On a sunny Thursday morning, Tian Tian, the male panda who just turned 16 on Aug 27, sat on the grass at the Smithsonian's National Zoo facing the throngs of visitors. He seemed to be deep in thought. Eventually he moved his 264-pound bulk into a nearby pond of water, still pondering.

What he didn't know was that just moments earlier, the zoo had announced that he was the father of both the surviving cub and another stillborn female cub delivered a week and a half ago.

The zoo confirmed on Thursday that the giant panda cub born there on Aug 23 was a female and was in good health.

A paternity examination showed that Tian Tian is the cub's father, while the second stillborn cub that Mei Xiang delivered on Aug 24 was also a female, and also sired by Tian Tian. The cubs were fraternal twins. (Mei Xiang had been artificially inseminated with semen from both Tian Tian and a second male giant panda, Gao Gao, in the San Diego Zoo.)

Zoo scientists used two tests to confirm the sex of the cubs. The first test was developed by scientists in China and analyzes a fragment of the zinc finger protein gene. The second test, also using a shorter fragment of the same zinc finger protein gene, was developed by the Smithsonian Conservation Biology Institute (SCBI) scientists and veterinarians and was used to verify the findings of the initial test, according to the zoo.

For the paternity tests, they compared genotype profiles of DNA samples from the cubs to profiles from Mei Xiang, Tian Tian and the Gao Gao.

The SCBI had blood samples from Mei Xiang, Tian Tian and Gao Gao on hand from previous research.

They compared a small sample of muscle tissue from the stillborn cub and a tiny sample of cheek cells from the cub born Aug 23 with the adult pandas' DNA samples for the test. Veterinarians obtained the cheek cell sample with a swab during a preliminary health check Aug 25, according to the zoo.

"The genetics laboratory conducts conservation research services for the zoo and the entire Smithsonian, including sexing animals, determining paternity, and disease testing," said Rob Fleischer, head of the SCBI's Center for Conservation and Evolutionary Genetics.

"This was a great opportunity to assist our reproductive and panda biologist colleagues to assess their artificial insemination methods in pandas," he said.

According to the zoo, Mei Xiang was artificially inseminated twice on March 30 after natural breeding efforts with Tian Tian failed.

A team of zoo scientists and veterinarians, including Tang Chunxiang, assistant director and chief veterinarian of the China Conservation and Research Center for the Giant Panda at Wolong, performed the artificial insemination.

During the first procedure she was artificially inseminated with a combination of fresh and frozen semen collected from Tian Tian. The frozen semen was from 2003.

The second procedure was performed with frozen semen collected from Tian Tian in 2003 and frozen semen collected from Gao Gao in 2003.

"We had never artificially inseminated Mei Xiang with semen from two males before this past breeding season," said Pierre Comizzoli, reproductive biologist at the Smithsonian Conservation Biology Institute.

"If Gao Gao had been the father of one or both cubs, that would have been very interesting because we would have known that the second artificial insemination was the one that was successful," he said.

DC resident Chris Cantelmi was visiting the giant pandas at the National Zoo on Thursday morning with his 5-year-old daughter and 2-year-old son. He was excited to learn that Tian Tian was the father of the cub. "Dad was here?" he said, but he seemed a bit disappointed that the indoor giant panda pavilion had been closed since Aug 2 and will stay closed to provide quiet for Mei Xiang and her cub.

Mei Xiang, whom people can now watch online via a round-the-clock webcam, has so far given birth to four cubs at the National Zoo. The first one, Tai Shan, born in 2005, was a sensation, but Tai Shan returned to China in 2010 for reproductive programs. According to the arrangement with Beijing, newborn cubs from parents on loan from Beijing belong to China.