ART field collaborate better than in many other scientific disciplines. “Everyone I spoke to about their unpublished work was incredibly open and helpful,” Pennington says.

The majority of the ART research so far has focused on the female ovulation cycle. Using ultrasound, scientists have been able to determine the ovulation cycle and induce ovulation in all four captive rhino species (no Javan rhinos are currently held in captivity).

Scientists have also been able to collect sperm from the four captive rhino species. Currently, the most common method used by scientists requires the rhino to be anesthetized — a risky procedure for the animal. A team of researchers from Salzburg Zoo in Austria was able to train a southern white rhino to undergo manual semen collection while fully awake, but the practice isn’t yet widespread.

The knowledge that reproductive scientists have gained has already proved invaluable. Roth and her team at Cincinnati Zoo were able to produce the first captive-bred Sumatran rhino in more than a century. Rhinos born at Cincinnati Zoo are now key members of a founding group of Sumatran rhinos in the Sumatran Rhino Project breeding program.

Despite these successes, the full potential of ART has remained tantalizingly out of reach for reproductive researchers. AI has been attempted in all four captive species but was only achieved in the greater one-horned rhino and the southern white rhino, with low success rates.

So far, attempts by researchers to produce a viable embryo in vitro (combining the sperm and egg outside of the uterus in a lab) have not been successful. This is a procedure that San Diego Zoo will need to perfect to produce a northern white rhino calf.

The trouble with rhinos

For researchers, understanding rhino reproductive science has been a slow process. “The number of animals you have to work on is much lower [than domestic livestock] and they are extremely valuable, so you have to proceed with great caution,” Roth says. A further difficulty is that the animals they get to work on are often older individuals that are struggling to breed naturally, making them poor candidates for reproductive research.

Roth says she believes natural selection is another key reason why ART practitioners working with wildlife species like rhinos haven’t found the same success as with domestic livestock. “Once [domestic livestock] come in and we start breeding them, we select artificially for fertility like crazy,” she says. By contrast, natural selection in wild animals has to account for a lot of other traits that an animal needs to survive in the wild.

A further complication for reproductive physiologists is the difficulty of working with such large and potentially dangerous wild animals. Luckily, rhinos are trainable, and because of their sheer size, Roth says she believes they don’t see humans as a threat. That makes them easier to work with than might be imagined. “I’d rather do a rectal exam on a rhino than a cow,” Roth says. “They’re just not fussed by it for whatever reason.”

ART: One part of the solution

Researchers around the world continue building their knowledge of rhino reproduction step-by-step and pioneering new techniques along the way. Only time will tell whether ART researchers can reverse the seemingly inevitable extinction of the northern white rhino. “We still have a long road ahead of us,” Pennington says, “but I do think there can still be hope for the northern white rhino.”

Roth says she’s also hopeful that the many small wins made by ART researchers can “add up to something huge in the end.” But she’s clear that ART shouldn’t be seen as a safety net, and that natural breeding is still the preferred option wherever possible. “That’s the best-case scenario, not just for reproduction but also for the behavior and experience of the animals.”

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