Willadsen Clones the First Farm Animal by Nuclear Transfer

Date March 10, 1984

Steen M. Willadsen improved on an earlier technique of cloning large mammals by developing a simple, easily used new method, making it possible for greater numbers of scientists to produce genetically identical animals for use in experimentation.

Locale Cambridge, England

Key Figures

• Steen M. Willadsen (b. 1944), veterinarian who developed cloning methods
• R. A. Godke (fl. late twentieth century), animal physiologist who collaborated with Willadsen
• C. Polge (fl. late twentieth century), animal physiologist who assisted Willadsen in studies of embryo division and growth in cows
• J. P. Ozil (fl. late twentieth century), French scientist who led a team working on splitting cow embryos to produce identical twins
• Steven A. Voelkel (b. 1957), reproductive physiologist who produced the first bisected embryo twin lambs in 1984

Summary of Event

Steen M. Willadsen and R. A. Godke reported in the March 10, 1984, issue of the journal Veterinary Record on their development of a new simplified technique for the rapid production of split embryos in sheep. This procedure improved on a previous splitting method first developed by Willadsen in 1979, which required several steps occurring over about three days. As with earlier techniques in use at the time, such splitting produced two separate embryos from a single embryo collected from a mother ewe. The resultant half embryos, called demi-embryos, were then implanted into the mother’s or a different ewe’s reproductive tract to allow for the development of identical twin lambs. The new procedure was considerably simplified, as it required only a single surgery on the mother, and the two demi-embryos were reimplanted into the same ewe within about an hour of the removal of the original embryo.

Work had been done since the 1950’s in removing embryos from both laboratory animals and large domestic animals and then alternatively replacing them, transferring them to new host mothers, or freezing them for storage and later transfer. In the 1970’s, this research led to the development of methods of separating the individual cells (blastomeres) from early two-cell, four-cell, or eight-cell embryos to allow the production of multiple individuals from the same original embryo. Such production of identical twins, triplets, and higher multiples is a means of artificial cloning. Work in this area was important to reproductive physiologists interested in how embryos develop, to researchers in other fields who needed twins for experiments in various areas, and to farmers and ranchers who wanted to increase the number of specifically bred animals.

In 1979, Willadsen published the description of his original method for micromanipulation of sheep embryos to divide an embryo in half at the two-cell stage. He first treated female sheep with hormones to cause superovulation (the production of more than the normal number of ova). The sheep were mated, and Willadsen surgically removed fertilized ova in the second day of the estrus cycle, when each embryo contained only two cells. Using a microscope, he held the embryo stationary with a capillary pipet and tore into the zona pellucida, the tough, clear outer coating that protects a mammalian egg. He separated the two cells and placed each into a new empty zona that had been collected previously. He then coated the demi-embryos with agar, a jellylike material derived from seaweed, to protect them within the torn zonas. A second layer of agar was used to coat the first, producing a small plug containing the two demi-embryos from a single original embryo.

Each cylinder produced in this manner was transferred into the oviduct of a recipient ewe that was synchronized at the same point in its estrus cycle as the mother, and the oviducts were ligated (tied off) to prevent the loss of the agar plug. After two and one-half days, the embryos were flushed out of the host mother’s oviduct and evaluated microscopically. Small hypodermic needles were used to manipulate the embryos out of the agar, and the freed structures were transferred to other synchronized ewes. Sixteen sets of monozygotic (identical) embryo pairs were produced in this way, and both embryos of each set were transferred into the same host mother. Ten of the ewes carried the resultant pregnancies to term, resulting in the birth of five single lambs (when only one of the demi-embryos survived) and five sets of monozygotic twin lambs.

Willadsen also found in 1980 and 1981 that embryos could be split in half or in quarters at both the four-cell and eight-cell stages of development, and the resultant demi-embryos were able to produce normal offspring. This was shown by production of identical quadruplets in sheep in 1980 and in cattle (work that Willadsen conducted with C. Polge) in 1981. For this work, the same agar technique was used, with the embryo divided into halves or quarters. Identical twins, triplets, or quadruplets were produced in these studies. Single cells (blastomeres) derived from eight-cell sheep embryos also were able to develop into lambs, but the recovery rate was very low, and either no offspring or only one lamb resulted from each of numerous attempts.

