This study represents the first detailed and comprehensive assessment of age-related, non-communicable diseases in adult cloned offspring of large animals. Working with sheep, four of which were cloned from the cell line that gave rise to Dolly, we undertook assessments of metabolic, cardiovascular and musculoskeletal health. Importantly, despite their advanced age (7–9 years), none of the clones showed any clinical signs of disease, being euglycaemic, insulin sensitive and normotensive. No animal was lame despite most showing radiographic evidence of mild OA in one or two joints, as would be expected in sheep of that age. None have required treatment for OA. In the absence of perfectly matched (by age, genotype and environment), naturally conceived controls, these conclusions were reached with reference to (a) a contemporary group of six-year-old sheep that were managed alongside the clones, and underwent the same metabolic and cardiovascular assessments, (b) published reference ranges for blood glucose, (c) pelvic radiographs of eight 5-year-old healthy sheep, and (d) the available scientific literature. Whilst contemporaneous controls would have been ideal, our data are nevertheless compelling, indicating no detrimental long-term adverse effects of SCNT on the health of aged adult offspring.

Because cloning by SCNT can lead to obesity and hyperinsulinaemia in aged mice18, and a single case of Type I (insulin dependent) diabetes was reported in a cloned calf41, we decided to measure glucose tolerance and insulin sensitivity in our aged cloned offspring. There are (infrequent) reports of naturally occurring type I diabetes in sheep42, where glucose concentrations for affected animals range between 16 to 19 mmol l−1. However, we are unaware of cases of type II (non-insulin dependent) diabetes, although insulin resistance has been extensively studied in this species43 where it is known, for example, that deficiencies in maternal periconceptional diet can lead to insulin resistance in adult offspring44. In humans, risk factors for insulin resistance leading to type II diabetes include advanced age and obesity; the latter risk factor is also related to enhanced insulin resistance in sheep43,45. Hence the current study sought to create an obese state in our experimental animals to fully test the extent of glucose tolerance and insulin sensitivity46. Hyperinsulinaemia in the four Finn-Dorset clones (Fig. 2c) was certainly influenced by the level of body fat, as weight loss reduced plasma insulin concentrations (Fig. 2f), consistent with improved peripheral insulin sensitivity which can be expected following weight loss in sheep46. However, peripheral insulin sensitivity is negatively related to visceral fat and intrahepatic lipid accumulation in humans47,48, which varies between ethnic groups49. It follows that genotype differences in regional fat distribution in sheep36 may at least partially account for the large difference in insulin response following glucose infusion observed between Finn-Dorset clones and ET-controls, which had a similar proportion of total body fat (Fig. 2a). Although this awaits final confirmation following post-mortem examination, previous studies indicate that the Finn-Dorset genotype has larger intra-abdominal fat depots than other common genotypes such as our ET-controls50,51.

Consistent with observations for cloned offspring from other species9,14,52, perinatal losses of cloned sheep have been associated with structural defects of the kidneys and heart, and with pulmonary hypertension10. Enlarged and abnormally developed kidneys were also evident in some of the six live-born Finn-Dorset clones that failed to survive to 3 months of age in the current study (Supplementary Table 6). We hypothesized that if subtle renal defects in surviving offspring were to persist late into adult life then this could lead to increased blood pressure, known as reno-vascular hypertension53. Hypertension has been described to ‘follow-the-kidney’ in cross-transplantation studies, suggesting factors intrinsic to the kidney mediate increases in blood pressure54,55,56. The renin–angiotensin–aldosterone system is a major target for long-term anti-hypertensive treatment as elevated intra-renal renin–angiotensin–aldosterone system action and/or sensitivity may directly cause increased blood pressure57,58. Here, for the first time using radiotelemetry blood pressure probes, we recorded circadian cardiovascular parameters continuously for individual cloned and ET-control sheep kept as a group in a stress-free environment. Ambulatory blood pressure of these cloned animals was similar to ET-controls and within the normal range for other sheep reported in the literature (that is, ∼120/80 mmHg; Supplementary Table 4). It was also similar to adult blood pressure in healthy humans. Classification of ‘hypertension’ in humans begins at a systolic blood pressure ≥140 mmHg (ref. 59) or ∼20 mmHg above resting. The elderly cloned sheep in this study could not, therefore, be considered to be hypertensive. Direct i.v. infusion of angiotensin II, to further challenge cardiovascular responsivity, failed to induce a greater pressor response than in ET-controls. We therefore conclude that cloning by SCNT had no detrimental, latent effect on the cardiovascular system of sheep in this study that survived into late adulthood.

Detailed musculoskeletal investigations were undertaken of all sheep, as Dolly had required treatment for OA from around 5 years of age, raising concerns over premature ageing in cloned animals22,23,24. Radiographs taken of Dolly showed OA in the left stifle (knee), however OA was evident in both stifles at post-mortem examination 18 months later (TJ King, Roslin Institute, personal communication). The consensus at that time was that, because the OA was localized rather than generalized, it was more likely of traumatic aetiology. However, the aetiopathogenesis of OA is accepted to be multifactorial, involving both genetic and acquired factors (such as joint trauma/overloading, and obesity). The prevalence increases with age (the strongest risk factor), but the course of the disease varies greatly between individuals60,61.

