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In the equine industry, horse breeders aim to produce one foal per mare per year. Thus, mares are bred while nursing. In high-yielding dairy cattle, concurrent lactation and conception may affect embryo quality, but effects on uterine fluid (UF) quality are unknown. No data are available in horses. The aim of our study was to analyse the effect of nursing on protein, metabolite and fatty acid (FA) compositions of UF at ~7.5 days post-ovulation. Anglo-Arab mares (multiparous (2.7 ± 0.9 foals), 10.8 ± 2.5 years old) that were either nursing (N; 105 ± 12 days of lactation) or barren (B) were inseminated with the semen of the same stallion at induction of ovulation with human chorionic gonadotrophin. Ovulation was confirmed by ultrasound within 48 h. At 7.4 ± 0.7 days post-ovulation, UF was collected with a human tampon that was left for 10 min in the uterus before embryo collection. Only mares for which no embryo was collected were selected (n = 5 in both groups). Trypsin digestion followed by liquid chromatography-tandem mass spectrometry analysis was used for proteomic analysis, with subsequent characterisation with the PANTHER platform using an overrepresentation test (Fisher's exact type with false discovery rate correction) with the PANTHER pathway database. An untargeted approach based on liquid chromatography-tandem mass spectrometry was performed for metabolomics (normalisation to 50 µg of protein), and data were analysed with the MS Peaks to Pathways tool of the MetaboAnalyst platform, using both the GSEA and mummichog algorithms (cut-off = 0.05). Fatty acid composition (% of total FA) was analysed using gas chromatography. Differences between groups were analysed using a linear model with permutation using R software with the pgirmess package (P = 0.05 for significance). Altogether, 2706 proteins with at least two peptides were identified in mares’ UF, with 164 being differentially expressed. Ubiquitin proteasome, involved in embryo-endometrium interactions, was the most enriched pathway in N mares (fold enrichment = 15.12; P