PS13-13. Mammary Gland Metabolomics Due To Feeding Rumen-Protected Methionine During A Heat Stress Challenge in Lactating Holstein Cows.

Abstract Heat stress (HS) can damage epithelial tissue, compromising absorption and nutrient uptake, and reducing production performance. We investigated the effects of rumen-protected Met (RPM, Mepron®, Evonik Operations GmbH) prior to and during a HS challenge using electric heat blankets (EHB) on mammary tissue metabolomics. After a 7-d adaptation on a basal diet without RPM, eleven lactating multiparous Holstein cows (711 ± 28 kg BW; 28 ± 2.0 kg milk/d) were assigned to a control (no RPM, n = 5) or an RPM-supplemented diet (0.10% of diet DM; RPM, n = 6) for 31-d including a HS challenge with EHB during the last 10-d. After completion of the last d of HS, cows were euthanized via captive bolt for collection of mammary tissue. Production data were analyzed using PROC MIXED in SAS. A total of 324 known and 2,324 putative metabolites were identified via LC-MS and analyzed using tools in MetaboAnalyst 6.0. Prior to HS, feeding RPM led to greater (P < 0.05) energy-corrected-milk (ECM, 35 vs. 29 kg/d), milk protein % (3.61 vs. 3.32) and yield (1.02 vs. 0.81 kg/d). During the HS challenge DMI was not affected, but ECM tended (P = 0.07) to be greater and milk protein % (3.51 vs. 3.33) and yield (1.13 vs. 1.01 kg/d) were greater (P < 0.05) due to RPM. Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) of known metabolites revealed distinct patterns between RPM and controls. At a conservative Variable Importance in Projection score (VIP) ≥1.0 there were >80 metabolites that contributed significantly to the unique profiles due to RPM. Analysis of biological functions among the top-15 discriminant metabolites, all of which decreased with RPM except for oleoyl-L-carnitine, suggested coordinated shifts in lipid metabolism, mitochondrial function, and one-carbon/propionate flux. The most striking feature was the marked increase with RPM of the phospholipids N-palmitoyl-D-sphingosine and ceramide (18:1 and 16:0), both of which are involved in barrier function and cell signaling. The increases in palmitoylcarnitine, oleoyl-L-carnitine, stearoyl-L-carnitine with RPM suggested higher fatty acid flux into mitochondria. Similarly, the marked increase in ursocholic acid with RPM indicated a potential beneficial effect of this bile acid against oxidative stress and inflammation, which are hallmarks of HS in cells. Preliminary integration of production and metabolomics data indicates that feeding RPM favorably remodels mammary tissue metabolism under HS, supporting epithelial barrier integrity, mitochondrial lipid flux, and redox balance, which likely contributes to improved milk protein synthesis and sustained production performance during HS.