No pride of ancestry, no hope of progeny. That's the reproductive life story of a mule.

Reproductive veterinarian Gordon Woods explained, "Mules result from breeding a male donkey (Equus asinus, 62 chromosomes) with a female horse (Equus caballus, 64 chromosomes) to produce a sterile foal with 63 chromosomes.

"Equine cloning," Woods continued, "has been hampered by a lack of optimal in vitro oocyte maturation and embryo culture systems. Previous reports of nuclear transfer pregnancies have been less than 65 days of development in utero," he added.

Woods, who calls himself a "horse doctor," is professor of animal veterinary science at the University of Idaho's Northwest Equine Reproduction Laboratory, which he founded in Moscow, Idaho. He is first and senior author of a one-page Sciencexpress report dated May 30, 2003. Its title: "A mule cloned from fetal cells by nuclear transfer."

"There are several key findings in this journal article," Woods observed. "One," he enumerated, "this is the first time an equine species has been cloned. Second, it's the first time that a sterile hybrid has been cloned. Third, the horse - the equine species - from the reproductive physiology standpoint is unique from other animals. It's certainly different from domestic farm herds, especially cattle. Meanwhile, successfully cloned animals include bovines, ovines, felines, rabbits, rodents and porkers."

Loosening In Vivo Fertilization Hang-Up

"In order to be successful in our cloning procedure," Woods told BioWorld Today, "we had to identify a specific difference in the horse system. If you look at in vitro fertilization, IVF, that technique is highly efficient in humans and in cattle. The human IVF fertility clinics, literally around the world, are standardized, as are cattle IVF. In marked contrast to the human and cattle IVF situations," Woods noted, "in horses only two foals have been born via IVF.

"In our cloning protocol," Woods continued, "we were actually working against - had not figured out - what the differences are between the horse and cattle systems of IVF. In our mule-cloning efforts we thought we may be facing some of the same problems. In preliminary experiments we identified that the intracellular calcium concentration in the horse is significantly lower than calcium concentrations in humans. So we measured an attenuated concentration of calcium response in a horse oocyte. We specifically went in and increased the calcium in our culture and activation media.

"In this cloning operation," he went on, "we were as close to the donor horse as we could be. Because of our concerns for IVF not working, we allowed the mare to mature the reproductive egg shortly before the expected time of ovulation. We removed the egg, placed it into a culture system, cultured the eggs for a short period of time, removed the DNA or genetic material from the egg, and replaced that with the genetic material from the mule cells. The oocyte that replaced DNA we then put through an activation process, cultured it for a short period of time, then placed it into a second, recipient, mare. That egg was actually outside the surrogate mother for approximately 24 hours."

"Of 334 manipulated oocytes," the journal article reported, "305 were transferred to recipient mares, resulting in 21 (6.9 percent) 14-day pregnancies." Only embryos maintained and activated in medium with elevated calcium concentrations established pregnancies maintained through day 45. The fetus developed to full term, 346 days, and weighed 46 kilograms at birth.

Racing Champion Mules Await Clone

"The result," Woods recounted climactically, "was that on May 4, 2003, at 3 in the morning, we had the natural unassisted birth of a live male mule [named Idaho Gem], color brown. At 12 minutes he stood, and as we were drying him off he bucked with his hind end. Idaho Gem was born with that capacity; Mom didn't have to teach it to him. Two additional mares carrying cloned mule fetuses were at gestational days 255 and 310 on May 4.We expect their births June 9 and Aug. 2, respectively.

"At 33 hours," Woods continued, "we had a photo shoot. We turned the baby mule and his mother [named Idaho Syringa] out into a grassy area, and this guy darted around like a little rabbit. At the end of the photo opportunity, it took six of us to scoot him and the Mom back into their stall - the pictures of health."

Asked what this particular animal will do in life, Woods observed, "His full brother is a champion racing mule - an athlete. Idaho Gem's function in life is going to be as an equine racing athlete. We'll put him into training and see how he performs.

"At the University of Idaho," Woods said, "a use patent for the method of increased calcium during the cloning procedure has been applied for, and we co-inventors have formed a separate spin-off company from the university, named ClonE 2 Inc. Its objective is to transfer earnings back into the university, to fund our future research. The source of the funding could be whatever purposes people have for the duplication of their animals.

"One goal," Woods suggested, "would be restoring or preserving other endangered equine species, using this approach. There are several endangered horse breeds at this point. One is a Mongolian horse," Woods concluded, "believed to be an ancestor of our modern Przewalski's horses and Somali wild asses."

Accompanying the Sciencexpress report is a brief Science editorial headed, "First cloned mule races to finish line." It points out that "Idaho Gem is a sibling of a world-champion racing mule named Taz."

"Some researchers," Woods commented, "suspect that the first clone, Dolly the sheep, aged prematurely because her DNA was derived from an adult cell. So our team rebred Taz's parents, took a somatic cell from the 45-day-old fetus, and fused it with an enucleated horse oocyte that we then inserted into a mare."

The same chemistry that led Woods to clone Idaho Gem may shed new light on specific cancer causes in humans. "The mortality rate for horses with metastatic cancer," he observed, "is 8 percent for all cancers and 0 percent for prostate cancer. Compare this to 13-14 percent for prostate cancer in humans."