From the barn in Sejra to Noah's ark in the desert

Bringing endangered species back to life, animal cloning, instant coffee, and the potato crop of the ancient Incas are linked in the fascinating research of Professor Amir Arab

Half of the 5,416 known mammal species in the world are in danger of extinction. In the last century, the extinction rate of various animal species, including mammals, increased 100 times to 1,000 times the natural rate. So what can be done to preserve biodiversity on Earth? This is exactly the question that Professor Amir Arab, from the Director of Agricultural Research in Rehovot, is trying to answer.

In my conversation with Professor Arab, several ways came up: preservation of natural habitats of endangered animals so that they can breed in them, artificial migration, i.e. moving animals at risk from one geographical area to another or artificially inseminating females in species whose population is not large enough for natural reproduction. Another direction is the preservation of sperm cells and fertilized eggs of animals in a "cell bank" in order to return them to our world if they become extinct.

Freezing is a common method of doing this. The "frozen zoo" in San Diego, California, for example, stores in a special facility the genetic material of 350 species of mammals, birds and reptiles. Freezing at the temperature of liquid nitrogen is very effective in cases such as preserving pieces of skin intended for transplantation, sperm cells and even tiny embryos, which are actually clusters of 8 cells formed from the division of the eggs after fertilization. Preservation in this way over time is very expensive, because it requires constant maintenance, and involves the risks of power outages, unexpected temperature rises and inadequate supervision. Apart from that, the method is not suitable for preserving unfertilized eggs, which are essential for the artificial fertilization of animals.

Here comes into the picture another, innovative method of preservation that answers these problems, and which Professor Arab deals with: freeze drying. The inspiration for the method comes, among other things, from potatoes that were completely dried after being frozen in ice and the cold, dry air of the mountains above Machu Picchu, the Inca capital in Peru, and remained vital after hundreds of years. Two-thousand-year-old dry date seeds found in excavations in Masada also germinated successfully when exposed to water. Dryness is therefore a very important condition for preserving cells, and if you dry them at a low temperature, you can seal them in a glass vial, bring them back to room temperature and place the vial on a shelf. The food industry has been using freeze drying for a long time, among other things in the production of instant coffee beans.

The idea of ​​preserving eggs, fertilized or unfertilized, through freeze drying came to Amir Arab's mind even before he was engaged in research for his doctorate at the University of California at Berkeley. As a naughty boy in Tiberias, Arab thought that studies were a waste of time. It is better to spend the time working in the barn and in the fields of Sjara, whose grandfather was among its founders and where his father was born. When he nevertheless turned to academic studies, after winning a prize for his thesis in high school in the field of biology and after completing military service in the paratroopers, of course, as befits a member of a farming family, he turned to the Faculty of Agriculture. A natural continuation was veterinary studies in Italy, during which he was exposed to the subject of embryo freezing. Even if a doctorate in medical engineering in Berkeley, California is not necessarily the accepted continuation of veterinary studies, Amir Arab realized that if he wanted to engage in the preservation of eggs and embryos through freeze drying, he must specialize in physics, and more precisely in thermodynamics, and understand in depth the processes involved in freezing and drying living tissues.

Lowering the temperature of living cells causes two damages: one is caused by the well-known anomaly of water. The volume of the freezing water increases, so the cells may be damaged by mechanical pressure. The other damage, no less serious, is caused by tiny ice crystals that form inside the cell, destroying its membranes and causing its death.

In nature, the body cells of fish living in the cold waters under the ice in the Antarctic region, for example, are rich in proteins called "antifreeze proteins". In the past, it was believed that the main function of these proteins is to prevent the water in the cell from freezing, similar to adding the substance ethylene glycol to the cooling water of cars in cold countries. Amir Arab managed to preserve eggs at a low temperature after soaking them in antifreeze proteins. He discovered that in fact the success was not due to the prevention of freezing but to the fact that the antifreeze proteins preserved the structure of the membranes. The scientific world had a hard time accepting the idea, but it turned out that membranes are damaged by cooling even if they don't reach the freezing temperature, so the role of these proteins is essential even above zero degrees Celsius. A similar reason also facilitates the preservation of sperm cells in freezing: sperm cells are rich in cholesterol which preserves the structure of the membranes in a similar way to antifreeze proteins.

Amir Arab's success in preserving the membrane structure of oocyte cells, as part of his postdoctoral work in the laboratory of Professor John Crowe at the University of California, Davis, opened the way to preservation through freeze drying. And this is how the method works: first, the membranes of the cells are protected with appropriate proteins, and the cells are immediately cooled quickly while significantly reducing the pressure in the freezing chamber. The reduction in pressure allows the frozen water to go directly from a solid state to a gas state and the water content drops from 90% to 40% and even below that. If the process is done under the appropriate conditions the cells maintain their vitality.

Upon his return to Israel 15 years ago, Professor Arab founded the IMT company that manufactures a device called Vitmaster, which enables rapid vitrification (turning a liquid into a solid without formation). The device cools the sample from room temperature to a temperature of 208 degrees Celsius below zero in just one-tenth of a second - a cooling rate of 50,000 degrees Celsius per minute, the fastest cooling known today.

Saving an unfertilized egg effectively and safely may have far-reaching consequences in human and animal fertility treatments. For example, this will allow women to save eggs before chemotherapy or even before mastectomy, and will pave the way for an egg bank. But is there a recipe here for preserving extinct populations like the tiger population in Ein Gedi?

The "Dried Noah Ark" project (Dried Noah Ark - DNA) in the antelope farm near Moshav Tzoper in the Arava is designed for exactly this purpose. Professor Arab and his team concentrate in the box cell samples that have been freeze-dried, sealed in vials and stored at ambient temperature. The goal of the project is to preserve the genetic heritage of Israeli species that are in danger of extinction, such as the harrod, the dishan and even the tiger. In the box, not only sperm or egg cells are preserved, but also normal cells from the body of the rare animal. The cloning of the cells, with the technologies developed after the cloning of Dolly the sheep, and the insertion of the cloned embryo into the womb of a surrogate mother of a related species, makes it possible to have children even after the species has become extinct. All that needs to be done before cloning is to add water. It is even possible to use cells sampled from the corpses of animals shortly after their death.

But even the possibility of cloning tigers, rhinoceroses and whales using their dried cells will not be useful if the number of individuals of any species is too limited. A single tiger in the Judean desert, or even a single pair of tigers will not be able to breed and establish a genetically healthy population. Dr. Yehuda Arab, the nephew of Amir Arab, therefore developed a mathematical model that makes it possible to calculate the necessary number of individuals and the ratio between the number of females and the number of males that will remove the danger of extinction from a certain species.
And as an interviewer I was left with one unanswered question: "How do you collect sperm cells and eggs from whales?

Dr. Deborah Jacobi is a member of the educational staff of Hamada, the center for scientific education in Tel Aviv, and a scientific editor at Scientific American Israel.