The onset of lactation in dairy cows is characterized by high output of methylated compounds in milk when sources of methyl group are in short supply. Methionine and choline (CHOL) are key methyl donors and their availability during this time may be limiting for milk production, hepatic lipid metabolism, and immune function. Supplementing rumen-protected Met and CHOL may improve overall performance and health of transition cows. The objective of this study was to evaluate the effect of supplemental rumen-protected Met and CHOL on performance and health of transition cows. Eighty-one multiparous Holstein cows were used in a randomized, complete, unbalanced block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) inclusion (with or without). Treatments (20 to 21 cows each) were control (CON), CON+Met (SMA), CON+CHOL (REA), and CON+Met+CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet (1.40Mcal of NE/kg of DM) with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NE/kg of DM) and were assigned randomly to treatments (CON, SMA, REA, or MIX) supplied as top dresses. From calving to 30 DIM, cows were fed the same postpartal diet (1.71Mcal of NE/kg of DM) and continued to receive the same treatments through 30 DIM. The Met supplementation was adjusted daily at 0.08% DM of diet and REA was supplemented at 60g/d. Incidence of clinical ketosis and retained placenta tended to be lower in Met-supplemented cows. Supplementation of Met (SMA, MIX) led to greater DMI compared with other treatments (CON, REA) in both close-up (14.3 vs. 13.2kg/d, SEM 0.3) and first 30d postpartum (19.2 vs. 17.2kg/d, SEM 0.6). Cows supplemented with Met (SMA, MIX) had greater yields of milk (44.2 vs. 40.4kg/d, SEM 1.2), ECM (44.6 vs. 40.5kg/d, SEM 1.0), and FCM (44.6 vs. 40.8kg/d, SEM 1.0) compared with other (CON, REA) treatments. Milk fat content did not differ in response to Met or CHOL. However, milk protein content was greater in Met-supplemented (3.32% vs. 3.14%, SEM 0.04%) but not CHOL-supplemented (3.27 vs. 3.19%, SEM 0.04%) cows. Supplemental CHOL led to greater blood glucose and insulin concentrations with lower glucose:insulin ratio. No Met or CHOL effects were detected for blood fatty acids or BHB, but a Met × time effect was observed for fatty acids due to higher concentrations on d 20. Results from the present study indicate that peripartal supplementation of rumen-protected Met but not CHOL has positive effects on cow performance.

The immunometabolic status of peripartal cows is altered due to changes in liver function, inflammation, and oxidative stress. Nutritional management during this physiological state can affect the biological components of immunometabolism. The objectives of this study were to measure concentrations of biomarkers in plasma, liver tissue, and milk, and also polymorphonuclear leukocyte function to assess the immunometabolic status of cows supplemented with rumen-protected methionine (Met) or choline (CHOL). Forty-eight multiparous Holstein cows were used in a randomized complete block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) level (with or without). Treatments (12 cows each) were control (CON), no Met or CHOL; CON and Met (SMA); CON and CHOL (REA); and CON and Met and CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet [1.40Mcal of net energy for lactation (NE)/kg of DM] with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NE/kg of DM) and were assigned randomly to each treatment. From calving to 30d, cows were on the same postpartal diet (1.71Mcal of NE/kg of DM) and continued to receive the same treatments until 30d. The Met supplementation was adjusted daily at 0.08% DM of diet, and CHOL was supplemented at 60g/cow per day. Liver (-10, 7, 21, and 30d) and blood (-10, 4, 8, 20, and 30d) samples were harvested for biomarker analyses. Neutrophil and monocyte phagocytosis and oxidative burst were assessed at d 1, 4, 14, and 28d. The Met-supplemented cows tended to have greater plasma paraoxonase. Greater plasma albumin and IL-6 as well as a tendency for lower haptoglobin were detected in Met- but not CHOL-supplemented cows. Similarly, cows fed Met compared with CHOL had greater concentrations of total and reduced glutathione (a potent intracellular antioxidant) in liver tissue. Upon a pathogen challenge in vitro, blood polymorphonuclear leukocyte phagocytosis capacity and oxidative burst activity were greater in Met-supplemented cows. Overall, liver and blood biomarker analyses revealed favorable changes in liver function, inflammation status, and immune response in Met-supplemented cows.

In nonruminants, nutrition during pregnancy can program offspring development, metabolism, and health in later life. Rumen-protected Met (RPM) supplementation during the prepartum period improves liver function and immune response in dairy cows. Our aim was to investigate the effects of RPM during late pregnancy on blood biomarkers (23 targets) and the liver transcriptome (24 genes) in neonatal calves from cows fed RPM at 0.08% of diet dry matter/d (MET) for the last 21 d before calving or controls (CON). Blood (n=12 calves per diet) was collected at birth before receiving colostrum (baseline), 24 h after receiving colostrum, 14, 28, and 50 d (post-weaning) of age. Liver was sampled (n=8 calves per diet) via biopsy on d 4, 14, 28, and 50 of age. Growth and health were not affected by maternal diet. The MET calves had greater overall plasma insulin concentration and lower glucose and ratios of glucose-to-insulin and fatty acids-to-insulin, indicating greater systemic insulin sensitivity. Lower concentration of reactive oxygen metabolites at 14 d of age along with a tendency for lower overall concentration of ceruloplasmin in MET calves indicated a lesser degree of stress. Greater expression on d 4 of fructose-bisphosphatase 1 (FBP1), phosphoenolpyruvate carboxykinase 1 (PCK1), and the facilitated bidirectional glucose transporter SLC2A2 in MET calves indicated alterations in gluconeogenesis and glucose uptake and release. The data agree with the greater expression of the glucocorticoid receptor (GR). Greater expression on d 4 of the insulin receptor (INSR) and insulin-responsive serine/threonine-protein kinase (AKT2) in MET calves indicated alterations in insulin signaling. In that context, the similar expression of sterol regulatory element-binding transcription factor 1 (SREBF1) in CON and MET during the preweaning period followed by the marked upregulation regardless of diet after weaning (d 50) support the idea of changes in hepatic insulin sensitivity during early postnatal life. Expression of carnitine palmitoyltransferase 1A (CPT1A) was overall greater and acyl-CoA oxidase 1 (ACOX1) was lower in MET calves, indicating alterations in fatty acid oxidation. Except forkhead box O1 (FOXO1), all genes changed in expression over time. Transcriptome results indicated that calves from MET-supplemented cows underwent a faster maturation of gluconeogenesis and fatty acid oxidation in the liver, which would be advantageous for adapting to the metabolic demands of extrauterine life.

