Morphokinetic-related response to stress in individually cultured bovine embryos.

The kinetics of in vitro-produced (IVP) bovine embryos is related to embryo viability, metabolism, and epigenetic patterns. Therefore, we believe that embryos with different speeds of development also respond differently to stress. In the present study, we performed global metabolic analysis (matrix-assisted laser desorption ionization time of flight mass spectrometry [MALDI-TOF]) of culture media, characterized apoptotic events (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] and caspase quantitation), and quantified transcript abundance of stress-related gene (real-time quantitative polymerase chain reaction [qRT-PCR]) in IVP bovine embryos with different developmental kinetics to investigate possible markers of stress response. For this purpose, embryos were considered "fast" if they presented four or more cells at 40 hours post insemination (hpi). Embryos presenting two cells at this time were classified as "slow". Evaluations were performed at 40 hpi, 112 hpi, and 186 hpi. Metabolome analysis revealed several metabolites differentially represented between groups at all time points related with energy, lipid and amino acids metabolism, and stress response. There was no difference in TUNEL positive cells between groups in any of the time points analyzed. Nevertheless, at 112 hpi, classified as a critical phase because of the genome activation, the amount of caspase 3 and 7 and total caspase were higher in slow when compared to fast group. Transcript abundance analysis of candidate genes (GRP78, HSP60, SOD1, and MORF4L2) was also different among groups. In conclusion, IVP bovine embryos of different development speeds respond differentially to the environmental stress leading to different metabolome patterns and apoptosis activation throughout the culture.

Rapid and noninvasive technique to assess the metabolomics profile of bovine embryos produced in vitro by Raman spectroscopy.

Morphological assessments are used to select embryos with the highest implantation potential, however it is still very limited. The development of new technologies, such as Raman spectroscopy have improved quantitative and qualitative analysis, and consequently led to a better characterization of embryos and improvements on the prediction of their potential. Therefore, we propose a method based on the conventional in vitro culture system of bovine embryos, and the subsequent analysis of the culture media drops by Raman spectroscopy. Our results obtained by PCA analysis clearly showed a separation of the spectral profiles from culture media drops with and without embryos.