ON MARCH 9, AN EVENT TOOK PLACE at California State University, Chico’s University Farm that, in other circumstances, would have passed without notice. That day, the farm saw the birth of three Charolais calves—a set of twins and one single—to two of the farm’s beef cows.

What set this otherwise commonplace occurrence apart was that the three calves were clones—the result of a year-long experiment undertaken by CSU, Chico and Cyagra, a Kansas-based scientific firm that specializes in cloning cattle. University farm staff and students named the groundbreaking trio Martie, Emily, and Natalie, in honor of the female country music band The Dixie Chicks. And, like their namesakes, the threesome soon found themselves in the media spotlight. Newspapers, television, radio—everyone wanted to see the calves. They gained national attention as, at the time, only a small number of cloned calves were known to have been born and survived.

But the initial firestorm of media coverage soon took a different course, from the curious to the critical. Over spring break, the calves were moved to the Bangor ranch of Professor Cynthia Daley, of CSU, Chico’s College of Agriculture and the cloning project’s coordinator, for their own safety. It was at that time that the calves began to show signs of illness. Daley immediately took them to the UC Davis Veterinary Hospital, a state-of-the-art facility. There, within two weeks of their birth, twins Emily and Natalie died.

While the media focused on the fact that two of the three calves had died, as a scientist, Daley says she had to look at the results in a different manner, investigating the cause of death but focusing on the surviving calf.

“We’re in the process of developing a technology that can be very useful to the [agriculture] industry,” says Daley. “We have made a contribution to the body of knowledge that will ultimately produce a standard protocol for handling cloned calves.”

Daley describes livestock cloning as “very doable,” as proven by her college’s experiment. “We’ve demonstrated that the cloning technology is indeed feasible,” she explains. “We’ve got some areas to work on. We’d like to improve pregnancy rates and calf livability.”

Cyagra’s director of operations, Audy Spell, oversaw CSU, Chico’s cloning project. At the time of the birth of the Chico calves, Cyagra had 10 other cloned calves that had survived in the United States, with some of those animals going on to produce their own offspring naturally, says Spell.

“It’s probably one of the most productive that we’ve done yet,” he says of the CSU, Chico study. “We’ve not had three calves born in any one situation anywhere else. We do gather very valuable data from this, even though we lost calves.”

Considering the early stage of cloning science, the project partners consider their work a success, and take special pride in the fact that Martie survived. Now, months later, Martie is growing and thriving. In late June, she traveled to the Byrd Cattle Company in Red Bluff. When she gets to the right age, she will be bred to see if she can reproduce a normal calf naturally.

CSU, Chico’s College of Agriculture undertook the cloning project through a $58,000 grant from the state Agricultural Research Initiative (ARI). Red Bluff cattle rancher Dan Byrd of Byrd Cattle Company and Cyagra (a division of Advanced Cell Technologies, based in Worcester, Massachusetts) provided the necessary matching funds. Additional funding came from ARI in 1999, when CSU, Chico was one of the four CSU campuses with agriculture programs—Chico, Fresno, Pomona, and San Luis Obispo—to receive $5 million. ARI’s funding supports a number of applied agricultural research pursuits, including agricultural business management, biodiversity, biotechnology, irrigation management, and natural resource management.

The project goals, says Daley, were to investigate if cloning cattle was a viable process, to see how the surrogate cows’ pregnancies progressed, and to determine whether calves born from cloning would be healthy. The value that the process has for agriculture is the ability to rapidly produce hundreds of animals with a superior set of genes, she says.

The best explanation for what a clone is? “Identical twins are clones—they started as one embryo, which splits at some early stage of gestation—thus their DNA is identical,” explains Daley. “Interestingly, identical twins are different in many ways even though their DNA is identical, similar to offspring generated by cloning technology. This is because of the dramatic effects of the environment on gene expression.”

Animals that have a specific set of chromosomes that express a desirable characteristic are candidates for cloning. While traditional animal breeding passes along only 50 percent of the DNA, and the recombination with the other half may or may not allow the desirable gene to be expressed, cloning will pass along 100 percent of the desired DNA combination.

“Candidates for cloning would truly be exceptional individuals with disease tolerance/resistance to foot-and-mouth disease or bovine spongiform encephalopathy [mad cow disease], or some other desirable trait, such as drought tolerance or feed efficiency,” says Daley. “Cloning can potentially create thousands of offspring with the right gene combination.”

In the spring of 2000, Daley assembled a team that included five CSU, Chico students—Allison Adams, Sadie Smith, Jennifer Taylor, BJ Macfarlane, and Stacey DePaul—and staff research associate, Andree Earley. Two cows, owned by Byrd Cattle Company, were the project’s tissue donors.

