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Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

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Abstract

Introduction

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

Objectives

To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.

Methods

Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.

Results

It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.

Conclusion

We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

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Acknowledgments

The authors would like to thank São Paulo Research Foundation (FAPESP) (grant number 2012/10351-2) and Coordination for the Improvement of Higher Education Personnel (CAPES) for financial support. The authors would also like to thank the Multiuser Mass Spectrometry Laboratory at Sao Paulo Federal University, Fernanda Bertuccez Cordeiro and Augusto Azzolini for the experimental support.

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Authors and Affiliations

  1. Center of Natural and Human Sciences, Universidade Federal do ABC, Rua Santa Adélia, 166, Bloco A, Torre 3, S642, Santo André, SP, Brazil

    Érika Cristina dos Santos, Kelly Annes & Marcella Pecora Milazzotto

  2. Department of Biomedical Sciences, University of São Paulo, Avenida Prof. Lineu Prestes, 2415, Edifício ICB III, Cidade Universitária, SP, Brazil

    Camila Bruna de Lima & Marcella Pecora Milazzotto

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  1. Érika Cristina dos Santos
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  2. Camila Bruna de Lima
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Correspondence to Marcella Pecora Milazzotto.

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All procedures and protocols were performed in accordance with the Ethical Principles in Animal Research set forth by the Brazilian College of Animal Experimentation, with approval from “Ethic Committee in the Use of Animal” of Universidade Federal do ABC (Protocol No. 008/2014).

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dos Santos, É.C., de Lima, C.B., Annes, K. et al. Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production. Metabolomics 12, 94 (2016). https://doi.org/10.1007/s11306-016-1029-2

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