Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid...

Refined profiling of conjugated estrogens and androgens during equine pregnancy using liquid chromatography-tandem mass spectrometry (LC-MS/MS) could provide accurate fetal-sex determination. Current methods for fetal-sex prediction remain limited by timing, accuracy, and operator expertise. This study investigated sex-specific differences in maternal conjugated steroid profiles to develop a reliable, non-invasive predictive method. Samples were collected from 141 mares of various breeds starting at sixteen weeks of pregnancy. The samples were pooled according to gestational stage, divided into 28-day blocks, and analysed using a validated LC-MS/MS method. Quantified analytes included estrone, estradiol, equilin sulfates, glucuronides, and dehydroepiandrosterone sulfate. Method validation encompassed linearity, trueness, precision, accuracy, uncertainty, quantification limits, recovery, matrix effects, carryover, sensitivity, and stability. The effects of fetal sex, breed, parity, and maternal age on steroid concentrations were investigated. Data were split into a Test group for model development and a Validation group for performance assessment. Fetal sex was the principal factor influencing conjugated estrogen and dehydroepiandrosterone profiles (p = 0.01-0.0001). Female pregnancies exhibited higher classical conjugated steroid concentrations at block 5, whereas male pregnancies showed delayed elevations persisting until term. Equilin derivatives were consistently lower in males. Estrone sulfate (E1S) showed reliable univariate performance for fetal-sex prediction (area under the curve (AUC) 0.710-0.865, blocks 5-10). Multivariate models using all conjugated steroids achieved superior accuracy (AUC 0.747-0.912, blocks 5-11), providing an alternative method to univariate model and ultrasonography. The validated LC-MS/MS method improves understanding of equine feto-placental endocrinology and offers a non-invasive tool for practical fetal sexing.