The tissue biopsies were shipped to Kansas, where Cyagra personnel began forming the embryos, a process accomplished by taking recipient eggs and replacing their existing DNA with that of the donor cells. DePaul, a senior in agriculture with an option in animal science, spent the summer of 2000 working as an intern at Cyagra, where she helped prepare the embryos. As DePaul explains the process, Cyagra scientists take oocytes, or eggs, from a cow and remove the egg’s nucleus. Then they take the cell they want to clone, place it in the oocyte, fuse them together with electric pulses, and chemically activate the egg to cause it to start dividing. At day seven or eight, during the blastocyst stage, the embryo is implanted in the cow chosen to carry the embryo until birth.

At Cyagra, DePaul aspirated oocytes from the original donor cows’ ovaries. She then watched the rest of the embryo production process and was there when the embryos were prepared and shipped overnight to CSU, Chico’s University Farm (The Paul L. Byrne Memorial Agricultural Teaching and Research Center), located a few miles from the Chico campus.

“They seem to have the micromanipulating part of it down, like taking the nucleus out and then starting the new nucleus,” says DePaul. “Now the major area of study is why there are problems with conception and with the animals after they’re born.”

At the farm, Precision Embryonics of Klamath, Oregon, implanted the embryos two at a time into two of the farm’s Hereford cows, which were specially selected (based on health, fertility, and status of estrous cycle at time of transfer) for the project. The embryos themselves are not genetically modified—the same 60 chromosomes are inserted into each donor egg, with no modification.

Once the implants had taken place, it wasn’t exactly clear sailing. The experiment met with several lost pregnancies before the two cows successfully completed their 283-day gestation periods and the three heifers were born. A total of 14 embryos—from both Charolais and black angus cows—were used in the experiment, with no pregnancies resulting from the black angus embryos. Nationwide, the cloned pregnancies that have so far taken place and been studied have been notable for their high rate of embryo loss.

“From each aspect of the research, students compared how this process contrasts with typical embryo transfer programs, and learned that cutting-edge research can also be the bleeding edge because we are navigating in uncharted water,” remarks Daley. “We didn’t know what to expect, so we tried to expect the unexpected.”

When it came time for the surrogate cows to give birth, the university decided to have the calves delivered by Caesarean section at the UC Davis veterinary laboratory, due to the possibility of difficulties during a vaginal birthing process. “I got to go down to Davis and help out, which was very hands on,” says Sadie Smith. “After the doctors did the C-sections, we cared for the calves. We dried them down, checked them out thoroughly, sucked the fluid out of their mouths and noses. We even bottle-fed them.”

With the birth of the three calves, the experiment partners had even more work before them. Daley, Earley, and the students monitored and studied the heifers. The students provided general support in terms of feed, daily temperatures, and treatments.

After the loss of Emily and Natalie, the cause of death was carefully investigated, and the students worked with the pathologists as they determined the cause of death. Shortly after the two calves died, Daley reported that they exhibited symptoms similar to conditions suffered by many of the calves involved in cloning experiments, including poor antibody transfer and abnormal internal organ formation. At this point, scientists believe that some of the malformations were the result of poor genetic expression during early embryo development.

Genes code for specific traits such as hair color, height, and hormone production. For a gene to be expressed, or for the trait to become reality, the gene is turned on within the cell, explains Daley. This gene actively produces a protein to express the trait. For example, hair color is expressed by a gene that codes for pigment protein which gives hair its distinctive coloration.

“One of the issues surrounding cloning is differential gene expression,” notes Daley. “The question is whether all the genes that are supposed to be turned on get turned on during early embryo development.”

Daley says the calves’ lower gastrointestinal motility wasn’t functioning normally. Emily and Natalie are believed to have died from a patent uracus, a condition where the umbilical cord is abnormally enlarged. This enlarged structure is prone to infection, an infection that led to septicemia and the death of the twins.

Martie, the surviving calf, was saved in part because she was larger and stronger at birth. With the loss of the twins, the clinicians decided to alter their treatment of Martie by changing antibiotics. She responded almost immediately to this new treatment with a drop in temperature and improved appetite. Within the week, she was on the mend. Once Martie returned home to the farm, the experiment resumed. Spell says the project partners plan to monitor the calf carefully, watching her growth rate, her immune systems, neurological functions, and more to ensure that her body is functioning normally.

DePaul—who was raised on a cattle ranch in northeastern California and plans on pursuing graduate studies in her field—had the opportunity to watch the experiment from start to finish.

“I got to learn the entire procedure of cloning—everything from learning how to culture the cells to activating the embryos,” says DePaul. “It was all brand new to me. It was amazing.”

Smith was a senior at CSU, Chico majoring in agriculture during the project and is now getting her master’s degree in animal science at the University of Connecticut. She hopes to do research in the area of animal biotechnology and cloning, and says she feels fortunate to have been able to work on the project.

“I think it just goes to show what the College of Agriculture does for us at Chico State as far as what kind of research is going on,” says Smith, who, like DePaul, was raised on a cattle ranch. “It’s really top of the line, at the forefront of what’s going on in the industry and around the world.”

