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Controlled cytoplast arrest and morula aggregation enhance development, cryoresilience, and in vivo survival of cloned sheep embryos

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posted on 2023-05-03, 19:04 authored by Zachariah McLean, Sarah ApplebySarah Appleby, Lisanne Fermin, Harold HendersonHarold Henderson, Jingwei WeiJingwei Wei, David WellsDavid Wells, Björn ObackBjörn Oback
Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

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Rights statement

© Zachariah Louis McLean, et al., 2021. Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Publication date

2021-02-02

Project number

  • Non revenue

Language

  • English

Does this contain Māori information or data?

  • No

Publisher

Mary Ann Liebert

Journal title

Cellular Reprogramming

ISSN

2152-4971

Citation

McLean, Z. L., Appleby, S. J., Fermin, L. M., Henderson, H. V., Wei, J., Wells, D. N., & Oback, B. (2021). Controlled cytoplast arrest and morula aggregation enhance development, cryoresilience, and in vivo survival of cloned sheep embryos. Cellular Reprogramming, 23(1), 14–25. doi:10.1089/cell.2020.0078

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