Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process

Abstract

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

Figure 1

Experimental design. Eight Holstein heifers enabled the production of in vitro and in vivo embryos by OPU-IVF and embryo collection procedures. After seven days of development, all grade 1 expanded blastocysts were biopsied and sexed. Half of each group underwent lipid extraction in a fresh state, the other half were first frozen before undergoing lipid extraction. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry and allowed the detection of 496 features.

Figure 2

(A) Principal component analysis plot (PCA) representing variance among in vivo and in vitro embryos according to principal component analysis. The orange dots show data for embryos with in vivo origin, and the blue dots show data for embryos with in vitro origin. (B) Multivariate analysis by orthogonal partial least square discriminant analysis (O-PLS-DA), discriminating the embryonic origins (vivo vs. vitro) according to the lipid profile of the embryos. (C) Univariate analysis by volcano plot based on fold-change and p value, highlight several lipids. Blue and orange dots correspond to significantly different lipids between in vitro and in vivo produced embryos. The grey dots represent the non-significant ones. Pink dots correspond to lipids with a significant p value but a fold-change between 0.66 and 1.5. All the lipids to the right of this area are over abundant in the in vitro produced embryos (blue dots), while those that are to the left of this area are over abundant in the in vivo produced embryos (orange dots). Statistical significance is determined at p < 0.05 and fold-change greater than 1.5 or less than 0.66. Significantly different annotated lipids are represented on the volcano plot by the following abbreviations, phosphatidyl-ethanolamine (PE), inositol (PI), serine (PS), glycerol (PG), triglycerides (TG) and oxidised glycerophospholipid (OHHdia-PS and PKHdiA-PS).

Figure 3

(A) Principal component analysis (PCA) plot representing variance among in vivo fresh and frozen embryos according to principal component analysis. The red dots show data for fresh in vivo produced embryos, and the green dots show data for frozen in vivo produced embryos. (B) Multivariate analysis by orthogonal partial least square discriminant analysis (O-PLS-DA), discriminating the state before and after freezing (fresh vs. frozen) according to the lipid profile of the embryos. (C) Univariate analysis via volcano plot based on fold-change and p value, highlighted several lipids. Red and green dots correspond to significantly different lipids between fresh and frozen in vivo produced embryos. Pink dots correspond to lipids with significant p values but a fold-change between 0.66 and 1.5. All the lipids to the right of this area are over abundant in the frozen in vivo produced embryos (green dots), while those on the left of the grey area are overabundant in the fresh in vivo produced embryos (red dots). Statistical significance is determined at p < 0.05 and fold-change greater than 1.5 or less than 0.66. Significantly different annotated lipids are represented on the volcano plot by the following abbreviations, lysophosphatidylcholine (LPC), phosphatidylglycerol (PG), monoacylglycerol (MG) and diacylglycerol (DG).

Figure 4

(A) Principal component analysis (PCA) plot representing variance among in vitro fresh and frozen embryos according to principal component analysis. The red dots show data for fresh in vitro produced embryos, and the green circles show data for frozen in vitro produced embryos. (B) Multivariate analysis by orthogonal partial least square discriminant analysis (O-PLS-DA), discriminating the state before and after freezing (fresh vs. frozen) according to the lipid profile of the embryos. (C) Univariate analysis via volcano plot based on fold-change and p value, highlighted several lipids. Red dots correspond to significantly different lipids between fresh and frozen in vitro produced embryos, particularly to those overabundant in fresh embryos, while the grey ones represent the non-significant ones. Statistical significance is determined at p < 0.05 and fold-change greater than 1.5 or less than 0.66. Significantly different annotated lysophosphatidylcholine (LPC) are represented on the volcano plot.

Figure 5

(A) Principal component analysis (PCA) plot representing variance among male and female embryos according to principal component analysis (B) Multivariate analysis by orthogonal partial least square discriminant analysis (O-PLS-DA), discriminating male and female embryos according to their lipid profile. The light-blue dots show data for male embryos, and the yellow dots show data for female embryos. (C) Univariate analysis via volcano plot based on fold-change and p value, did not highlight any lipids. The grey dots represent the non-significant lipids. Statistical significance is determined at p < 0.05 and fold-change greater than 1.5 or less than 0.66.

Figure 6

(A) Representation of differential lipids between fresh in vivo and in vitro embryos through a Venn diagram. In vivo produced embryos are represented by an orange ellipse and in vitro produced embryos are represented by a blue ellipse. (B) Representation of lipid differentials between fresh (red ellipse) and frozen (green ellipse) in vivo produced embryos through a Venn diagram. (C) Representation of lipid differentials between fresh (red ellipse) and frozen (green ellipse) in vitro produced embryos through a Venn diagram.