We derived equine fibroblast cell lines from skin biopsies taken from one Arabian thoroughbred male and one Haflinger female. Oocytes were obtained by in vitro maturation of follicular oocytes recovered from equine ovaries retrieved from abbatoirs. After removal of the nucleus and zona material, embryos were reconstructed by cell fusion with the fibroblasts. Only fused embryos (97%) were activated and cultured in vitro to the blastocyst stage; these were then transferred into recipient mares (see supplementary information).

We cultured 513 and 328 successfully fused reconstructed embryos from the male and the female cell lines, respectively. In total, 22 blastocysts developed, 8 from the male and 14 from the female cell line. Of these, eight blastocysts from the male cell line and nine from the female line were transferred non-surgically into nine recipients. Two of the cloned embryos that were reconstructed with female cells were transferred into the Haflinger mare from which the cell line was derived.

Overall, four single pregnancies were diagnosed by ultrasonography after 21 days of gestation. Two were lost soon afterwards and one aborted at 187 days of gestation; the fourth, however, was carried to term (336 days) and a healthy female foal was born naturally on 28 May 2003 (birth weight, 36 kg). Remarkably, this cloned foal was born from the same Haflinger mare who was the original cell-line donor (Fig. 1).

Figure 1: The cloned foal, named Prometea, with its genetically identical mother seven days after birth.
figure 1

The cells used for cloning were derived from a skin biopsy of the mare. The enucleated oocytes were obtained by in vitro maturation and removal of the nucleus from oocytes recovered from horse ovaries from an abattoir. After embryo reconstruction by micromanipulation and cell fusion, the cleaved embryos were cultured in vitro to the blastocyst stage. Two of the derived embryos were transferred into the mare donor of the cell line. A single pregnancy was established and was carried to term with the birth of a female foal who is genetically identical to her surrogate mother.

To confirm the genetic identity of the foal and her mother, we analysed DNA from blood leukocytes from both animals and from the placenta. Comparison of 12 equine-specific microsatellite loci (Stock-Marks kit, Perkin-Elmer, Applied Biosystems) confirmed that DNA from the foal and from the placenta was identical to that of the recipient mare.

Our cloning procedure was relatively efficient, as one live foal was produced from four pregnancies, although there was high developmental failure from the cleavage stage to blastocyst (8 of 467 and 14 of 286 developed in the male and female cell lines, respectively) and early implantation. This success was aided by advances in assisted reproduction in the horse2, particularly at the oocyte-activation stage, when protein synthesis and phosphorylation must both be inhibited3, and in the refinement of the zona-free manipulation technique4,5.

The remarkable birth of a live foal from its genetically identical recipient is at odds with the idea that the maintenance of gestation depends on immunological recognition of the pregnancy by the mother, based on evidence that abortion can result from inadequate recognition of fetal antigens6.

Furthermore, our result adds the horse to the list of mammals that have so far been successfully cloned from an adult somatic cell. In principle, cloning could enable gelding champions to contribute their genotype to future generations, as well as opening up an opportunity to verify the reproducibility of traits such as character and sporting performance.