Daley notes that every aspect of the project was a learning experience for the students. “There are very few undergraduates who get to look through a microscope and see a set of blastocyst-stage cloned embryos ready for transfer, see the transfer, watch the fetuses grow, and then help care for the first set of cloned calves to be born in California,” she asserts.

Daley says that the students had to apply what they learned in reproductive physiology, livestock production, and general animal biology to fully appreciate what was taking place. “This is where the classroom material comes to life,” she says. “When they can read it, see it, and touch it, they will understand it.”

As those closest to Martie point out, she still remains in the eye of the storm, or, more precisely, several storms—medical, ethical, philosophical, and moral. There are many who object to cloning on the grounds that it is unethical, that it interferes with nature. CSU, Chico philosophy professor Becky White, who specializes in moral theory and bioethics, says that cloning is an abuse of the animals involved and should be avoided. “Evidence suggests that the vast majority of cloned animals that are born alive are born with serious defects; most of these die quickly, often in pain. Those that survive often are grossly overweight, leading to painful joint degeneration and limited or even loss of mobility.”

Daley says: “I understand Dr. White’s position completely and believe that the ethicists should be heard. However, obesity and degenerative disease has not been reported in domesticated species (cattle, sheep, pigs, goats) that have been produced through the more recent advances in cloning technology. An example of this is Martie, who is a normal calf—not grossly overweight, nor is she in pain. She is gaining at a rate typical of other calves her age.

“But there are many reports of other abnormalities. Typically, these are the animals that do not make it to term. Successful application of this technology to production agriculture will hinge on the elimination of these abnormalities through further developments and research.”

What many people don’t realize is that cell research is nothing new, says Daley. It was 30 years ago that the first research with embryo transfer—now a commonplace practice—began. Like cloning now, embryo transfer was also greeted with protest and controversy. Daley says that transfer of sexually produced embryos has been used in cattle since the 1970s.

“At that time, the application of this new technology was also viewed skeptically and raised questions of practicality and economic feasibility,” notes Daley. “We now have embryo transfer technology down to a science with excellent results, commonly used by the seedstock industry to improve the genetic merit of livestock. I suspect that it will take a similar length of time to bring the cloning technology to commercial practicality.”

The technique’s most notable successes have come in the last decade. In 1995, Ian Wilmut and his colleagues from the Roslin Institute, near Edinburgh, Scotland, used the technology to clone two lambs—Megan and Morag—from embryo-derived cells that had been cultured in a laboratory. The birth of the two lambs was the first time cultured cells had been used to successfully create live animals. That success would lead, a year later, to the Roslin Institute and its collaborator, PPL Therapeutics, creating Dolly, a sheep cloned from a mammary gland cell taken from a 6-year-old sheep. When Dolly’s birth was announced in 1997—it was later named “Science Breakthrough of the Year”—the interest, and controversy, surrounding cloning procedures exploded. Now the heated debates revolve around stem cell research and human cloning, with some pointing to animal cloning research as having paved the way.

“I used to think that scientists and health care providers would be reluctant to undertake human cloning until the problematic results in animals were eliminated—but that was before the three scientists/physicians [Panayiotis Zavos, Dr. Severino Antinori, and Brigitte Boisselier] announced in August that not only were they ready to begin (on a ship in international waters, if necessary, to avoid bans in several countries), but they had hundreds of couples willing to ‘give it a try,’ ” says White.

Daley points out that while animals have served as models for testing and development of treatments destined for human use, there are significant species differences between humans and livestock. “Our project was focused on applications to the livestock industry, not human cloning,” says Daley. “In a broad sense, much of what is learned in animal cloning can be applied to human cloning in theory, although the actual methods will probably be quite different. I realize that the idea of human cloning may conjure thoughts of Frankenstein and other such horrors instilled in society by the popular press; however, the applications to the livestock industry should not necessarily be limited because someone somewhere may learn how to apply this technology to human reproduction.”

Insights gained during the CSU, Chico project involved several areas of cloning science, including low pregnancy rates, embryo gene expression, and neonatal calf survival. How long before cloning is as common as other technologies, such as artificial insemination? Spell says it’s hard to say. “We hope that it may be within five years,” he says, noting that it took a long time for artificial insemination to become efficient and economical enough in a commercial situation.

Daley is in the process of applying for another grant for a cloning project at CSU, Chico. “We would like to focus the next step on improving the pregnancy rates to the cloned embryos,” says Daley. “Our first study has gone a long way toward improving calf livability. The next calves would be handled accordingly, therefore significantly reducing death loss.”

DePaul says that when she saw Martie in late June, she looked “great.” “She’s a normal, 3-month-old calf,” she says.

About the author

Elizabeth Larson is managing editor of the Lake County Record-Bee in Lakeport, California, as well as a freelance writer.