Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos

Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selection. This study compared metabolomic profiles of culture media from viable Day 4 ICSI-derived equine embryos progressing to the blastocyst stage with non-viable embryos arrested at the morula stage. Thereby, hypothesizing distinct metabolic signatures associated with developmental competence. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), amino acids, lipids, and other metabolites in individually cultured embryo media were analyzed. Results showed viable Day 4 embryos consumed significantly more dihydroxyphenylalanine (DOPA) than non-viable embryos. Increased secretion of amino acid derivatives (e.g., hippuric acid, glutamate, homoarginine), glycerolipids (e.g., diglyceride DG 18:0_20:0, triglyceride TG 17:0_36:4), phosphatidylcholines (PC O-34:3, PC O-36:3), cholesteryl ester (CE 22:2), ceramides (Hex2Cer d18:1/24:1, Hex2Cer d18:1/20:0, Cer d16:1/23:0), dehydroepiandrosterone sulfate (DHEAS), and glycodeoxycholic acid (GDCA) were observed in viable early embryos. These findings suggest metabolomic profiling can identify biomarkers of equine embryo viability as early as Day 4, potentially enabling early embryo selection, shorter culture period and improved vitrification and transfer outcomes. This study represents one of the first comprehensive metabolomic analyses of equine ICSI embryos, laying a foundation for developing non-invasive, biomarker-based assays to enhance assisted reproductive technologies (ART) efficiency.