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Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

¹ Monash Institute of Medical Research, Centre for Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia² Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Melbourne, Victoria 3000, Australia³ Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 7, Frederiksberg C DK-1870, Denmark⁴ Monash Immunology and Stem Cell Labs, Monash University, Clayton, Victoria 3800, Australia⁵ Stemagen Corporation, 4150 Regents Park Row, La Jolla, California 92037-1417, USA

Correspondence should be addressed to N I Alexopoulos; Email: natalie.alexopoulos{at}med.monash.edu.au

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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