Just five years ago, Sudan’s death shocked the world of biology and environmental conservation. At age 45, the old rhino was euthanized to avoid suffering from ailments typical of old age. went andThe last male northern white rhino, and although two females survived, there are no specimens that could fertilize them.

In practice, it is an extinct species.. Or not?

In 2015, a scientific project was born with the aim of going around what had already been written and it came to fruition on March 19, 2018: the disappearance of a large subspecies of mammal. the consortium BioRescuemultidisciplinary and international, has since then worked on the resurrection of the Sudanese lineage and its subsequent reintroduction into the wild.

The method they apply is assisted reproduction. Successful with the most varied animal species since it was developed at the end of the 19th century, its use in the conditions presented by the white rhinoceros implies a great challenge.

ovular fertilization in vitro and the subsequent surrogacy to resurrect this subspecies uses genetic material from a missing male. The methodology is absolutely current due to the ‘Obregon case’, when it is known that it is the same one that the actress and presenter resorted to to have a girl who will be the biological daughter of her late son Aless. But, far from the ethical and legal concerns that these techniques pose in humans, their application in the veterinary world is fully accepted. And, in cases like that of the northern white rhinoceros, it is the only way to recover a lost heritage of universal biodiversity.

“What we do is repair nature using cutting-edge science,” explains Dr. thomas hildebrandt, director of the BioRescue program. “Right now, humanity is facing a serious problem. If nothing changes in the next eight years, we will be at the point of no return. We’re creating a plan to save species on the verge of extinction“.

The northern white rhinoceros (Ceratotherium simun cottoni) is one of two subspecies of white rhino: the other is the southern white rhino. Both are originally from Africa: the first lived in vast regions south of the Sahara, in Uganda, Sudan, Central African Republic and Chad; the second still survives south of the Equator, in South Africa, Botswana, Zambia, Zimbabwe, Angola, Namibia and Mozambique.

The northern species differs by having shorter legs and wider hooves than the southern one, an adaptation to the marshy areas where it lived. The fur on the tips of the ears is also thicker, the only place, along with the tip of the tail, where rhinos have fur. Some biologists consider that, rather than subspecies, they are two different species..

What we do is repair nature using cutting-edge science.

In the 1970s, there were over 700 northern white rhinos in the wild. A decade was enough to see its population reduced to just 15 individuals, with nothing being done for its conservation.. Along with the loss of their habitats and wars, the main reason for the disaster was poaching. They were killed to cut off their horns.

The population was also diminished by the captures of specimens destined for zoos. The one in San Diego, one of the most prestigious of these establishments, once held up to eight wild-caught northern white rhinos. Sudan himself was captured in 1975, when he was two years old, along with five other specimens. His destination was the Czech zoo Dvr Králové.

The current status of the species is limited to two unique specimens of northern white rhinoceros, two females. The question is whether we still have time to save species from extinction. BioRescue scientists have been working for seven years to find a positive answer.

The project is in the final stages of development: surrogacy time. It’s about the development of an embryo in the womb of a female southern white rhino. The implant comes from the genetic material of a couple of the extinct subspecies, whose initial stages of development were obtained in the laboratory.

Fatu (left) and his mother Najin graze at Ol Pejeta sanctuary in Kenya. THOMAS MUKOYA

So far, other paths have been tried. Attempts to insert hormonal implants into live females or to arrange for them to be artificially inseminated have failed. The transport to Kenya of two of the last living males along with the two females mentioned also did not work, in an attempt to make their original ecosystem favor reproduction more than the habitat of zoos.

It was then that scientists increased the collection of eggs and sperm from the few specimens still alive. Aside from Sudan, the only four male northern white rhinos alive in various zoos around the world have been subjected to multiple electroejaculations.. With them, doses of semen useful for reproduction were obtained, totaling 300 milliliters, which were cryopreserved in liquid nitrogen.

The next step was to obtain genetic material from the two live females. They are Najin and Fatu, daughter and granddaughter from Sudan, born in captivity at the Dvr Králové Zoo in the Czech Republic. The first, 32 years old, was very old and her oocytes were of poor quality for the project. Not Fatu’s, whose 21 years give them enough vigor to develop into viable embryos.. The process of extracting the oocytes from the ovaries is performed using an ultrasound-guided probe while the animal is asleep under general anesthesia.

