96 Embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles) on porcine embryos

Egg yolk constitutes about one-third of the whole chicken egg structure and is enriched with the metabolites and nutrients that are essential for chicken embryonic development. However, the effects of egg yolk-derived lipid membraneous vesicles on embryo development were not yet elucidated. In the current study, for the first time, egg yolk nanovesicles (vitellovesicles [VVs]) were isolated, characterised, and used as a supplement for porcine embryo culture. The egg yolks of 10 freshly oviposited eggs were filtered and subjected to ultracentrifugation at 100,000 × g for 3 h to obtain a pellet. The pellet was examined through Cryo-TEM and nanoparticle tracking analysis and revealed bilipid membranous vesicles with an average diameter of 158.6 ± 20.5 nm and 1.6 × 10¹⁰ particles/mL. The protein contents of the pellet were treated with trypsin to obtain tryptic peptides that were further analysed through MS and MS/MS (LTQ-Velos ESI ion trap mass spectrometer, Thermo Scientific), and MASCOT 2.4 was used to analyse MS/MS data. The top protein cargo contents of the VVs revealed with proteomics included albumin (58%), ovotransferrin (7.2%), apovitellenin-1 (16.9%), apolipoprotein A (5.3%), apolipoprotein B (4.5%), and vitellogenin-2 (1.2%). VVs (1.6 × 10⁸ /mL) were supplemented with the in vitro culture medium of Day-7 hatched parthenogenetic blastocysts (n = 160, 8 replicates of 10 blastocysts from each control and VVs-supplemented group). After two days of blastocyst culture, the total cell count was significantly increased in VVs-supplemented embryos when compared with the control, nonsupplemented embryos (62.2 ± 5.3 vs 21.4 ± 4.6, respectively; P < 0.05, Student’s t-test). Moreover, the TUNEL assay showed that apoptotic cells were increased in control groups when compared with VVs-supplemented group (23.1 ± 3.8% vs 2.4 ± 0.9%, respectively; P < 0.05, Student’s t-test). Furthermore, reduced glutathione was 2.5-fold increased in VVs-supplemented group while reactive oxygen species (ROS) were 5.3-fold increased in control groups. qPCR analysis showed that VVs significantly increased the expression of lipid metabolism-associated genes (MGLL and LIPE), anti-apoptotic gene (BCL2), and superoxide dismutase (SOD-1), and significantly reduced apoptotic gene (BAX) (P < 0.05, Student’s t-test). Culturing embryos on Matrigel basement membrane matrix indicated that VVs significantly enhanced embryo attachment and embryonic stem cell outgrowths when compared with the control group (77.7% vs 38.8%; P < 0.05, chi-squared test). To the best of our knowledge, this is the first evidence that indicates an embryotrophic effect of the VVs on mammalian embryos, and this effect might be attributed to the protein cargo contents of the VVs; however, further transcriptomic and metabolomic characterisation of VVs cargo contents is still required. The VVs can be used for the formulation of a novel in vitro culture medium for supporting mammalian embryo development.

This work was supported by NRF grants # 2021H1D3A2A02040098, 2022R1I1A1A01065412, and 2021R1A2C2009294.