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Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium.
Journal of Chromatography. A 2016 November 26
Metabolic differences between early male and female embryos can be reflected in culture medium (CM). We used a single bovine embryo culture step (24h) supporting improved birth rates under chemically defined conditions (CDC) to investigate biomarker detection of embryonic sex in contrast to classical BSA-containing medium. In vitro matured slaughterhouse oocytes were fertilized in vitro with a single bull. Embryos were initially cultured in synthetic oviduct fluid with BSA. On day-6, morulae were cultured individually in droplets with (BSA) or without protein (CDC). On day-7, expanded blastocysts were sexed (amelogenin gene amplification) and CM was stored at -145°C until metabolomic analysis by UHPLC-TOF MS. N=10 embryos per group (i.e. male-protein; female-protein; male-non-protein; female-non-protein) were produced. Statistical analysis revealed N=6 metabolites with different concentrations in CM, N=5 in male embryos (methionine, tryptophan, N-stearoyl-valine, biotin and pipecolic acid), N=1 in female embryos (threonine) (P<0.05 in BSA; P<10-7 in CDC). Only the clear threshold between males and females in CDC allowed correct classification of 100% males and 91% females within 5 out of 6 biomarkers (one female outlier showing the male biomarker profile). The use of CDC represents a critical aspect in the efficient detection of embryonic sex biomarkers by metabolomics.
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