Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, andIn VivoSurvival of Cloned Sheep Embryos

Abstract

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 metaphasearrested 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.

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach

“…Among these documents, 10 had no record of citations. The most recent document was by McLean et al [42] who studied the effect of embryo aggregation during the vitrification process of cloned sheep embryos. They observed that the aggregated embryos had an in vivo survival rate similar to that of the group that was not vitrified.…”

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach

“…Among these articles, 10 articles had no record of citations. The most recent article was by McLean et al [42] who studied the effect of embryo aggregation during the vitrification process of cloned sheep embryos. They observed that the aggregated embryos had an in vivo survival rate similar to that of the group that was not vitrified.…”

infer: An R package for tidyverse-friendly statistical inference

“…For one, the principled approach of the infer package has made it an especially good fit for teaching introductory statistics and data science (Baumer et al, 2020;Çetinkaya-Rundel & Ellison, 2021;Ismay & Kim, 2019) and research in data pedagogy (Fergusson & Pfannkuch, 2021;Loy, 2021). Further, the package has already seen usage in a number of published scientific applications (Ask et al, 2021;Fallon & Hinds, 2021;McLean et al, 2021). Finally, the package integrates with the greater tidymodels collection of packages, a burgeoning software ecosystem for tidyverse-aligned predictive modeling used across many modern research and industrial applications (Kuhn & Wickham, 2020).…”