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Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5'-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 μM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 μM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase β (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine β-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (-EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase β-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase β-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower β-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Polymorphonuclear leukocytes (PMNL) are the first responders upon pathogen invasion and hence play an important role in inflammatory and immune responses. Rumen-protected methionine (MET) and choline (CHOL) during the peripartal period affect the immune response and inflammatory status in dairy cows to different extents. We aimed to examine the effect of MET and CHOL supply on expression of genes regulating key PMNL functions and associations with whole-blood immune challenge. Thirty multiparous Holstein cows from a larger cohort randomly assigned from -21 to 30 d relative to parturition to a basal control (CON) diet, CON plus MET at a rate of 0.08% of dry matter, or CON plus CHOL at 60 g/d were used. Blood was sampled at -10, 7, and 30 d relative to parturition for inflammatory biomarker analyses and PMNL isolation. Neutrophil and monocyte phagocytosis and oxidative burst in vitro were assessed in whole blood at 1, 7, and 28 d. Although neutrophil and monocyte phagocytosis did not differ, oxidative burst in neutrophils and monocytes was greater in MET-supplemented cows relative to CON cows. Compared with CON, PMNL adhesion and migration-related genes (ITGAM, ITGB2, ITGA4) were downregulated in response to MET and CHOL. Expression of CADM1 and SELL was also lower in MET-supplemented cows compared with CON cows but not in CHOL cows. In contrast, compared with CON cows, the expression of ICAM1 was lower in CHOL but not MET cows. Similar to adhesion and migration-related genes, cows receiving MET- or CHOL-supplemented diets had lower expression of inflammation-related genes (IL1β, IL10RA, NFKB1, STAT3, TLR2). However, expression of IRAK1 and TLR4 was lower in MET- but not CHOL-supplemented cows. Plasma taurine concentration was greater in MET cows compared with CHOL and CON cows, suggesting a better redox status in plasma. In agreement with plasma taurine, oxidative stress-related genes (CBS, CTH, GPX1, GSS, SOD2) in PMNL were lower in response to MET and to CHOL supply. Overall, immunometabolic gene expression profile and blood biomarker analyses suggest an overall better redox status in PMNL during the transition period in response to MET and CHOL supply. These adaptations in PMNL might be beneficial for mounting a better bactericidal response upon challenge., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Pregnancy and early life are critical periods during which environmental factors such as nutrition can affect development. Rumen-protected methionine (Met; RPM) supplementation during the prepartum period improves not only performance but immune responses in dairy cows. We investigated the effects of enhanced maternal supply of Met via feeding RPM on whole-blood in vitro lipopolysaccharide (LPS; 0, 0.01, or 5 μg/mL of blood) challenge and targeted microRNA and mRNA abundance in calf blood polymorphonuclear leukocytes (PMNL). Calves (n = 12/maternal diet) born to cows fed RPM at 0.08% of diet dry matter (DM)/d (MET) for the last 21 ± 2 d before calving or fed a control diet with no added Met (CON) were used. The PMNL were isolated at birth (before colostrum feeding) and d 1 (24 h after colostrum intake), 14, 28, and 50 of age. Maternal blood was collected at -10 ± 1.3 d relative to calving. Cows in the MET group had greater DM intake and lower prepartal haptoglobin concentration. In CON cows, haptoglobin was positively correlated with proinflammatory and host-defense mRNA abundance in CON calves. Except for NOS2 and NFE2L2, abundance of CASP8, MPO, ZBP1, and TNF was lower at birth in MET calves. Interleukin 1β concentration in response to LPS challenge in CON and MET calves was greatest at birth, underscoring the role of this cytokine for lymphocyte activation. Compared with 1 d of age, the interleukin-1β response to incremental doses of LPS was greater at 14 through 28 d, suggesting that the neonatal calf can mount a robust response to inflammatory stimuli. Greater abundance in CON calves of NOS2, CADM1, and TLR2 coupled with lower SELL from 1 through 50 d of age suggested a chronic activation of the PMNL. There was a marked upregulation over time of MIR125b, MIR146a, MIR155, and MIR9 in both CON and MET calves, suggesting that these microRNA could affect gene transcription associated with differentiation and inflammatory function in PMNL. Regardless of maternal diet, the gradual downregulation of MIR223 (the most abundant microRNA in PMNL) is in line with the progressive increase over time in the proinflammatory signature of the PMNL. Data revealed the potential for maternal supply of Met during late pregnancy through either greater DM intake or Met to elicit some changes in PMNL function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine ® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ± 2.0 μM) than cows in MET (30.39 ± 1.6 μM). Cows in CON had greater cystine plasma concentrations (3.62 ± 0.3 μM) than cows