Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium.
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<10-7in 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.
Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Centro de Biotecnología Animal, Área de Genética y Reproducción Animal, Camino de Rioseco 1225, 33394 Gijón, Spain, Spain.
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Gómez
Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Centro de Biotecnología Animal, Área de Genética y Reproducción Animal, Camino de Rioseco 1225, 33394 Gijón, Spain, Spain. Electronic address: mmunoz@serida.org.
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Muñoz
Foodomics Lab & Metabolomics Platform, CIAL, CSIC, Nicolas Cabrera 9, 28049 Madrid, Spain.
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Simó
Foodomics Lab & Metabolomics Platform, CIAL, CSIC, Nicolas Cabrera 9, 28049 Madrid, Spain.
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Ibáñez
Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Centro de Biotecnología Animal, Área de Genética y Reproducción Animal, Camino de Rioseco 1225, 33394 Gijón, Spain, Spain.
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Carrocera
Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Centro de Biotecnología Animal, Área de Genética y Reproducción Animal, Camino de Rioseco 1225, 33394 Gijón, Spain, Spain.
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Martín-González
Foodomics Lab & Metabolomics Platform, CIAL, CSIC, Nicolas Cabrera 9, 28049 Madrid, Spain.
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A
Cifuentes
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D001755:Blastocyst / Q000737:chemistry; D002417:Cattle; D003470:Culture Media; D004622:Embryo, Mammalian / Q000737:chemistry*; D047108:Embryonic Development; D005187:Fallopian Tubes / Q000737:chemistry; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D055432:Metabolomics / Q000379:methods*; D009865:Oocytes / Q000737:chemistry; D011247:Pregnancy; D012732:Sex Determination Analysis / Q000379:methods*
D000596:Amino Acids; D015415:Biomarkers; D003470:Culture Media
Biomarker; Bovine; Embryo; Metabolomics; Sex; UHPLC-TOF MS