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In the dairy cow, late gestation and early lactation are characterized by a complexity of metabolic processes required for the homeorhetic adaptation to the needs of fetal growth and milk production. Skeletal muscle plays an important role in this adaptation. The objective of this study was to characterize the metabolome in skeletal muscle (semitendinosus muscle) and in serum of dairy cows in the context of the physiological changes occurring in early lactation and to test the effects of dietary supplementation (from d 1 in milk onwards) with conjugated linoleic acids (sCLA; 100 g/d; supplying 7.6 g of cis-9,trans-11 CLA and 7.6 g of trans-10,cis-12 CLA per cow/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). The metabolome was characterized in skeletal muscle samples collected on d 21 and 70 after calving in conjunction with their serum counterpart using a targeted metabolomics approach (AbsoluteIDQ p180 kit; Biocrates Life Sciences AG, Innsbruck, Austria). Thereby 188 metabolites from 6 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and hexoses) were quantified in both sample types. In both groups, dry matter intake increased after calving. It was lower in sCLA than in CTR on d 21, which resulted in reduced calculated net energy and metabolizable protein balances. On d 21, the concentrations of dopamine, Ala, and hexoses in the skeletal muscle were higher in sCLA than in CTR. On d 21, the changed metabolites in serum were mainly long-chain (>C24) diacyl phosphatidylcholine PC (PC-aa) and acyl-alkyl phosphatidylcholine (PC-ae), along with lysophosphatidylcholine acyl (lysoPC-a) C26:1 that were all lower in sCLA than in CTR. Supplementation with CLA affected the muscle concentrations of 22 metabolites on d 70 including 10 long-chain (>C22) sphingomyelin (SM), hydroxysphingomyelin [SM(OH)], PC-aa, and PC-ae along with 9 long-chain (>C16) lysoPC-a and 3 metabolites related to amino acids (spermine, citrulline, and Asp). On d 70, the concentrations of lysoPC-a C18:2 and C26:0 in serum were higher in the sCLA cows than in the CTR cows. Regardless of treatment, the concentrations of Ile, Leu, Phe, Lys, His, Met, Trp, and hydroxybutyrylcarnitine (C4-OH) decreased, whereas those of ornithine, Gln, and trans-4-hydroxyproline (t4-OH-Pro) increased from d 21 to 70 in muscle. The significantly changed metabolites in serum with time of lactation were 28 long-chain (>C30) PC-ae and PC-aa, 7 long-chain (>C16) SM and SM(OH), along with lysoPC-a C20:3 that were all increased. In conclusion, in addition to other significantly changed metabolites, CLA supplementation mainly led to changes in muscle and serum concentrations of glycerophospholipids and sphingolipids that might reflect the phospholipid compositional changes in muscle. The metabolome changes observed in sCLA on d 21 seem to be, at least in part, due to the lower DMI in these cows. The changes in the muscle concentrations of AA from d 21 to 70, which coincided with the steady energy and MP balances, might reflect a shift of protein synthesis/degradation balance toward synthesis., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)