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Non-surgical embryo collection and transfer succeeds in smallest mammal yet -- the domestic ferret Cornell zoologist's procedure offers hope for endangered species

Cornell University animal scientists may have a way to help rebuild populations of endangered mammalian species, now that they have succeeded in the first live births by non-surgical embryo collection and transfer in domestic ferrets.

Their new procedure with specially designed, miniature instruments is expected to offer a less invasive way to harvest and transfer embryos. Surgical procedures pose health risks to the animal and may compromise its ability to reproduce naturally -- particularly important considerations when working with endangered species.

Two healthy domestic ferrets (Mustela putorius furo) were born April 15 in an animal science laboratory at Cornell's College of Agriculture and Life Sciences, 49 days after 12 seven-day-old embryos were non-surgically collected and transferred to a surrogate mother of the same species. The scientists plan to further refine the technique with domesticated ferrets, which are plentiful as pets, and hope to apply the technique to black-footed ferrets (Mustela nigripes), one of the rarest mammals in North America.

"We believe this is the first non-surgical embryo collection and transfer resulting in live young in small- to medium- sized mammals," said Robert H. Foote, the Jacob Gould Schurman Professor of Animal Science and Physiology Emeritus who, with Cornell colleague Xiangzhong Yang, pioneered many of the cloning procedures used today in cattle. Embryo transfer is now an everyday occurrence in cattle, but it is still in its infancy -- so to speak -- in exotic animals. Collecting and transferring embryos non-surgically has been one of the stumbling blocks, Foote noted. The non-surgical procedure was developed in ferrets and rabbits, as a model for eventual application to other small mammals, including endangered species.

Until now, surgical procedures required entering through an animal's abdomen to flush embryos from -- or transfer them to -- the oviduct or uterus. Non-surgical embryo collection and transfer has been tried experimentally in larger wild animals in zoos, but the ferret was a greater challenge because its reproductive anatomy is on a much smaller scale.

"The ferret's cervix is not much bigger than a pinhead, and it is sometimes covered by tissue, so finding the entrance to the uterus is not easy," said Jeffrey D. Kidder, the zoology doctoral student who developed and performed the non-surgical embryo collection and transfer. "I had help from cardiologists and urologists in developing the procedure, but even their instruments were too large."

The ferret embryos are harvested from the donor animal with an array of custom-built instruments, including a 0.86- millimeter catheter. While Kidder watches a video monitor connected to a fiber-optic endoscope on loan from Olympus Corp., a fluid medium is injected into the donor ferret's uterus. The fluid flushes embryos out through the catheter and into collection dishes. Harvested embryos are selected by microscopic examination for viability and held in a nutrient solution.

Embryo transfer in ferrets reverses the process. The embryos are transferred, using catheters of similar size, to a surrogate mother who has been hormonally treated with human chlorionic gonadotropin so that her uterus is in synchrony with that of the embryo donor.

Marshall Farms Inc. of North Rose, N.Y., donated domestic ferrets and provided technical assistance in ferret husbandry. Michael Simkin of Cornell's Department of Animal Science and Paul Roberts in the College of Veterinary Medicine contributed technical expertise in various phases of the experiments. The experiments were supported by the New York Cooperative Fish and Wildlife Research Unit and the Department of Animal Science at Cornell. Additional equipment and support was provided by the cardiology and urology departments of the Cornell University Medical College in New York City.

Kidder spent two years developing the transfer procedure with rabbits before attempting ferrets. The first live birth of rabbits from non-surgical embryo collection and transfer occurred April 25. Although the rabbit's reproductive tract is somewhat larger, its different shape presented other challenges and required the development of special instruments as well, he noted.

The Cornell zoologist expects to perform about 30 transfer procedures in domesticated ferrets before trying the technique with a species as endangered as the black-footed ferret. One question still to be answered is whether the domesticated species, which originally came from Europe, is closely enough related to serve as a surrogate mother for black-footed ferret embryos.

Taking reproductive science a step further, a single ferret embryo at the appropriate stage of early development could be cloned with techniques now being perfected by Yang and colleagues in the Cornell Embryo Bioengineering Program and at other laboratories. Using the rabbit as a model, the ferret embryo may be able to be separated into as many as 32 totipotent cells -- each with the potential of growing into a genetically identical animal.

When cloning cattle, the nucleus of each separated embryo cell is put in a special "nurse egg" cell that already has its nucleus removed, Foote explained. Those reconstructed embryos are then activated, cultured and grown to a stage called the blastocyst, which are transferred to synchronized recipients. Although 32 clones from one embryo are theoretically possible, that number has not yet been achieved because of the difficulty of the multistep process, Foote said.

The cloning studies hold hope for even greater achievements, Foote said, referring to the possibility of culturing special cells in the blastocyst that will grow into embryos and produce millions of identical cells. Cloning would not be attempted in humans for ethical reasons, he said, but it makes sense for cattle and other animal species where multiple copies with superior characteristics -- for disease resistance, for example -- are desirable. The technology to culture embryos also has considerable application in humans for whom in vitro fertilization is now a widely used procedure to aid infertile couples.

However high-tech the test-tube cloning becomes, the embryos still need a mother -- or at least a surrogate mother -- and that requires embryo transfer. Animals as small as ferrets didn't have much hope for non-surgical embryo transfer until a student zoologist with a steady hand, a lot of patience and a gentle touch came along.