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of β-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

Irisin, a newly discovered myokine, is considered as a promising candidate for the treatment of metabolic disturbances and cardiovascular diseases. In the present study, we used two animal models, apolipoprotein E-deficient mice fed on a high-cholesterol diet and a mouse carotid partial ligation model to test the anti-atherosclerotic effect of irisin. Irisin treatment (0.5 μg/g body weight/day) significantly reduced the severity of aortic atherosclerosis in apolipoprotein E-deficient mice fed on a high-cholesterol diet and suppressed carotid neointima formation in a carotid partial ligation model. It was associated with decreased inflammation and cell apoptosis in aortic tissues. In addition, in a cell culture model, irisin restored ox-LDL-induced human umbilical vein endothelial cell dysfunction by reducing the levels of inflammatory genes via inhibiting the reactive oxygen species (ROS)/ p38 MAPK/ NF-κB signaling pathway activation and inhibiting cell apoptosis via up-regulating Bcl-2 and down-regulating Bax and caspase-3 expression. Our study demonstrated that irisin significantly reduced atherosclerosis in apolipoprotein E-deficient mice via suppressing ox-LDL-induced cell inflammation and apoptosis, which might have a direct therapeutic effect on atherosclerotic diseases.

Sirtuin 1 (SIRT1) regulates adipocyte and osteoblast differentiation. However, the underlying mechanism should be investigated. This study revealed that SIRT1 acts as a crucial repressor of adipogenesis. RNA-interference-mediated SIRT1 knockdown or genetic ablation enhances adipogenic potential, whereas SIRT1 overexpression inhibits adipogenesis in mesenchymal stem cells (MSCs). SIRT1 also deacetylates the histones of sFRP1, sFRP2, and Dact1 promoters; inhibits the mRNA expression of sFRP1, sFRP2, and Dact1; activates Wnt signaling pathways; and suppresses adipogenesis. SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus and thus induces the transcription of genes that block MSC adipogenesis. In mice, the partial absence of SIRT1 promotes the formation of white adipose tissues without affecting the development of the body of mice. Our study described the regulatory role of SIRT1 in Wnt signaling and proposed a regulatory mechanism of adipogenesis.

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The liver functionality index (LFI) measures the changes of albumin, cholesterol, and bilirubin concentrations between 3 and 28 d postpartum. This composite index, based on variables with direct relevance to liver-specific plasma protein synthesis (albumin), hepatic/intestinal lipoprotein synthesis (cholesterol), and clearance of breakdown products of heme catabolism (bilirubin), provides a tool for evaluating manifestations of hepatic disease. Both energy and protein metabolism are likely to be affected by various physiological challenges in this period but have not been tested systematically. The present study was conducted to profile AA in cows with high or low LFI during the peripartal period and relate this to production outcomes. Eighteen multiparous cows were used from -21 through 28 d around parturition and divided retrospectively into the high or low LFI group. Blood samples were obtained on -21, -14, -7, 1, 3, 7, 10, 14, 17, 21, and 28 d relative to calving, and biomarkers and AA in plasma were measured. Grouping based on LFI resulted in 8 cows with high LFI (HLFI) and 10 cows with low LFI (LLFI). Although the temporal response in dry matter intake (DMI, 16.3 kg/d) and body condition score (2.56) did not differ, cows with high compared with low LFI had greater overall milk production (37.9 vs. 32.9 kg/d) although energy-corrected milk yield did not differ (42.6 vs. 38.7 kg/d). As expected, cows grouped as LLFI had lower cholesterol and albumin but greater bilirubin after calving compared with HLFI animals. Despite similar temporal responses in DMI between groups, concentrations of total AA were greater in HLFI, particularly after calving. Although concentrations of total essential AA (EAA) and branched-chain AA did not differ with LFI status, cows in HLFI had greater concentrations of Thr and Ile postpartum. Nearly all plasma AA concentrations followed the general trend of a nadir at 1d after calving followed by a gradual increase to prepartal levels before 28 d. Glycine was the only AA exhibiting a gradual increase in concentration through the transition, with a maximum at 7d postpartum followed by a gradual decrease. We detected no effect of LFI status on plasma Lys, which decreased markedly from -21d to calving, followed by an increase to prepartal values by d7. In contrast, concentrations of Met and His decreased markedly between -21 and 10d and did not reach prepartal values by 28 d. The marked decrease in Gln concentration after calving regardless of LFI might compromise immune function during this period. Overall, the results indicate the existence of an association among inflammation, liver function postpartum, and AA plasma concentrations, irrespective of temporal differences in DMI. Cows with better indices of liver function produced more milk and maintained greater concentrations of total AA and some EAA such as Thr and Ile. Whether these AA played a direct role in the greater milk production remains to be determined.