204 Rumen-protected methionine modulates plasma and follicular fluid metabolites of beef cows

Methionine is an essential amino acid required for protein synthesis, DNA methylation, lipid metabolism, cell signaling, and mitochondrial function. Methionine is rapidly degraded by rumen microbes. This study aimed to evaluate the effect on the plasma and follicular fluid metabolome from supplementing a rumen-protected methionine (RPM) in the diet of Senepol cows. A total of 11 cows were randomized to receive a roughage-based diet with 15 g of RPM or no RPM (CON) in a crossover design experiment with two replicates. Cows were supplemented individually for 14 days before follicular aspiration and ovum pickup (OPU). At the time of OPU, plasma and follicular fluid were collected. A subset of six animals (three per group in both replicates) were selected randomly for quantitative targeted metabolomics analysis using the Biocrates MxP Quant500 kit (Biocrates Life Sciences, AG), which measures 630 metabolites. Individual metabolites were analyzed using the PROC MIXED procedure of SAS (SAS/STAT 15.1, SAS Institute), and a pathway analysis was conducted using Ingenuity Pathway Analysis (IPA, Qiagen) with an FDR < 0.05. The replicate did not affect metabolites concentration in plasma and follicular fluid. As expected, plasma methionine concentration increased in the RPM group (CON, 29.6 ± 1.8; RPM, 47.8 ± 1.8 µM; P < 0.01); however, glycine decreased in the RPM group (CON, 233.3 ± 16.9; RPM, 164.8 ± 16.9 µM; P < 0.05). Other metabolites that increased (P < 0.05) in RPM were acetyl carnitine (CON, 2.1 ± 0.2; RPM, 3.0 ± 0.2 µM), hydroxy valeryl carnitine (CON, 0.03 ± 0.005; RPM, 0.06 ± 0.005 µM), ceramide d18:0/24:1 (CON, 0.08 ± 0.01; RPM, 0.13 ± 0.01 nM), and triacylglycerol (TG 20:4_35:3; CON, 0.005 ± 0.0002; RPM, 0.013 ± 0.0002 µM). In the follicular fluid, methionine concentration increased in the RPM group (CON, 10.8 ± 3.6; RPM, 22.4 ± 3.6 µM; P = 0.05). Additionally, follicular fluid ceramide (CON, 0.02 ± 0.004, RPM: 0.04 ± 0.004 nM) and diacylglycerol (DG 16:1_18:2; CON, 0.001 ± 0.0007; RPM, 0.01 ± 0.0007 µM) concentration increased (P < 0.05) in RPM, while diacylglycerol concentration decreased (DG 16:0_20:4; CON, 0.10 ± 0.01; RPM, 0.01 ± 0.01 µM; P < 0.05). The pathway analysis results showed that cows in the RPM group had a positive z-score (upregulation) in 10 and 35 different pathways in the plasma and follicular fluid, respectively. Interestingly, methionine was involved in six of them in both plasma and follicular fluid (transport of bile salts, transport of inorganic cations/anions and amino acids, choline metabolism, tryptophan catabolism, sulfur amino acid metabolism, and phase II conjugation of compounds). Moreover, a negative z-score (downregulation) was observed in one pathway (tRNA charging) in plasma and follicular fluid. In conclusion, supplementation of RPM altered the metabolite composition of plasma and follicular fluid. Interestingly, different pathways were regulated by RPM in both plasma and follicular fluid.