Bring in the clones

Scientists at UC Davis are circumventing nature by attempting to replicate cows, and it can get messy.

Lucy, the cloned calf, was put on oxygen in the first hours of her short life.

Lucy, the cloned calf, was put on oxygen in the first hours of her short life.

Photo by Larry Dalton

The cow lay on her side on the operating table. Surgical drapes covered her huge hide and hooves so that all dirt would be kept away from the newborn, and out of the incision.

Cindy Batchelder, a Ph.D. student at Davis, had more invested in the birth of this calf than any of the veterinarians, residents or students who crowded around the enormously pregnant, knocked-out cow. But instead of accepting invitations to get involved, Batchelder hung back. She didn’t want her inexperience with medical procedures, like giving oxygen, to endanger the calf, so she watched like a nervous father afraid to touch his own newborn.

In her case, the metaphor is surprisingly apt.

Batchelder had prepared for a year before attempting her first cloning experiment. And in August 2001, nine months after inserting the first cloned embryos into the wombs of the cows that would shelter them as they grew, she watched her experiment come to life. She observed the birth of the first cloned calf ever produced in California. It was in fact one of the first cloned cows ever produced anywhere. Batchelder was in awe, not so much at her own ability to genetically engineer a calf, but at the idea that something like a calf could be engineered at all.

Batchelder watched as the surgeon made an incision almost two feet long in the cow’s abdomen. It wasn’t big enough. Another cut was needed to release not just the calf but also all the fluids surrounding it.

For the Animal Sciences Department at Davis, the Caesarian section delivery, managed by the university’s Veterinary Medical Teaching Hospital, was to be an unveiling, the scientists’ first opportunity to observe and to study a living cloned animal, one that was proving to be unusually large.

Dr. Lisle George, the head veterinarian, eased the slimy calf out headfirst. As Batchelder explains it, the red Hereford calf was so heavy that the veterinarian couldn’t hold her by himself. “It wasn’t necessarily the most gentle landing,” says Batchelder, “but they’re born in the field all the time, dropping out of the cow.”

Cloned calves are often abnormally large; it’s one of the mysteries that cloning experiments like this are trying to unravel. They often share other problems as well. Sometimes they’re born with under-developed lungs that take anywhere from an hour to a couple of days to fully mature. Sometimes they have trouble regulating body temperature. Patterns were emerging, but why? That’s the question driving everybody to distraction.

Scientists love to remind us that Dolly, the first cloned animal, was only born five years ago.

Batchelder had tried to reach everyone who’d ever created a live cloned calf. What should she expect? What can go wrong? Based on their answers, she’d requested oxygen, a heating pad, and precisely measured milk rich in antibodies to help the newborn fight off disease.

Dr. George helped the animal onto the heating pad. With cautious optimism, Batchelder and the rest of the team looked her over. There was a little bit of edema, a swelling under the skin around her face, but that was common with large calves.

Team members dried the calf off with towels, stimulating her the way the mother usually would with her tongue. Others used a rubber bulb to suction fluids from her nose.

Surrounded by curious scientists, the calf began to stir. She tried to lift her head, and then to vocalize, bawling like a regular calf. She was feisty enough to try and stand, but the veterinary team kept her down while they checked her vital signs. There was a little congestion, so the calf was put on oxygen, and then she was moved, with oxygen tubes and tank, into her own stall.

She was named Lucy in commemoration of the fiftieth anniversary of I Love Lucy. They were both adorable redheads.

“At the end of the day,” remembers Batchelder, “we were actually very optimistic, because it seemed like she was really pretty normal.”

But Lucy’s condition began to deteriorate. At the end of the second day, the calf started spiking a temperature. She had already been on antibiotics as a preventative, but they didn’t seem to be working. “They thought pneumonia,” remembers Batchelder.

For all the advanced biology that went into cloning Lucy, science had still taken only the first steps in creating healthy cloned animals.

