By David N. Leff

When a Japanese host wishes to impress — or honor — his dinner guests, he puts beef on the table.

Japan's cattle-raising industry is highly protected from foreign imports, which accounts in part for the fact that the choicest super-premium beef roasts cost about $100 per pound. A contributing factor is the extravagant upbringing lavished on superior animals, bred and raised to end up on the plates and palates of the wealthy.

"A genetically ideal calf is just the starting point," according to a "News Focus" commentary in the current Science, dated Dec. 11, 1998. "The animals are fed beer and given daily massages as part of a regimen that results in fine flecks of fat uniformly scattered throughout the meat."

To those tactics of good breeding and tender loving care now add adult somatic-cell nuclear transfer.

The same issue of Science carries a research report titled "Eight calves cloned from somatic cells of a single adult." Its senior author is molecular geneticist Yukio Tsunoda, professor of animal reproduction at Kinki University, in Nara, Japan.

The "News Focus" commentary on this achievement carries the headline: "Bid for better beef gives Japan a leg up on cattle." It goes on to note that, galvanized by the birth of Dolly, the Scottish cloned sheep, in early 1997, "at least seven Japanese groups set out to replicate the experiment in cattle."

Tsunoda and his co-authors broke away from the pack to finish first, but herds of runner-up Japanese cloned calves are in various stages from gestation to live births.

"Five groups," the Science editorial noted, "have reported the births of 19 calves cloned from adult cows, and so many more surrogate mothers are carrying cloned cow embryos, that the Ministry of Agriculture, Forestry and Fisheries has given up trying to track them all."

Until Dolly, the Ministry had frowned on attempts to use adult cells, but reversed that guideline in mid-1997.

Of the Kinki group's eight cloned calves, half were alive and well, as of Nov. 1, 1998. Two had been born 120 days before that date; two others, 85 days prior. The older pair developed from primordial oviduct epithelial cells; the younger two from cumulus cells — the jelly-like epithelial mass that enwraps and nurtures the ovum.

The four calves that failed to survive had died neonatally of natural causes, Tsunoda's paper explained. "Postmortem analysis did not reveal any abnormality; however, environmental factors appeared to account for their deaths." One succumbed to heatstroke; the other three to obstetrical complications during delivery.

The front-running Japanese feat, like that of Dolly's Scottish creators, drew its acclaim from the fact that its four-footed nuclear-transfer progeny were cloned from an adult cow and sheep, respectively, rather than from undifferentiated embryonic cells, as in previous practice. (See BioWorld Today, Feb.2, 1997, p. 1.)

Recipe For Cloned Calf On The Hoof

A single anonymous cow from a nearby slaughterhouse — her stage of estrus unknown — provided the Kinki co-authors with their starting material. From her ovaries they collected and cultured cumulus oöphorus cells, and from her Fallopian tubes, oviductal cells. They then fused these differentiated cells, by electroporation, with enucleated eggs from a different cow. Another jolt of electricity ensured their reactivation.

Then, after eight or nine days in culture, 20 cumulus-derived and 20 oviduct-derived nuclear transplants had developed into blastocysts. The team transferred 10 of these proto-embryos from both cell types into surrogate mother cows. They seeded six cumulus-derived blastocysts into three recipient females, and two oviductal ones into two others. All five cows became pregnant. (See BioWorld Today, Nov. 6, 1998, p. 1.)

Of those 10 blastocysts, eight cloned female fetuses completed gestation, and were born alive. This 80 percent survival rate was far higher than that achieved in the Dolly experiment, in which only one of 13 blastocysts came to term. The Japanese survival rate, the Science commentary observed, "is far higher than that of any other group attempting to clone large mammals." Seven of them were delivered vaginally; the eighth by Caesarian section. All eight were genotypically identical to their original cell donor and phenotypically to each other.

The two living oviduct-derived neonates were born on day 242 of gestation, and averaged just under 18 kilograms. The two cumulus calves, born on days 276, weighed in at a 25-kilogram average. Significantly, one cumulus calf, which died three days after delivery, had a whopping birth weight of 32 kilograms. Such outsize fetuses are a frequent problem in nuclear-transfer cloning.

Questions Waiting For Answers

"Nuclear transfer of adult somatic cells from farm animals," the Science paper observed," is the most efficient technique for obtaining large numbers of genetically identical animals ... [They] can be selected from animals already proven to be ideal milk or meat producers."

One problem awaiting investigation, the co-authors pointed out, "concerns the cytoplasmic contribution of the oocyte to the properties of the clone," inasmuch as that bovine ovaries obtained from a slaughterhouse often come with no knowledge of the eggs' genetic background.

Another pending question concerns damage to cells during freezing and thawing. In their own experiment, "both donor cell types were freshly prepared and used before freezing. Yet to be determined is whether other adult cell types can be reprogrammed to direct the development of fertile animals."

That determination should be forthcoming this spring. "A second batch of cloned calves," Tsunoda told Science, "still in utero, has been produced using cells from 20 different tissues, including the liver, kidney and heart. "We are at the starting point to study the reprogramming [of cells]," he concluded. *