A research mare at Texas A&M carrying a cloned foal recently lost her pregnancy. "We lost it at nine months of gestation via premature separation of the placenta and placentitis (placental infection), which we treated for three weeks before she slipped, said Dr. Katrin Hinrichs, professor in TAMU's College of Veterinary Medicine.

A University of Idaho and Utah State University team made the announcement that they had produced the first successful equine clone--a mule--on May 29. The Texas fetus would have been the first cloned foal to be born in the United States. The University of Cambridge and Louisiana State University also have equine cloning projects, but have not announced success in their quests.

Hinrichs and other TAMU researchers aren't sure if the failed pregnancy was because of an inapparent problem with the cloned fetus, or placentitis, which is a leading cause of pregnancy loss even with normal foals. The mare had a prior dystocia (difficult birth) that might have compromised her ability to harbor a pregnancy.

"The bottom line is, we don't know the cause," said Hinrichs. "The mare had signs of placentitis and the premature separation for about three weeks but we could see that the foal was still alive. Then when she slipped, we saw that the foal had contracted front legs and an umbilical hernia, but grossly (visibly to the unaided eye) it was otherwise normal." She added that the fetus had no malformations of its organs, but she hasn't received a histology (cell and tissue study) report yet. Contracted forelegs can result from the uterus or the amnion (a thin, avascular lining on the inside of the placenta) restricting movement of the legs as the foal grows

Embryo Research
Cloning a foal wasn't the focus of Hinrichs' research when she transferred five cloned embryos into mares. At the time, she was studying in vitro fertilization by intracytoplasmic sperm injection (ICSI), or injection of one sperm into an egg, in the laboratory. "We have been concentrating on in vitro culture of horse embryos, and what we can do to make that more efficient," she said. This research is directly applicable to making the cloning process more efficient as well.

"Nobody has worked out the best method to figure out how to grow a fertilized egg to an embryo in the laboratory," said Hinrichs, whose team has managed to get their ICSI success rate with blastocyst formation (embryos that are big enough to transfer to mares) to 20-35%. Procedures that manipulate the embryo in the laboratory increase the chances of early fetal loss or only trophoblast forming (a placenta without a fetus). Hinrichs wants to determine the best method for embryo culture, to optimize the number of pregnancies maintained and the number of mares that go to term and have a normal foal.

During the ICSI studies, Hinrichs team decided to transfer five cloned embryos that were produced in the lab (which had resulted from more than 100 cloning procedures). Of those five, one resulted in the pregnancy that was lost.

The Idaho/Utah team does their embryo transfer process differently than researchers at TAMU. When the Idaho/Utah team performs the cloning procedure, it immediately takes multiple cloned embryos and puts them into the oviducts of a recipient mare through a surgical incision (113 cloned embryos in 2003 resulted in three full-term mule colts). TAMU researchers perform the cloning procedure and wait to see which fertilized eggs become embryos in the laboratory, and then transfer the viable ones into the uteri of recipient mares through the cervix (non-surgically).

"We are planning to do more transfers of cloned embryos this year to determine the proportion of foals that go on to term successfully," said Hinrichs.

When successful, would the cloning process become commercial for TAMU? "A lot of people approach you, but right now it's a very expensive procedure. Most people are interested in cloning, but not interested enough to fund research in it," she said.

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