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Two limitations currently restrict the acceptability and adoption of bovine cloning as a commercial reality. The first of these is its low rate of efficiency and the second is that some of the cloned calves are not healthy. Abnormalities in the placenta are thought to contribute to many of the losses in early gestation; however, less is known of the pathology of clone deaths in the perinatal period. To date, the majority of perinatal deaths have been attributed to the “large offspring syndrome” characterized by increased birth weight and a range of morphological abnormalities thought to be associated with in vitro culture and manipulation. This report describes multi-systemic abnormalities in aborted, stillborn, and neonatal genetically modified and unmodified cloned calves weighing less than 60 kg at birth and aged between 6 months gestation and 3 weeks postnatal, generated in various experiments. Three of 14 genetically modified cloned calves had cystic renal dysplasia and osteopetrosis. All three and a fourth had irregular nodular, fibrotic livers with biliary abnormalities. Another two had marked flexion of the fetlock joints. Eleven calves derived from an unmodified cloned cell line by nuclear transfer had nodular, fibrotic livers with biliary anomalies, 9 of 11 had cystic renal dysplasia and cardiomegaly, two had osteopetrosis, and two had contracted tendons. In addition, three calves had polymicrocerebral gyri, two had retinal dysplasia, and one had an aortic aneurysm. Only one calf from a second unmodified cloned cell line produced by nuclear transfer had no significant congenital abnormalities. All calves were negative for bovine virus diarrhea virus (BVDV) by competitive-antigen ELISA, and by virus isolation and no BVDV antibodies were detected by AGID assay. Furthermore, all cell lines and media used were negative for BVDV by virus isolation. Two calves were tested and found to be negative for Akabane virus and Aino virus. There are very few reports of the pathological abnormalities of cloned animals. Similar multi-systemic abnormalities have not been found in non-cloned calves, but several analogous conditions occur in humans, including Simpson-Golabi-Behmel and Zellwegers syndromes. Further ultrastructure studies and genetic analysis are needed to investigate the mechanisms of these multi-systemic disorders, which may ultimately elucidate mechanisms for improved reprogramming and increase the efficiency of generating cloned animals with somatic cells by nuclear transfer.

74 ANATOMICAL ABNORMALITIES IN CALVES PRODUCED BY NUCLEAR TRANSFER