Other investigators in this area were also working on producing cloned animals. J. P. Ozil and his colleagues in France thought that Willadsen’s agar-coating method was more complex than necessary and devised a technique that eliminated that aspect of the procedure. In experiments with cattle, they used later-stage multicellular embryos, called gastrulas, and omitted the protective agar-coating step. With no agar around the embryos, it was not necessary to retrieve them three days later to scrape off agar so that implantation could take place in the host mother’s uterus. In this process, the demi-embryos were separated microsurgically and then placed in empty zonas. These “naked” demi-embryos were inserted directly into the synchronized host-mother recipients, with resultant rates of pregnancy (64 percent) and of twinning (67 percent) that were lower than Willadsen’s rate in sheep (75 percent), but still reasonably high.

Willadsen decided in 1984 to try the later, multicellular embryos as the source of demi-embryos. Rather than collecting embryos at day two of the estrus cycle, in the two-cell stage, or day three, in the four-cell or eight-cell stage, the new method reported by Willadsen and Godke harvested embryos at day six, seven, or eight of the cycle. At day six, a sheep embryo is in the morula stage of development, and at day seven or eight, it is in the blastocyst stage. At these stages, there are hundreds of small cells, and the zona pellucida has disintegrated, no longer necessary for protection. Two specific areas develop in the blastocyst: an inner cell mass that becomes the actual individual and the outer layer (trophectoderm) that forms fetal membranes that attach to the mother’s uterus.

In Willadsen’s new procedure, these embryos were collected surgically and then cut in half using a capillary pipet and a fine glass needle. The hollow ball of cells was held so that the inner cell mass was at the top, and division was accomplished by passing the needle into the ball and moving it up and down to divide the embryo, cutting it against the pipet. The two demi-embryos were not exactly the same, but each contained about the same amount of inner cell mass and trophectoderm, enough to produce a new individual. The mother ewe from which the original embryo was taken remained under anesthesia for approximately an hour while the procedure was performed, then the two resultant demi-embryos were replaced into her uterus.

This rapid removal, bisection of the embryo, and replacement was much more efficient than the previous method. Of the eighteen ewes implanted with a pair of demi-embryos, pregnancy continued to term in sixteen ewes (85 percent pregnancy). Seven produced single lambs, one produced nonidentical twins (one lamb was from an embryo that had not been recovered and split), and eight produced identical twins (50 percent twinning). All twin production was from the bisection of blastocysts, with no twins developing from morula-stage embryos. Unpublished studies in cattle by Willadsen and colleagues also showed high viability of similarly split and transferred embryos.

Significance

A major reason Willadsen’s original technique was important was that it made increased numbers of genetically identical animals available for use in experimental studies. Such animals are desirable research subjects because scientists can assume that differences in responses to particular treatments are the result of the treatments and not of genetic differences between test organisms. It had been estimated that the use of one pair of twins in an experiment in cattle, as the experimental animal and its untreated control, was the informational equivalent of using twenty-two unrelated animals under the same experimental conditions. Identical twins occur naturally in cattle and sheep very infrequently, in less than 1 percent of births, and such births occur at random in the population. Willadsen’s method of producing identical twins from specific animals and specific matings was able to increase the accuracy and efficiency of experiments and greatly reduce the number of animals needed to obtain useful results.

Willadsen recognized that his earlier technique was rather complicated for general use in the burgeoning market for production of twin cattle and sheep, because production was desired on the ranch as well as in the laboratory. Use of the agar-coating method required that the user be experienced in embryo transplantation and in micromanipulation for division of the original embryo. The publication of the simplified new procedure provided a significant improvement over the previous method, making it possible for even relatively inexperienced users to produce reasonably successful results in twin production. The procedure developed by Willadsen opened up the production of identical twins in sheep and cattle to many more research investigations and agricultural uses.

