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Natalie I Alexopoulos Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Poul Maddox-Hyttel Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Pernille Tveden-Nyborg Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Nancy T D'Cruz Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Tayfur R Tecirlioglu Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Melissa A Cooney Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Kirsten Schauser Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Michael K Holland Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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Andrew J French Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research, Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Department of Basic Animal and Veterinary Sciences, Monash Immunology and Stem Cell Labs, Stemagen Corporation, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia

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In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

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