When Dolly was born in Edinburgh, Scotland, the cloned sheep took the scientific world by storm, kicking off a flurry of international research in the cloning of various species.

Batchelder and her amazing microscope.

Photo by Larry Dalton

In the years since Dolly’s birth in 1996, research institutions like Davis, that focus on agriculture and already have veterinary teaching hospitals, found they could do their own cutting-edge cloning research. The year Dolly was born, Batchelder walked into the office of Dr. Gary Anderson, chair of the Animal Sciences Department, and said that she wanted to be the first person to clone a cow.

The science is time-consuming and woefully inefficient at this early stage, but Batchelder begins fresh each week with at least 100 cow eggs shipped Fed Ex from South Dakota. Removed from adult female cows, the unfertilized eggs include only the DNA of that adult. Batchelder removes the DNA from the egg and replaces it with a cell from the cow she wants to replicate. When the experiment is successful, the cell fuses with the rest of the egg and the egg is “activated,” meaning it’s put through chemical baths that tell it to start growing as if it had been fertilized by the sperm of a bull. If after a week the genetically altered egg, or embryo, seems to be developing normally, it’s placed into the uterus of another adult cow. If it develops regularly through gestation, which is another very big if, it is then born as an exact genetic replica of the cow that gave up the implanted cell, with all that cow’s genetic characteristics.

That’s how it’s supposed to happen.

Some species clone fairly easily. Others, like horses, do not. We don’t know why. Batchelder’s embryos are smaller than average when they’re implanted into the cow, but larger when they’re born. We don’t know why. And calves that make it through the first few days of life tend to develop normally into adulthood. Another mystery.

The abnormalities in cloned calves are extremely difficult to study, partly because a clone is so difficult and time-consuming to create. Just like in humans, gestation in cows takes nine months. And of all the genetically manipulated eggs that are prepared for transfer into waiting cows, only a few make it that far. Some eggs haven’t matured correctly before Batchelder gets them. Some are ruined during the sensitive and tedious lab work. Others don’t survive the activation process that tells them to grow.

So far, around the world, only about 100 cloned calves have been born. Only a small handful of them have grown to bare calves of their own. So far, Davis has only produced Lucy.

In a laboratory on the second floor of Meyer Hall at Davis, Batchelder looks into the microscope to examine skin cells from the ears of adult cows. One of the goals of the experiment is to determine which kinds of adult cells produce the healthiest clones.

The skin cells are attached to the bottom of petri dishes and covered in a nutrient-rich pink fluid. Just like any living organism, these cells not only ingest nutrients, but they excrete waste, so the fluid, the medium, is regularly siphoned off and replaced with fresh fluid.

“I’m from a dairy farm, whole animal background,” says Batchelder, looking at a variety of petri dishes inside an incubator, “but that’s kind of my little herd of cells, and sometimes it takes me as long to take care of them, and do all the things I have to do to keep them healthy and growing well, as it does to feed my 30 cows at the feedlot.”

Batchelder’s cloning experiment has inserted her directly into the center of the life cycle. She’s fiddling with details that only nature has attempted to fiddle with before her. In spite of the enormity of it, Batchelder doesn’t focus on her God-like powers over the genetics of life and death. “I’m completely awed by the process,” she says, speaking specifically of Lucy, “not that we did it.”

Batchelder siphons the pink fluid out of the petri dish and replaces it with an orange-ish fluid. “I have them in culture with enzymes to remove them from the bottom of the dish,” she says. Under the microscope, the dish shows the cells still clumped together, perfectly round. “That’s how they lift off,” says Batchelder, “in sheets, and then I take them and spin them … ”

She transfers the cells and the fluid into a slender test tube, which she presses to the center of an electrified foam pad that whirls the orange-ish liquid into a small cyclone. Batchelder then slips a narrow glass needle into the test tube and begins to gently suck out the cells and the fluids, and then let them fall back into the tube, suck them up, let them fall, further loosening the cells.

