Non-Invasive Identification of Sex in Cultured Bovine Embryos by UHPLC-MS/MS Metabolomics.

Introduction: Different gene expression between male and female bovine embryos leads to metabolic differences.

Objective: We used UHPLC-MS/MS to identify sex metabolite biomarkers in embryo culture medium (CM).

Methods: Embryos were produced in vitro under highly variable conditions, i.e., fertilized with 7 bulls, two breeds, and cultured with BSA or BSA + serum until Day-6. On Day-6, embryos were cultured individually for 24 h. CM of Day-7 embryos (86 female and 81 male) was collected, and Day-6 and Day-7 embryonic stages recorded.

Results: A study by sample subsets with fixed factors (culture, bull breed, and Day-6 and Day-7 stages) tentatively identified 31 differentially accumulated metabolites through 182 subsets. Day-6 and Day-7 stage together affected 13 and 11 metabolites respectively, while 19 metabolites were affected by one or another stage and/or day. Culture supplements and individual bull changed 19 and 15 metabolites, respectively. Single bull exerted the highest influence (20 metabolites with the significantly highest p values). Lipid (93 subsets; 11 metabolites) and amino acid (55 subsets; 13 metabolites) were the most relevant classes for sex identification.

Conclusions: Single biomarker led to inefficient sex diagnosis, while metabolite combinations accurately identified sex. Our study is a first in non-invasive sex identification in cattle by overcoming factors that induce metabolic variation.