Despite mare’s milk being highly nutritious and beneficial to human health, the effects of different dietary regimens on the lipid profile of mare’s milk remain to be completely understood. Herein we employed targeted multiple reaction monitoring-based lipidomics with ultra-high performance liquid chromatography–quadrupole time-of-flight–mass spectrometry to compare the milk lipid profiles of mares fed: pasture grass (P), corn stover and concentrate (H), or cornsilage and concentrate (S). Overall, 461 lipids belonging to 17 subclasses were identified; 261 significantly different lipids were identified on applying the following criteria: false discovery rate-adjusted P < 0.05, variable importance in projection ≥ 1.0, and fold change ≥ 2.0 or ≤ 0.5. The number of significantly different lipids decreased in the order of P > S > H. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis using differential lipids led to the identification of 34, 32, and 31 metabolic pathways on pairwise comparisons between the P and H, P and S, and S and H groups, respectively. The milk of mares in the P group showed significantly higher levels of specific lipids, including triacylglycerols, phosphatidylcholines, and phosphatidylserines, which are known to participate in polyunsaturated fatty acid and lipid digestion and absorption-related metabolic pathways. Altogether, our data provide comprehensive insights into the correlations between feeding systems and quality of mare’s milk in terms of bioactive profile.

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