“And now I’m going to put them in a centrifuge. They’ll come to the bottom and we’ll have a clump of cells at the bottom.” The centrifuge machine is a big white bowl with a black lid. The test tube goes in, the lid closes, the centrifuge whirls everything around and the cells separate to the bottom. The cells are collected into three small thimble-sized tubes for the next step of the transfer.

Each tiny cell includes all the DNA Batchelder needs to create a clone that matches exactly the adult cow from which the sample was taken. Though the process of separating the cells is sensitive, it’s nothing like removing and replacing the genetic material in an egg, which is the next phase.

The next Tuesday, Batchelder sits in her walk-in closet-sized office in the Cole Facility. A portable heater keeps the room toasty enough to protect the eggs from temperature shock.

The shiny black counters are covered with machines and suction devices. Boxes hold disposable supplies that can be attached to tools, used, detached and thrown away without ever touching human hands.

A small red heart is taped to the door. In white script, it says, “Be nice to me today.” Above that, Campbell’s Maxim reads: “Hell is the place where everything tests perfectly and nothing works.”

In this room, Batchelder spends her Tuesdays scooping the DNA out of eggs and replacing it with skin cells. At the end of the day, maybe 20 can be stored for a week in hopes that they will grow into transferable embryos. For each hundred eggs delivered for Batchelder’s experiments each week, between two and five will survive to be transferred to the uterus of a cow. Most of these will be lost in the first month.

Batchelder sits on her stool before a large and impressive microscope. Besides its ability to peer into the tiniest egg, it also provides Batchelder with long narrow flexible arms that can apply the minutest amount of suction, holding an almost infinitesimal egg in place. Batchelder manipulates the arms by twisting dials and knobs and pushing around nubby snub-nosed joysticks. The microscope rumbles like the engine of the smallest imaginable bulldozer.

Marcelo Bertolini explaines how an ultrasound provides black-and-white images of a developing calf inside the womb.

Photo by Larry Dalton

With the help of the microscope, Batchelder will remove the DNA from an egg and replace it with a single skin cell, giving the egg the DNA of the donor cow.

Batchelder has attached a monitoring screen to the microscope. The eggs appear one at a time, filling the screen with circles within circles. Inside the round cytoplasm at the center of the egg is everything a cell would need to function. One small round bead called a polar body nestles against the outside membrane of the cytoplasm.

Batchelder manipulates the microscope’s mechanical arms, applies a tiny bit of suction, and picks up an egg. The other arm of the microscope holds a tiny glass tube called a pipette. The pipette is beveled to an ultra-sharp tip that punctures the skin of the egg and applies a little bit of suction, removing the polar body and a section of the cytoplasm right under the polar body. The polar body and the clump of cytoplasm below it, called the metaphase plate, include all the DNA of the cow that produced the egg. Removed, they leave the egg a perfect cushion for different DNA.

On the screen, the egg looks as if the smallest possible ice-cream scooper has whisked away about a quarter of the cytoplasm.

Trying to bioengineer the genetic construction, the fertilization and the development of the embryo is hard enough, but it’s complicated by the fact that the eggs have a short life span. From the moment the eggs arrive at the lab, Batchelder has only a few hours to reconstruct them and activate them before they become too old to work with.

Batchelder looks at the skin cells under her microscope. Sucking one cell up into her pipette, she then drops it into the hollowed-out scoop of one of her eggs. She repeats the process over and over and over again.

Seven days later, the surviving embryos, maybe only one or two, will be ready for implantation into the cows, which are as big and messy and difficult to work with as the eggs are small and clean and difficult to work with.

Batchelder’s is not the only cloning experiment going on at Davis. Graduate student Kara Hoffert also plants cloned 7-day-old embryos into cows, though on day 30 of development, her embryos are harvested for study.

