For three years, from 1998 to 2000, the team worked without apparent success. After transferring the nuclei from the mule cells into 134 horse eggs and implanting them into mares, two apparently "false pregnancies" resulted, but both failed to proceed past four weeks.
In 2001, the team began to focus on the calcium levels in the fluid surrounding the eggs during the cloning procedure. The change led to the first fetal heart beat, signifying the team had crossed a significant hurdle in the experiment. That year, researchers transferred 84 eggs, establishing five apparent pregnancies.
"The results were impressive and immediate," Woods said. The first change led to a significant advance in the development of cloned embryos.
In 2002, Woods, White and Vanderwall continued to adjust the calcium levels in the fluid surrounding the egg during the cloning procedure. That change dramatically increased the team's success.
The team established 14 pregnancies using mule DNA in 113 attempts. Eight of the pregnancies continued to at least the 40-day stage when heartbeats were detected.
To test whether mule DNA could be limiting success, the team also made 61 attempts to use horse DNA. The test resulted in seven apparent pregnancies, two of which developed heartbeats. Neither of the horse clone pregnancies developed past the critical 60-day threshold, however.
The UI-Utah State team is the first to succeed among several teams worldwide attempting to clone a member of the horse family. The 2002 preliminary testing showed the method developed by the researchers to successfully clone a mule should work equally as well with a horse, Woods said.
"It basically came down to a matter of numbers, and we wanted to focus most of our attention on cloning a mule, which was our original objective," Vanderwall said.