Factors relating to expression of <i>IGF2R</i>, <i>IFNΤ</i>, <i>AKR1B1</i>, and <i>PLAC8</i> at the blastocyst stage.

<p>Relationship between <i>IGF2R</i> and timing of the first cleavage (<b>A</b> and <b>B</b>), <i>IFNΤ</i> and timing of the first cleavage (<b>C</b> and <b>D</b>), <i>IFNΤ</i> and the number of blastomeres at the end of the first cleavage (<b>E</b>), <i>AKR1B1</i> and the presence or absence of multiple fragments at the end of the first cleavage (<b>F</b>), and <i>PLAC8</i> and oxygen consumption at the blastocyst stage (<b>G</b> and <b>H</b>) are shown. Relative expression is shown as the fold change from the mean value of <i>in vivo</i>–derived blastocysts, which was set at 1. **Coefficient of determination, <i>r</i>, was statistically significant based on simple regression analysis (<i>P</i><0.01) (<b>A</b>, <b>C</b>, and <b>G</b>). Different letters indicate significant differences between groups based on one-way ANOVA followed by Fisher's PLSD (^A,B<i>P</i><0.01) (<b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b>). Gene expression in blastocysts classified based on timing, blastomere number, the presence or absence of multiple fragments at the end of the first cleavage, and oxygen consumption of blastocysts at 168 hpi (<b>I</b>). Embryos displaying similar patterns of expression for these five genes were grouped together on closely connected branches of the dendrogram with the same colors. The color map indicates normalized expression values that were based on <i>in vivo</i>–derived blastocysts for each gene examined. Red represents high expression; black, approximately equal expression; and green, low expression as compared with <i>in vivo</i>–derived blastocysts.</p

The effect of the number of blastomeres at the onset of the lag-phase on cell numbers, ICM ratio, and apoptosis incidence.

<p>The number of total, ICM, and TE cells, the ICM ratio, and the apoptotic incidence in blastocysts with different numbers of blastomeres at the onset of the lag-phase are shown in <b>A</b> and <b>D</b>, respectively. Embryos were separated into groups based on high (<b>B</b> and <b>E</b>) or low oxygen consumption (<b>C</b> and <b>D</b>) with a cut-off of 0.84×10⁻¹⁴ mol s⁻¹. Data are presented as the mean ± SD. Different letters indicate significant differences between groups based on one-way ANOVA followed by Fisher's protected least signicicant difference (PLSD) (^A,B<i>P</i><0.01; ^a,b<i>P</i><0.05).</p

Correlation between oxygen consumption at the blastocyst stage and number of total (A), ICM (B), and TE (C) cells and the percentage of ICM cells relative to total cell number (A4).

<p>**Coefficient of determination, <i>r</i>, was statistically significant based on simple regression analysis (<i>P</i><0.01). Relationship between oxygen consumption at the blastocyst stage and hatching after 48 additional hours of culture (<b>D</b> and <b>E</b>). The level of oxygen consumption was considered high or low with a cut-off of 0.84×10⁻¹⁴ mol s⁻¹ (<b>E</b>). Double asterisks indicate significant differences between groups based on the χ² test (<i>P</i><0.01).</p

Classification matrices for selected regression models illustrating the prediction accuracy of variables examined either individually or in combination.

a<p>The percentage of pregnancies correctly classified as a proportion of those blastocysts predicted to result in a successful pregnancy.</p>b<p>The percentage of embryos correctly classified as resulting in no pregnancy and pregnancy (the hit ratio).</p>c<p>Conventional variables based on the stage and morphological quality code of blastocysts at the time of transfer.</p>d,e<p>Different letters indicate a significant difference (P<0.05). Data were analyzed with the Yates' corrected χ² test.</p

Overview of the experimental scheme.

<p><i>In vitro</i> development of bovine IVF embryos in the developed microwell culture dish with identification code (<b>A</b> and <b>B</b>) was tracked with time-lapse cinematography (TLC) for 168 h post-insemination (hpi). SECM was used to measure oxygen consumption in embryos that developed to the blastocyst stage at 168 hpi (<b>C</b>). Timing of the of the first cleavage; duration of the second, third, and lag-phases resulting in developmental arrest at the fourth or fifth cell cycle; number of blastomeres; presence or absence of multiple fragments; unevenness or evenness of division at the end of the first cleavage and at the onset of the lag-phase; cell cycle observed lag-phase (fourth or fifth cell cycle); and oxygen consumption at 168 hpi were examined in relation to blastocyst qualities such as cell number (n = 173), apoptosis incidence (n = 74), hatchability (n = 195), karyotype (n = 111), and gene expression (n = 75) with multivariate analysis. Based on the identified prognostic factors that reflected the various blastocyst qualities, OPU-IVF blastocysts (n = 52) were then assessed and transferred to recipient cows (n = 52). (<b>D</b>) The number of blastomeres at the end of the first cleavage was categorized as 2 and 3/4 blastomeres. The numbers of blastomeres at the onset of the lag-phase were categorized as 4/5, 6–8, and 9–16 based on the result from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036627#pone.0036627.s001" target="_blank">Fig. S1</a>. Bar = 10 mm (<b>A</b>), 300 µm (<b>B</b>) and 100 µm (<b>C</b>).</p

Involvement of cytokinesis during <i>in vitro</i> development on chromosome complementation.

<p>(<b>A</b>) Two sets (2n = 60), one set (n = 30), and more than two sets of chromosomes (3n, 4n, 5n etc.) were considered diploid, haploid, and polyploid, respectively. The arrowheads indicate the X chromosome. Bar = 10 µm. (<b>B</b>) The timing of the first cleavage in diploid embryos (defined as blastocysts in which all nuclei were diploid) and non-diploid embryos (defined as blastocysts with more or fewer than two sets of chromosomes in the analyzed nuclei). Different letters indicate significant differences between groups based on one-way ANOVA followed by Fisher's PLSD (^A,B<i>P</i><0.01). (<b>C</b>) Relationship between timing of the first cleavage and the incidence of diploid embryos. Blastocysts were divided into two groups, fast and slow, based on the timing of the first cleavage with a cut-off of 27 hpi (<b>C</b>). Double Asterisks indicate significant differences between groups based on the χ² test (<i>P</i><0.01). Ploidy (<b>D</b>–<b>F</b>) and incidence of abnormal chromosomes per embryo (<b>G</b>–<b>J</b>) in blastocysts derived from embryos with 2 and 3/4 blastomeres at the end of the first cleavage (<b>D</b> and <b>G</b>); rapidly cleaving and slowly cleaving embryos at the end of the first cleavage (<b>E</b>, <b>H</b>, and <b>I</b>); and embryos with 4/5, 6–8, and 9–16 blastomeres at the onset of the lag-phase (<b>F</b> and <b>J</b>) are shown. **Coefficient of determination, <i>r</i>, was statistically significant based on simple regression analysis (<i>P</i><0.01) (<b>H</b>). Different letters indicate significant differences between groups based on one-way ANOVA followed by Fisher's PLSD (^A,B<i>P</i><0.01) (<b>I</b>). The incidence of diploid blastocysts (<b>K</b>) and the incidence of abnormal chromosomes per blastocyst (<b>L</b>) were classified according to the timing and blastomere number at the end of the first cleavage and the blastomere number at the onset of the lag-phase.</p