Clinical examination of cloned sheep in the present study revealed only mild lameness in the left foreleg of one animal, which may have been associated with her slightly abnormal forelimb conformation. None of the Finn-Dorset clones had an abnormal gait or showed lameness. Several clones had soft tissue swelling or fibrous thickenings of various parts of their limbs, as would be expected in a random sample of sheep. However, these did not appear to be associated with any discomfort. Subsequent radiography revealed mild OA in various joints in several sheep. It is not surprising that no sheep showed marked lameness, as radiographic changes indicative of OA do not necessarily correlate with the extent of clinical disease62. Mild OA (scored by two independent reviewers) was seen most often in the hip (13 of 13 sheep) and stifle (12 of 13 sheep) joints. Scott63 reported that the stifle and elbow joints are most commonly affected by joint trauma. However in the present study, only 5 of 13 sheep showed elbow OA, which was bilateral in most cases. Only Clone 2262 had significant OA in multiple, but not all, joints. The majority of sheep appeared to have a mild increase in bone density of the acetabular rim, however none had significant osteophytosis or remodelling of the acetabulum or femoral head and neck as would be expected with OA. Similar findings were apparent in pelvic radiographs of eight healthy 5-year-old sheep (Supplementary Fig. 2), enroled in an unrelated study, made available courtesy of Professors Allen Goodship and Gordon Blunn (University College London). Thus it may be a normal radiographic finding in healthy ageing sheep, and not, in isolation, indicative of OA. This will be further assessed when post-mortem examination of the present cohort is undertaken at a future time.

Radiographic changes appear relatively late in the disease process and so MRI of the stifles (knees) of the four Finn-Dorset clones was undertaken, as MRI is more sensitive in detecting early structural changes60. To minimize the duration of sedation required, MRI scans were taken only of the stifle joints, because these were among the most commonly affected joints in our population, and were affected in Dolly23. T2* and proton density, fat-saturated MRI images provided the most useful diagnostic information, showing periarticular osteophytosis primarily on the trochlear ridges and caudal tibia. However, the extent of the disease remained mild, and not unexpected in aged sheep.

Reports of OA in non-experimental sheep are rare in the literature. A case series was reported by Scott64, describing elbow OA in 10 sheep (aged 2 to 5 years) that had shown forelimb lameness of at least 3 months duration. A post-mortem study of the stifle joints of 65 clinically normal sheep aged 6 months to 11 years by Vandeweerd et al.65 identified cartilage defects in 66% of the sheep, the severity of the lesions increasing with age. These authors proposed that OA exists in normal ageing sheep, and is not always associated with clinical signs. Pathological changes were also found in the femoral head cartilage of clinically normal 2.5- to 3-year-old sheep by Zilkens et al66. Thus there is no evidence of an increased incidence or severity of OA in the clones in this study. However, a single cloned sheep in the present study had obvious OA in multiple joints, although it varied in severity between joints, and not all joints were affected. It is not surprising that the joints were variably affected, as studies in humans have shown, for example, that a factor such as obesity increases the risk of knee OA by a factor of three, but has less effect on hip OA60. As discussed previously, a more homogenous distribution might have been expected, both within and between sheep, if cloning by SCNT was a direct causative factor in the development of OA.

From the current series of assessments we conclude that there are no long-term detrimental health effects of cloning by SCNT for a long-lived species such as the sheep. This conclusion is consistent with less detailed longevity studies in cattle17, and suggests that the ageing process in surviving clones of large animal species is not accelerated. On initial inspection, our data in sheep may appear at odds with the health status of Dolly and predictions of premature ageing, which were based on terminal restriction fragment analyses of her genomic DNA20. While telomere length was reduced in SCNT clones relative to age-matched controls in that and subsequent studies in sheep67, these effects did not manifest following SCNT in cattle21. Further inconsistent reports of shorter telomeres in cloned offspring from other species68 have led to the consensus that telomere length is generally restored during nuclear reprogramming69. The extent of telomere restoration in turn is dictated by intricate epigenetic alterations to telomeric and sub-telomeric chromatin, variation in which could explain discrepancies between species and donor cell types within species. It follows that the relationship between telomere length, health and longevity in multicellular organisms is complex and, for our current cohort of animals, awaits organ-specific cell enrichment and analysis following post-mortem at a future date.

In conclusion, although the efficiency of SCNT has improved in recent years, its overall efficiency remains low, with high embryonic and gestational losses compared to natural mating and assisted reproduction. A relatively high proportion of clones also fail to successfully make the transition to extra-uterine life, some harbouring congenital defects, such as observed in the kidney. For those clones that survive beyond the perinatal period, however, the emerging consensus, supported by the current data, is that they are healthy and seem to age normally.