On July 28, 2022 they were collected 23 oocytes from the granddaughter from Sudan. The operation took place in Ol Pejeta, where he and his mother are under close surveillance by armed guards and 24-hour dogs to scare away poachers. It was the tenth collection, which allowed obtaining 158 oocytes, of which 148 belong to Fatu and 10 to Najin.

Once extracted, the immature eggs were transported by air to the Avantea Biotechnologies laboratory in Cremona (Italy). Oocytes are large cells that cannot be frozen under ideal conditions, so they must be fertilized when freshly obtained. In the facilities of the Italian company, the valuable genetic material was matured and fertilized. Its embryonic development has also been achieved. Everything is also cryopreserved.

Northern white rhino sperm is very important and we don’t want to waste it.

In total, it was possible to produce 22 embryos, all from oocytes extracted from Fatu. Half of them were created with thawed semen from Suni, a male who died at Dvr Králové, while the other half was created with seminal material from Angalifu, who lived at the San Diego Zoo (USA).

“Right now, we are in the process of transferring pure southern white rhino embryos to produce a pregnancy,” says Hildebrandt. “Various transfers are being made in different countries. We had a successful implementation, but it ended up failing”

BioRescue’s director believes that the expected results will be achieved in the coming months. The cryopreserved genetic material of the northern white rhinoceros will then be used. “The spermatozoa of this species are very important and we don’t want to waste them”, he insists.

A new problem arises in the process: the inability to conceive of the only two female northern white rhinos. To Najin’s, due to his advanced age, we must add that of Fatu, who doesn’t seem able to support a pregnancy due to the weakness of his hind legs.

It’s a race against time: we are a small entity, with few resources and we need financial support

The solution is obvious: the search for a mother rented. For that, Hello Pejeta Conservancy it’s the best place. Thirty southern white rhinos reside in this African wildlife sanctuary. Among them, two females have genetic characteristics that bring them closer to those of their northern cousins. They are candidates to carry this surrogate pregnancy in their bellies.

At the same time, BioRescue works on a second approach: the use of stem cells to produce gametes. They have already produced primordial germ cells from stem cells induced in the laboratory from rhino skin. The first are precursors of eggs and sperm, which are obtained with a new development in the laboratory.

This complex process was successfully completed for the first time in 2016, when a team from Osaka University, led by Katsuhiko Yahashi, successfully gave birth to healthy mice. Using material from the northern white rhino, Japanese researchers managed to reproduce it in December 2022 as well. A world first in large mammals.

These cutting-edge techniques open the door to increasing the number of individuals in a future population of the extinct subspecies, with significantly greater genetic diversity and in greater quantity than conserved sperm, which belong to just five males.

From BioRescue they are hopeful with the progress of a program that, they guarantee, will soon reach its objective, although they know that they are working at the limit. “The groundbreaking scientific work we do will lay the groundwork for future conservation and rescue initiatives”, concludes Professor Hildebrandt. “But it is a race against time: we are a small entity with limited resources and we need financial support to complete it successfully”,

Sudan disappeared five years ago. With it, another species was wiped off the face of the planet. Today it seems that science will make it possible for their descendants to step back on Earth. A question as surprising as it is paradoxical. While some strive and dedicate enormous resources to resurrect extinct forms of life, others strive to annihilate those that exist.

In vitro and post mortem fertilization

fertilization in vitro It has been applied in the veterinary world since its discovery 133 years ago. It was first performed by British embryologist and zoologist Walter Heape, who successfully transferred two fertilized eggs from an Angora rabbit to a Belgian hare. On May 29, 1890, four rabbits were born, two of the Angora breed and the other two of the Belgian hare.

Effective in different animal species, it took almost a century to be applied to humans. On July 25, 1978, Louise Brown, the first test-tube baby in history, was born in the United Kingdom. The birth of the first human being from artificial fertilization is considered one of the greatest events in the history of medicine.

A step further is the one carried out with the northern white rhinoceros, the same used by Ana Obregón: ‘in vitro’ post mortem fertilization. It consists of using the semen of a deceased male that is kept cryofrozen to fertilize the egg of a fertile female that will carry another female without any genetic relationship with the resulting embryo.