On the days her embryos are to be implanted, Hoffert is assisted by veterinarian Marcelo Bertolini, a recent graduate of Davis and now a volunteer member of the cloning team. “We’re lab mates,” he says of Hoffert and Batchelder, who used to help him with his own research. As a favor, he now implants their embryos and performs the ultrasounds, the internal X-rays that show the scientists how the potential calves are developing.

Bertolini cautiously hypothesizes that the embryos’ development is retarded while they’re kept in the lab’s incubators, causing later developmental problems. That retardation, he says, also affects the placenta, which then fails to regulate and control the fetus’s growth during gestation.

After a week in the incubators, the embryos tend to be smaller than average 7-day-old embryos, but the resulting calves are consistently larger than average.

Bertolini explains his theories, in his warm Brazilian accent, as he and Hoffert prepare to drive out to the feedlot in Hoffert’s white sedan with the broken passenger side door. “Grad student salary,” she says.

At the feedlot, Hoffert and Bertolini pull out rolled-up one-piece coveralls. They slip them over T-shirts and jeans and then pull on big rubber wading boots.

The cows watch them. Their faces are warm, relaxed-looking cow faces. Their hides are covered in dung and mud. Their tails whip around lazily.

The lab out here consists of an uncovered concrete slab with a rolling metal worktable, a counter and a huge double sink. Bertolini flips open a plastic box of supplies and pulls out a beautiful red wood tobacco pipe. It helps keep him calm, he says, but as he works, his casual pace grows almost feverish. He pulls out the guns for transferring the embryos, fills syringes, whips out paper towels and protective gloves, and lays out vials of medicines.

The gun consists of tubes within tubes. About 2 feet long and flexible, the first tube holds the needle with the embryo in it. That tube is encased in another, which is encased in another of plastic. The gun, when all put together, is maybe half as big around as a pencil. A sheath of plastic provides the final layer, keeping the embryo stable and clean.

Nothing else about the process is clean.

Hoffert and Bertolini whistle and hoot to drive a number of cows forward into a narrow curved concrete alley. At the end is a confining chute that will hold each cow in place during the procedure.

“Zebu cows,” Bertolini says ominously of the muddy cows in the alley, “they’re crazy.”

When she’s not outside with live cows, Kara Hoffert is in the lab working with their DNA.

Photo by Larry Dalton

Zebu breeds, originally from India, are known for being more disease resistant, and more heat resistant, but much more wild at heart.

The first cow enters the chute and metal doors swing shut heavily against her neck. The sides of the chute squeeze against her stomach.

Bertolini gives the cow a numbing shot in the back and moves her tail to the side, trapping it between her hip and the rungs of the chute. He takes a large plastic milk jug of water and cleans around the cow’s vagina and rectum. He then draws a blue plastic glove over his right arm, all the way up to his shoulder.

Periodically, the cow throws a mild cow fit, slamming her legs against the cage. Then she settles down again.

After picking up the gun with his left hand, Bertolini slowly works his entire right arm into the cow’s rectum. The cow stands nearly still. He “palpates” her, feeling around the reproductive organs through the wall of the rectum. His right hand acts as a guide while the left moves the gun through the vagina, through the narrow cervix and into the uterus. The vet’s face becomes visibly strained as the cow slowly lowers her rump, putting a good deal of her weight on his arm.

A cow’s uterus is divided into two horns. A pregnancy can take on either side. For the sake of the experiment, Bertolini places an embryo into each horn. The guns puncture through the final sheath of plastic to shoot the embryo deep. Bertolini extracts his left hand. The blue plastic glove is covered in dung and blood.

A second cow heads into the chute. She too is caught by the neck, but with less force. She responds well to a numbing shot to the backside, but then grows agitated. The longer she’s in the chute, the fiercer she becomes. She starts to kick and bellow, splashing dung everywhere, her fat gray tongue lolling. Her head is released and she rears up, twisting herself roughly and trapping her right front hoof behind her in the rungs of the chute at about shoulder height. She bellows, forced against the top of the chute, and slowly lowers her weight. The hoof is trapped and the shoulder and leg bend obscenely while Hoffert pokes at it. Finally the cow makes one big lunge and frees it herself. She hollers furiously.

