Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

Abstract

In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, using a 5-h activation delay, the blastocyst rate was significantly higher using oocytes after 20 h IVM than after 24 h IVM; however, only 28% of oocytes were in metaphase II (MII) at 20 h. In experiment 4, oocytes were denuded of cumulus at 20 h, and those in metaphase I (MI) were returned to culture for 3 h (20+3H treatment); blastocyst rates were 30% and 27%, respectively (8-h and 5-h delay to activation, respectively). Four live foals resulted from the transfer of 17 blastocysts (24%) produced using MII oocytes and a 5- or 8-h activation delay. Use of equine oocytes immediately after reaching MII, combined with a longer delay from reconstruction to activation, increased developmental competence after equine NT.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

“…In the horse, no differences in cleavage was reported after comparing 30 min and 2 h time windows between fusion to activation, in contrast to our findings [ 46 ]. However, Choi et al [ 15 ] have recently demonstrated that the use of horse oocytes immediately after reaching MII, combined with longer time periods (5- or 8-h) from reconstruction to activation, increased developmental competence after cloning. These results contrasts with those reported in the bovine, which determined that too prolonged exposure to arrested MII oocyte cytoplasm may result in a high incidence of structural abnormalities in nuclear material [ 11 ].…”

“…For cloning to be successful, the differentiated state of the donor cell needs to be reset to an embryonic state within a relatively short period of time between embryo reconstruction and activation [ 11 , 12 ]. Some reports have focused on this step, which has demonstrated to be beneficial to epigenetic reprogramming and normal embryo development in different mammalian species [ 13 , 14 , 15 ]. However, when this interval was prolonged embryo development was compromised [ 16 , 17 , 18 ], probably due to oocyte aging [ 19 ].…”

Assisted reproductive techniques in mares

“…Somatic cells have been successfully isolated from frozen‐thawed equine semen (Brom‐de‐Luna, Canesin, Wright, & Hinrichs, ) but cloning was not attempted. Cloned foals have been produced from oocytes recovered from immature follicles by TVA (Choi, Velez, Macias‐Garcia, & Hinrichs, ; Choi et al., ) including production of a mitochondrial‐identical cloned foal via recovery of oocytes from two mares maternally related to the donor horse. Currently, few breed registries allow registration of cloned horses, however, many competitions that do not require strict breed registry allow cloned horses to compete, including the Fédération Equestre Internationale (FEI), which oversees equestrian competitions worldwide, including the Olympics.…”

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses

“…Until now, healthy foals were born only by using fetal and adult fibroblasts as nuclear donors, 5 , 16 , 29 – 32 with high rates of embryonic losses and postnatal mortality as typical outcomes, though. The most common defects observed in cloned foals included limb deformities, umbilical abnormalities and failure of passive transfer, 16 , 31 , 33 – 35 probably due to inefficient reprogramming of the donor cell.…”