Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium

•UHPLC-TOF-MS metabolomics discovered new biomarkers for embryonic sex determination.•The same biomarkers were found under different culture media.•Chemically defined conditions provided uniform analytical responses.

Metabolic differences between early male and female embryos can be reflected in culture medium (CM). We used a single bovine embryo culture step (24 h) 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.