Bertolini assesses the situation and decides that the cow is too wild and angry to go through the procedure. Her will wins out and she’s released.

Hoffert runs her hands through her short brown hair and pulls out pieces of dung.

Hoffert attempts to identify potential problems in the early phases of development by harvesting the 30-day-old cloned embryos for study. That may seem a tricky process, and it would be if the cows lived through it, but Davis’ cows are raised for meat. When the 7-day-old embryos they are transferring grow to be 30 days old, Hoffert’s cows are slaughtered, providing what she calls flippantly, “a good steak.” Then the embryos are retrieved.

After placing the two remaining embryos in a different cow, Bertolini and Hoffert clean up slowly, wiping the dung off their skin, scrubbing the bottoms and sides of their boots and letting them dry in the last warm yellow daylight. Bertolini smokes his pipe. The end of the day brings on a pleasant pastoral laziness.

Both Hoffert and Bertolini foresee a day when experiments like theirs might vastly improve farming. Bertolini gives a recent example of the possibilities. Pigs regularly excrete dangerous levels of phosphorous into the environment, so a pig was developed that could absorb more phosphorous and excrete less, lowering contamination in water and soil. “Eco-pig,” he says to Hoffert. She laughs.

Within the general public, cloning is often discussed in terms of ethical and philosophical concerns. But apart from the national and international debates over human cloning and stem cell research (using human embryos for research), animal cloning may become, with time, a cutting-edge tool for the creation of better meat and milk products and healthier cows.

Asked about human cloning, Bertolini says outright that it’s a ridiculous and selfish idea. Hoffert agrees, but foresees a day when someone, though no one she would ever work with, will probably try it. Like every other scientist interviewed for this report, Hoffert and Bertolini both believe that science isn’t ready.

Batchelder later admits to wrestling with the idea of human cloning, though she agrees that the science is far too immature right now. But she’s studied multiple ways in which the cloning of cattle and other animals can benefit human health in the long run.

A transgenic pig (a pig with cells from another species) might be able to produce a heart that could be transplanted into a human without rejection. And cows and other animals with four stomachs have been found to have traces of an anti-carcinogen in their milk and meat. Cloning could exactly reproduce the cows with the highest levels of this anti-carcinogen, quickly improving the health benefits of the products derived from that herd.

Batchelder also imagines bringing endangered species back from the brink of extinction. She tells of a species very similar to a cow that had exactly one specimen left. Cells were taken from the live adult female and 13 new animals were cloned. Each of them, at maturity, will be impregnated with frozen sperm from males that no longer exist anywhere on this earth. From that sperm and the clones of the female, a new herd will hopefully be born.

But some wonder if cloning will dangerously decrease genetic diversity.

“I guess you have to ask yourself,” says Batchelder, “would you rather have a hundred of the same panda or no pandas?”

Genetic material from inside a tiny cow egg is removed and replaced with the genetic material from a different cow.

Photo by Larry Dalton

It sounds like a well-rehearsed platitude, but it addresses people’s biggest concerns. The idea of a herd of genetic replicas makes a lot of people woozy, but Batchelder reminds us that genetics is only part of the puzzle. Environment provides the stimulus for development.

No living calf, no matter how similar genetically to an adult cow, will ever replicate its life experience. Environment and upbringing and nutrition and other outside influences will all determine how the animal matures during gestation and after birth. Therefore, no two living things will ever be exactly the same.

Batchelder hopes to eventually observe the effects of environment on cloned calves, but currently, only one of Batchelder’s cows is past the most critical stages of pregnancy. She’s the only chance Davis has for a second cloned calf by summer. But clones have been lost as late as a day before birth, so Batchelder carefully monitors the development of the 4-month-old fetus through ultrasounds.

