The male, called Suni, “was probably the last male capable of breeding”, according to Dvur Kralove zoo in the Czech Republic, where the rhino was born in 1980.
There are only six of the very rare rhinos left, having been hunted by poachers in central and east Africa for their horns, which are highly prized for traditional Chinese medicine.
The Czech zoo is the only one in the world to have succeeded in breeding the sub-species in captivity.
Suni — who is thought to have died from natural causes in the Ol Pejeta reserve — was one of two males and two females from Dvur Kralove zoo reintroduced into the wild in Kenya in 2009, in an operation dubbed “the last chance of survival”.
It was hoped that the females’ hormones would normalise in the wild, but even attempts at assisted conception failed.
“One can always believe in miracles but everything leads us to believe that hope they would reproduce naturally has gone,” the zoo’s spokeswoman Jana Mysliveckova told AFP.
Sperm from the males born at Dvur Kralove has been conserved at the Leibniz Institute for Zoo and Wildlife Research (IZW) in Berlin.
Another pair of the rhinos, too old to reproduce, live at the Wild Animal Park in San Diego in the United States, with another aged female remaining at Dvur Kralove, close to the border with Poland.
“The number of rhinos killed by poachers has increased incredibly in the past few years,” Mysliveckova said. “According to some scenarios, there will be no rhinos left in the wild in Africa in 10 years or so.”