In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of "pharming" whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

A major limitation of cloning adult farm animals using somatic cell nuclear transfer is the extreme inefficiency of producing live offspring. For example, Dolly was one of 277 cloned embryos that developed to term (0.3% efficiency). "Millie" was just one offspring that resulted after 95 attempts. Similar inefficiencies regardless of species or somatic cell type continue to be reported. Death of cloned embryos and fetuses occur throughout pregnancy. Moreover, a high proportion of cloned offspring are generally larger than normal (large offspring) and die soon after birth. In general, there have been at least five periods of loss observed with clones derived from adult animals. The first and perhaps most dramatic occurs during preimplantation development. In cattle as well as other species including goats, sheep, and rabbits, >65% of one-cell cloned embryos fail to develop to compact morula or blastocyst (Table I). Approximately 50% of bovine cloned embryos (compact morula or blastocyst) establish pregnancy after transfer of a single embryo into a surrogate recipient (i.e., presence of an embryo proper with a heart beat between days 29�32; Table II).

Table I. Developmental Potential of Clones (Constructed with Adult Somatic Cells Ovarian/Granulosa and Skin Fibroblasts), Parthenotes or In Vitro Produced (IVP) Embryos (Edwards et al.)

Development of cloned bovine embryos, at least for the first 29�32 days, parallels that of those embryos produced after in vitro maturation, fertilization and culture (IVMFC). However, beginning at approximately 30 days and continuing through day 60 of pregnancy, embryonic death may occur in 50�100% of cloned pregnancies (absence of heartbeat and detachment of placental membranes; Table II). Others have reported similar losses. Pregnancy losses in cattle of this magnitude are significantly higher than expected in animals bred by natural service (2�10% loss or developed using IVMFC procedures (16%;). Examination of placentae originating from cloned embryos between days 40�50 of gestation reveal placentae that are hypoplastic, partially developed with rudimentary cotyledons, or those that are essentially normal when compared with IVF derived embryos. The third period of loss that has been noted is associated with an increased incidence of spontaneous abortions during the second trimester of pregnancy. Complete macroscopic and histopathologic examinations of aborted fetuses reveal few abnormalities. However, placentae are oftentimes grossly abnormal with a marked reduction in fetal cotyledons (fewer than 20 compared with the expected 70�120). Moreover, fetal membranes are generally thickened and edematous. The fourth period of loss noted for cloned bovine pregnancies occurs during the third trimester between days 200�265 (280 days ¼ term). Loss during this time period is generally characterized by a marked increase in the incidence of hydrallantois and fetal death. Hydrallantois is accompanied by a marked reduction in placentomes (often fewer than 70, and in some cases <20), marked hypertrophy of many cotyledons, adventitial placentation and severe edema of the intercotyledonary placental membranes. Fetal anasarca with generalized edema and marked edema of the umbilicus is usually present

Table II. Developmental Potential of Cloned Bovine Embryos Constructed with Quiescent-Induced Versus Proliferating Adult Ovarian/Granulosa Cells After Embryo Transfer

^* M/B ET. Total number of morulae and blastocysts transferred to individual recipient animals.
^** Pregnancy loss is described as the absence of embryonic heartbeat followed by detachment of fetal membranes.

Death of late term cloned fetuses is primarily the result of inadequate placentation. Thus far, complete gross and microscopic examinations of late term and early neonatal fetuses have not identified any known genetic or inherited abnormalities. The majority of fetal lesions observed can be attributed to changes secondary to inadequate placental development. Amniotic squames and meconium are generally present in the lungs of all late term fetuses indicating some degree of stress in utero before death.

In some cases, a few cloned embryos will develop to term. However, the majority are not born without complications. In particular, cloned calves derived from adult animals are usually larger at birth and have a lower postnatal survival rate when compared to in vitro counterparts. Most calves derived from abnormal placentae require intensive monitoring and therapy after birth to treat a whole plethora of complications that may include lung dysmaturity, pulmonary hypertension, respiratory distress, hypoxia, hypothermia, hypoglycemia, metabolic acidosis, enlarged umbilical veins and arteries, and/or development of sepsis in either the umbilical structures or lungs. Severity of complications may not be evident for several months. Gibbons et al. lost one calf at approximately 60 days of age and necropsy revealed adhesions in the lungs consistent with pneumonia, and digestive problems consistent with vagal indigestion. Similar complications have been described for clones derived from fetal and embryo cells. In spite of the extreme inefficiency of SCNT, there are some clones born "normal and healthy" requiring little if any veterinary care after birth. Pace et al. reported similar growth rates, reproductive performance and lactational characteristics of clones compared with non-cloned dairy cattle. Moreover, Enright et al. demonstrated that cloned heifers were not different in estrous cycle length, ovulatory follicle diameter, number of follicular waves, or profiles of hormonal changes (leutinizing hormone, follicle stimulating hormone, estradiol, and progesterone). Concentrations of growth hormone, IGF-I and IGFBP3 values recorded for clones derived from a 13-year-old Holstein were all within the range reported for non-cloned calves of similar ages. However "normal and healthy" cloned animals may appear, it is possible that undiagnosed pathologies may develop later in life as a result of subtle changes in chromatin structure and/or gene expression. Miyashita et al. noted differences in telomere lengths among cloned cattle derived from different cell types. Moreover, X-chromosome inactivation may (mice) or may not (cattle) be normal. Wrenzycki et al. noted aberrant expression of genes thought to be of importance in stress adaptation, trophoblastic function, and DNAmethylation during preimplantation development in cloned bovine embryos. Yet, many mice and other animals have survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. The ultimate consequences of epigenetic aberrations of the genome in cloned animals remain unclear but may result in an early death.