The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets

Abstract

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1-simultaneous electrical pulses and activation group (SFA group); and Group 2-electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p<0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p>0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p>0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.

Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases theIn VitroDevelopmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

“…In vitro cultured fibroblast cells, which had been derived from the dermointegumentary tissue of fetuses and adult specimens, are the commonly used source of nuclear donor cells in the pig cloning procedure [ 14 – 18 ]. The degree of molecular and epigenetic differentiation of these cells that is related both to the advanced methylation profile of DNA cytosine residues and to the lysine deacetylation profile of histones forming nucleosomal core of nuclear chromatin often seems to make the converting of the abovementioned covalent modifications back to a totipotent state of embryonic (zygotic) cells impossible.…”

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

“…Exogenous nucleoplasmic and cytoplasmic factors derived from the nuclear donor cell, which are responsible for modulating the epigenetic status of genomic DNA, are incorporated together with oocyte mRNA transcripts and proteins, into the remodeled nucleus of the somatic cell (the so-called pseudo-pronucleus). The pseudo-pronucleus is formed following artificial activation of the embryonic developmental program of the reconstructed oocyte [1,[128][129][130][131][132][133][134][135][136][137]. In turn, an overabundance of the somatic cell-derived agents modulating the epigenetic profile of the donor nucleus may remarkably reduce the concentration and activity of the oocyte's epigenetic factors.…”

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development

“…For activation, several effective protocols for artificial oocyte activation have been developed. However, most protocols require prolonged exposure to non-specific inhibitors with high variability in results (Bang et al, 2013;Liu et al, 2015;Li et al, 2017). Hence, it has been shown that the use of agents that mimic the physiological activation events, such as the zinc chelator TPEN, through the manipulation of Ca 2þ and Zn 2þ , improved chemical activation and developmental rates of porcine oocytes (Lee et al, 2015;Macedo et al, 2019).…”