Changes in plasma and milk choline metabolite concentrations in response to the provision of various rumen-protected choline prototypes in lactating dairy cows.

Choline feeding in the form of rumen-protected choline (RPC) has been shown to increase milk production and improve measures of metabolic health (e.g., liver triglyceride) in dairy cows. The objective was to characterize changes in plasma and milk choline and choline metabolite concentrations, including microbial-derived trimethylamine N-oxide (TMAO), in response to increasing ruminal spot-doses, different types of RPC, and ruminal stability of RPC in lactating cows. For experiment 1, 12 mid-lactation (121 ± 16.3 d in milk) Holstein cows were balanced by total plasma choline concentrations and milk yields. Cows were assigned to 1 of 3 lipid-encapsulated RPC products (main plots): prototypes P1, P2, and P3 (containing 59, 56, and 30% choline chloride, respectively). Within each main plot, cows were assigned to a sequence of doses in a 4 × 4 Latin square design: 0, 18, 36, or 54 g of choline chloride. Treatments were preconditioned with ground corn and administered as a single ruminal bolus once per experimental period 1 h postfeeding of a total mixed ration. For experiment 2, we compared a control (0 g of choline chloride) versus P2, and P4 and P5 (60 and 62% choline chloride, respectively) in a repeated 4 × 4 Latin square design. Experiment 2 followed a similar design as experiment 1 with modifications: 12 late-lactation (228 ± 7.10 d in milk) Holstein cows were used; treatments were administered as part of a premeal; and cows received a daily allowance of a total mixed ration as equal provisions every 4 h within 24 h before and after treatment. For both experiments, plasma and milk samples were collected for choline and choline metabolite quantification. Data were analyzed using a mixed model including fixed effects of treatment, period, and time. Contrast statements were used to test for linearity of dose and differences between prototypes for experiment 1 and 2, respectively. Plasma and milk TMAO concentrations increased with RPC dose (peak by h). Milk choline and betaine yields increased with RPC dose in a quadratic manner; albeit, dependent upon RPC type. Milk phosphocholine (PCho) and glycerophosphorylcholine (GPC) yields changed by select RPC dose (experiment 1), however Met, PCho, GPC, phosphatidylcholine, and total choline concentrations in milk, and plasma Met and sphingomyelin concentrations were not responsive. We conclude that plasma or milk choline, betaine, and TMAO concentrations are responsive to RPC type, dose, and stage of lactation evaluated.

Intravenous trimethylamine N-oxide infusion does not modify circulating markers of liver health, glucose tolerance, and milk production in early-lactation cows.

In rodents and humans, the gut bacteria-derived metabolite trimethylamine N-oxide (TMAO) has been implicated in the progression of cardiovascular disease, chronic kidney disease, fatty liver, and insulin resistance; however, the effects of TMAO on dairy cattle health and milk production have not been defined. We aimed to determine whether intravenous TMAO infusion modifies measures of liver health, glucose tolerance, and milk production in early-lactation cows. Eight early-lactation Holstein cows (30.4 ± 6.41 d in milk; 2.88 ± 0.83 lactations) were enrolled in a study with a replicated 4 × 4 Latin square design. Cows were intravenously infused TMAO at 0 (control), 20, 40, or 60 g/d for 6 d. Washout periods lasted 9 d. Intravenous glucose tolerance tests (GTT) occurred on d 5. Blood was collected daily. Milk was collected on d -1, 0, 5, and 6. Urine was collected on d -1 and 6. Circulating metabolites, milk components, and TMAO concentrations in milk, urine, and plasma were quantified. Data were analyzed using a mixed model that included the fixed effects of treatment. Concentrations of TMAO in plasma, milk, and urine increased linearly with increasing dose. Dry matter intake and milk production were not modified by treatment. Daily plasma triacylglycerol, fatty acid (FA), and glucose concentrations were not modified. Serum albumin, total protein, globulin, total bilirubin, direct bilirubin, aspartate aminotransferase, γ-glutamyl transferase, and glutamate dehydrogenase concentrations were also not modified by treatment. Serum GTT glucose, FA, and insulin concentrations were not modified by treatment. Plasma total, reduced, and oxidized glutathione concentrations were also not modified by treatment. We conclude that a 6-d intravenous infusion of TMAO does not influence measures of liver health, glucose tolerance, or milk production in early-lactation dairy cows.

Effects of serine palmitoyltransferase inhibition by myriocin in ad libitum-fed and nutrient-restricted ewes.

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, 12 non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from day 1 to 14 and were nutrient-restricted (straw only) from day 15 to 17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on day 17. Plasma was analyzed for free fatty acids (FFAs), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by day 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in the liver and omental and mesenteric adipose, VLC ceramide and DHCer in the pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.

Palmitate and pyruvate carbon flux in response to choline and methionine in bovine neonatal hepatocytes.

Choline and methionine may serve unique functions to alter hepatic energy metabolism. Our objective was to trace carbon flux through pathways of oxidation and glucose metabolism in bovine hepatocytes exposed to increasing concentrations of choline chloride (CC) and D,L-methionine (DLM). Primary hepatocytes were isolated from 4 Holstein calves and maintained for 24 h before treatment with CC (0, 10, 100, 1000 µmol/L) and DLM (0, 100, 300 µmol/L) in a factorial design. After 21 h, [1-14C]C16:0 or [2-14C]pyruvate was added to measure complete and incomplete oxidation, and cellular glycogen. Reactive oxygen species (ROS), cellular triglyceride (TG), and glucose and ß-hydroxybutyrate (BHB) export were quantified. Exported very-low density lipoprotein particles were isolated for untargeted lipidomics and to quantify TG. Interactions between CC and DLM, and contrasts for CC (0 vs. [10, 100, 1000 µmol/L] and linear and quadratic contrast 10, 100, 1000 µmol/L) and DLM (0 vs. [100, 300 µmol/L] and 100 vs. 300 µmol/L) were evaluated. Presence of CC increased complete oxidation of [1-14C]C16:0 and decreased BHB export. Glucose export was decreased, but cellular glycogen was increased by the presence of CC and increasing CC. Presence of CC decreased ROS and marginally decreased cellular TG. No interactions between CC and DLM were detected for these outcomes. These data suggest a hepato-protective role for CC to limit ROS and cellular TG accumulation, and to alter hepatic energy metabolism to support complete oxidation of FA and glycogen storage regardless of Met supply.