On a recent Monday, Batchelder took her kids out to the beef barn. “They think anybody can clone anything,” she says, “because their mom does it.”

It’s pouring rain, but the boys scatter happily in their raincoats and wading boots.

For Bertolini, the ultrasonography and the embryo transfer are similar, but this time he’ll insert a probe that can send back pictures of the developing pregnancies.

With his right arm, Bertolini moves the probe around inside the rectum of a cow that received a transferred embryo a week earlier. On the screen, he and Batchelder examine the black-and-white images. They find an ovary and look for the kidney-sized embryo that should be nearby. They find nothing.

Batchelder looks visibly disappointed. Each embryo transfer is the final product of so many hours’ worth of work. In spite of all she’s done to manipulate the process, she can’t force the cow’s body to recognize the transferred embryo as a pregnancy. In this case, the cow’s body flushed out the embryo as part of its normal monthly cycle.

The final cow, with her almost 4-month-old pregnancy, is brought forward. Curly, black and white, this gentle Hereford walks easily into the chute. The doors close against her neck and the cow looks around with only slight curiosity.

With his right arm, Bertolini inserts the probe and works his way down to the fetus. Batchelder looks into the black-and-white image on the screen and pokes at a small divided white spot. “See it,” she says, “that’s the hoof.”

On the screen, the bones of the calf’s tiny leg are barely visible as shades of light gray against dark gray. Bertolini moves the probe around, and the calf’s face appears. The animal is lying on its back. A dark spot appears on the screen in a field of delicately curved white. It’s the eye cradled in the crevice of the skull. The tiny little calf opens its mouth and closes it again as if it’s practicing chewing. It shakes its head a little. The scientists seem both relieved and delighted.

Lucy had looked like a success at this stage too, and on through the long months of gestation. Twice the size of a normal calf at birth, she was vocal, feisty and very well-formed, but only on the outside. Inside, it later became obvious, Lucy had problems so severe that she couldn’t possibly have lived.

Batchelder remembers panicking near the end of the third day, when Lucy’s temperature started to soar.

“So then they switched antibiotics and were giving her some things for the fever,” says Batchelder, whose voice develops a strange faraway wavering tone, “which didn’t seem to be very effective in bringing the fever down, and then it went even higher. … I’ve never seen a calf with a fever of 107,” she whispers.

Anderson and others rallied around her. We can handle this, they tried to assure her.

When Batchelder went to check on Lucy at about 10:00 p.m. on the third day, she didn’t like the way the calf looked. Batchelder hadn’t spent any time with her children in days, but she decided to stay with Lucy just a little longer. She applied extra alcohol baths, and the calf revived, but only slightly.

At about 4:00 in the morning, remembering a promise to be there when her children woke up, Batchelder forced herself to get in the car and drive.

“I hadn’t even made it home when they called and said that she had died,” remembers Batchelder, a flush rising up from her throat.

An autopsy showed that the calf had serious developmental problems. “Her liver was basically scar tissue,” says Batchelder, “she had circulatory problems. We’re not sure where the fever came from.”

It’s only been five years since Dolly, everybody reminds everybody.

Months later, Batchelder sits in the lab waiting for a brand new batch of eggs to arrive Fed Ex. “It’s kind of overwhelming at times,” she says, remembering her three days with Lucy. “I’ve been amazed at every step of my project at what we’re able to do to the eggs and the cells and still have them live at the end of a day, let alone a week later, let alone nine months later when we get a calf on the ground. … I mean to actually see the calf on the ground, you know, living, breathing … and to think I made it from one of these cells.” Batchelder looks incredulous.

“I was so thrilled,” she says excitedly. Her voice drops, “ … and then I was just praying, ‘please let her be OK.’ ”

Batchelder is interrupted by one of her team members. The newest shipment of eggs has arrived. The clock is ticking. She only has a few hours to manipulate the eggs. If she’s lucky, one of the eggs she reconstructs today will become another Lucy in nine months.