Integrating morphological embryo quality scoring and untargeted metabolomics identifies candidate biomarkers in Bali cattle embryos

Abstract

Background and Aim: Embryo quality is a major determinant of the efficiency of in vitro embryo production in cattle. Although morphological assessment is routinely used for embryo selection, it may not fully reflect the underlying metabolic state. Metabolomics may complement morphological evaluation by identifying biochemical characteristics associated with embryo quality. This study aimed to evaluate Bali cattle embryo quality and identify candidate metabolite biomarkers by integrating multi-parameter morphological scoring with liquid chromatography–high-resolution mass spectrometry (LC–HRMS)-based untargeted metabolomics. Materials and Methods: Thirty Bali cattle embryos produced through in vitro fertilization were evaluated using six morphological parameters: general embryo structure, blastomere uniformity, fragmentation, cellular compaction, zona pellucida condition, and cellular degeneration. Embryos were classified as good- or poor-quality according to their composite quality scores. Three embryos from the same quality group were pooled per metabolomic sample, generating 10 analytical pools (five/group). Untargeted LC–HRMS metabolomics, differential metabolite analysis, multivariate analysis, Kyoto Encyclopedia of Genes and Genomes-based functional analysis, receiver operating characteristic analysis, and correlation analysis were performed. Candidate biomarkers were prioritized using differential patterns, variable importance in projection scores >1, pathway involvement, area under the curve values >0.8, and significant associations with embryo quality parameters. Results: Good quality embryos had significantly higher general embryo structure, blastomere uniformity, cellular compaction, zona pellucida condition, and total quality scores. Metabolomic profiles differed between quality groups. Nine metabolites were prioritized as candidate biomarkers. Pyruvate, citrate, (S)-malate, 2-oxoglutarate, nicotinamide adenine dinucleotide phosphate, and L-glutamate were higher in good quality embryos, whereas succinate, oxidized glutathione, and L-tyrosine were higher in poor-quality embryos. These metabolites were principally associated with energy metabolism, amino acid metabolism, and redox regulation and showed significant associations with morphological embryo quality parameters. Conclusion: Integrating morphological scoring with untargeted metabolomic profiling revealed coordinated metabolic differences associated with Bali cattle embryo quality. The nine-metabolite panel represents a set of candidates, rather than validated, biomarkers because the metabolomic analysis was based on pooled embryos and a small discovery dataset. Individual embryo analysis and validation in larger independent cohorts are required before predictive or practical application. Keywords: Bali cattle, candidate biomarkers, embryo quality, energy metabolism, in vitro fertilization, metabolomics, oxidative balance, untargeted metabolomics.

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