Effect of Embryo Density on <i>In Vitro</i> Development and Gene Expression in Bovine <i>In Vitro</i>-fertilized Embryos Cultured in a Microwell System

Abstract

To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development

“…In the present study, there was no reduction in blastocyst rate and quality in a WOW dish. This result is in agreement with previous findings that the development of embryos to blastocysts was independent of the total number of embryos in a WOW dish (Sugimura et al, 2013). We speculated that diffusible factors such as autocrine/paracrine growth factors released by embryos can be diffused in a droplet and influence the growth of their adjacent embryos (Stokes et al, 2005).…”

Prediction of blastocyst development and implantation potential <i>in utero</i> based on the third cleavage and compaction times in mouse pre-implantation embryos

“…The time of the first cleavage alters gene expression associated with implantation potential, and slowly cleaving embryos exhibit reduced viability, chromosomal abnormalities, and decreased oxygen consumption at the blastocyst stage, thereby lowering hatching and pregnancy rates [50, 51]. A higher mitochondrial DNA copy number is also associated with a higher rate of zygotic cleavage in humans and pigs [52, 53].…”

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

“…[111][112][113] Expression levels of both IFN-tau and IGF2R depended on embryo density when the embryos were maintained in droplet culture. 114 Up-regulated IFN-tau expression and down-regulated IGF2R expression were observed when embryos were cultured in groups of 25 embryos, while no differences were found in the well-of-the-well (WOW) system culture. 114 Increased embryo density appears to enhance the accumulation of toxic by-products of embryo metabolism such as ammonium.…”