Not only did it become possible to produce twins for use in experiments, but it also became easier to produce many offspring of a desirable mating. Through superovulation of the mother before mating, cloning of the resultant embryos, and implantation of the demi-embryos into host mothers, the actual mother could then be used repeatedly for ovum production, rather than being pregnant herself. Many offspring could be produced in this way for use in experiments or for agricultural applications, such as further breeding or milk or meat production.

Another use of the technique was that one of a pair of demi-embryos could be transferred to a host mother immediately, while the other was placed in frozen storage and later thawed and implanted. One of Willadsen’s published research reports includes a photograph of identical twins produced in such a way, with one of them two weeks old and the other two and one-half months old. Such methods allow scientists to carry out an experiment on genetically identical individuals at the same time, but with the individuals at different ages.

The embryo-splitting technique developed by Willadsen soon became widely used in agriculture. Researchers such as Steven A. Voelkel and Godke in Louisiana began using it in 1984 in studies on cattle, sheep, and pigs. Voelkel was involved in the development of a modification of the technique that allowed automatic splitting of the embryo using a micromanipulator. At Southwestern Louisiana University’s animal research center, the method was also applied to the embryos of rhesus monkeys in an attempt to develop a colony consisting of identical twins for use in research. Given that monkeys are closely related to humans genetically, such a colony would be of great help in studies related to human health, such as development of vaccines against malaria and acquired immunodeficiency syndrome (AIDS).

Bibliography

Bavister, Barry D., ed. The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in Vitro. New York: Plenum Press, 1987. Collection of papers discusses embryonic development in a variety of animal species. No specific discussion of twin production, but good background information on in vitro embryo culture in two appendixes. Chapter 12 is of particular interest on the growth of early embryos of domestic animals, mostly pigs.

Beier, H. M., and H. R. Lindner, eds. Fertilization of the Human Egg in Vitro. Berlin: Springer-Verlag, 1983. Contains a paper by Willadsen and Fehilly on the developmental capacity of separated cells from two-, four-, and eight-cell sheep embryos.

Brackett, Benjamin G., George E. Seidel, and Sarah M. Seidel. New Technologies in Animal Breeding. New York: Academic Press, 1981. Collection of articles of general interest concerning animal breeding. Includes a chapter titled “Parthenogenesis, Identical Twins, and Cloning in Mammals” that references work done by Willadsen and coworkers from 1979 to 1981. Also discusses other cloning methods.

Klotzko, Arlene Judith, ed. The Cloning Sourcebook. New York: Oxford University Press, 2001. Collection of essays by scientists and bioethicists addresses both the science of cloning and the social issues raised by the possibilities of cloning technology. Includes a chapter by Willadsen that describes developments in the area of mammalian nuclear transplantation since his original research.

Kolata, Gina. Clone: The Road to Dolly and the Path Ahead. New York: William Morrow, 1998. A science journalist provides a clearly written history of cloning as well as discussion of the implications of the technique for humankind.

McKinnell, Robert Gilmore. Cloning: A Biologist Reports. Minneapolis: University of Minnesota Press, 1979. A professor of cell biology provides a clear scientific explanation for the general reader of what is involved in cloning. Discusses the cloning of frogs in depth (frogs were the highest animals that had been cloned at the time the book was written).

Willadsen, S. M. “A Method for Culture of Micromanipulated Sheep Embryos and Its Use to Produce Monozygotic Twins.” Nature 277 (January 25, 1979): 298-300. Willadsen’s first paper on splitting sheep embryos discusses his original technique, which involved coating the split segments with agar for protection.

Willadsen, S. M., and R. A. Godke. “A Simple Procedure for the Production of Identical Sheep Twins.” Veterinary Record 114 (March 10, 1984): 240-243. Presents Willadsen’s simplified procedure for splitting an early sheep embryo. Describes the collection, splitting, and return of the embryos to the original or host mother as well as the results.

Willadsen, S. M., and C. Polge. “Attempts to Produce Monozygotic Quadruplets in Cattle by Blastomere Separation.” Veterinary Record 108 (March 7, 1981): 211-213. Describes the techniques and results of a procedure for dividing cow embryos into four parts and attempting to produce a calf from each quarter embryo.