in MET (2.8 ± 0.3 μM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amino acids not only serve as precursors for protein synthesis but also function as signaling molecules that can regulate the mammalian target of rapamycin (mTOR) pathway. Methionine and Lys are the most-limiting AA for milk production and a ratio of ∼3:1 Lys:Met in the metabolizable protein has been determined to be ideal. Besides Met and Lys, recent studies have evaluated Ile, Leu, Val, and Thr as potentially limiting for milk protein synthesis. The objective of this experiment was to determine if varying the ratio of Lys:Thr, Lys:Ile, Lys:Val, and Lys:Leu while maintaining an ideal ratio of Lys:Met and fixed ratio of other essential AA (IPAA) elicits changes in intracellular metabolites, gene transcription related to protein synthesis, and phosphorylation status of mTOR pathway proteins. Immortalized bovine mammary epithelial cell line (MAC-T) cells were incubated for 12 h (n = 5 replicates/treatment) with IPAA (2.9:1 Lys:Met; 1.8:1 Lys:Thr; 2.38:1 Lys:His; 1.23:1 Lys:Val; 1.45:1 Lys:Ile; 0.85:1 Lys:Leu; 2.08:1 Lys:Arg) or IPAA supplemented with Thr, Ile, Val, and Leu to achieve a Lys:Thr 1.3:1 (LT1.3), Lys:Ile 1.29:1 (LI1.29), Lys:Val 1.12:1 (LV1.12), or Lys:Leu 0.78:1 (LL0.78). Compared with IPAA, metabolomics via gas chromatography-mass spectrometry revealed that increases in availability of Thr, Ile, Val, and Leu led to greater concentrations of essential AA (Leu, Ile, Thr), nonessential AA (Gly, Glu, Gln, Ser, Pro, Asp), and various metabolites including uric acid, phosphoric acid, N-acetylglutamic acid, and intermediates of glycolysis and the tricarboxylic acid cycle. Compared with other treatments, LV1.12 led to greater phosphorylation status of serine/threonine kinase B (Akt), mTORC1, and ribosomal protein S6 and lower phosphorylation of α subunit of eukaryotic translation initiation factor 2. In addition, LV1.12 upregulated abundance of CSN2 and both the abundance and promoter methylation of CSN1S1. Although LI1.29 led to the second highest response in mTORC1 phosphorylation status, it resulted in the lowest phosphorylation of Akt and eEF2 and mRNA abundance of CSN2 and various AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, SLC43A1). Overall, data indicate that an increase in Val at an ideal ratio of Lys:Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).)

The liver functionality index (LFI) represents an assessment of transition cow metabolic health by measuring changes in biomarkers associated with liver plasma protein synthesis (albumin), lipoprotein synthesis (cholesterol), and heme catabolism (bilirubin). The present analysis was conducted to determine the role of peripartal rumen-protected Met or choline (CHOL) supplementation on LFI groupings, and to assess relationships with performance, inflammation, oxidative stress status, and plasma AA profiles. A cohort of 40 multiparous Holstein cows that were part of 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) were used. From -21 d to calving, cows received the same close-up diet and were assigned randomly to each treatment. From calving to 30 d, cows were on the same postpartal diet and continued to receive the same treatments until 30 d. Addition of Met was adjusted daily at 0.08% dry matter of diet and CHOL was fed at 60 g/cow per day. Liver (-10, 7, 20, and 30 d) and blood (-10, 4, 8, 20, and 30 d) samples were harvested for biomarker analyses. Cows were ranked retrospectively and assigned to low (LLFI, LFI <0) and high (HLFI, LFI >0) LFI groups regardless of Met or CHOL supplementation. Compared with cows in LLFI, close-up and lactation DMI, milk yield, milk fat yield, and milk protein yield were greater in HLFI cows. As expected, cows in LLFI had lower plasma cholesterol and albumin but greater bilirubin concentrations around parturition. Plasma haptoglobin concentration was also lower in HLFI cows, but plasma paraoxonase and hepatic total and reduced hepatic glutathione concentrations were greater. Although higher concentrations of His, Met, and Trp, as well as a tendency for greater Ile, were observed in HLFI cows, overall essential AA concentrations did not differ with LFI status. In contrast, overall concentrations of nonessential AA were greater in HLFI cows due to greater circulating concentrations of Ala, Asn, Gln, Pro, and Ser. Similarly, overall concentrations of total AA and total sulfur-containing compounds were greater in cows with HLFI. Feeding Met compared with CHOL led to a tendency for more cows classified as HLFI. Overall, results support the broader application of the LFI in the management of transition cows. In that context, the fact that precalving concentrations of compounds such as reduced glutathione, total sulfur-containing compounds, Met, Tau, and homocysteine differed between HLFI and LLFI independent of Met or CHOL feeding also underscores their potential for monitoring cows that might be at a greater risk of developing health problems after calving. Further studies on the applicability of these biomarkers to monitor transition success appears warranted., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Multiparous Holstein cows were assigned in a randomized complete block design into four treatments from 21 d before calving to 30 d in milk (DIM). Treatments were: MET [n = 19, fed the basal diet + rumen-protected methionine at a rate of 0.08% (w/w) of the dry matter, Smartamine ® M], CHO (n = 17, fed the basal diet + choline 60 g/d, Reashure ® ), MIX (n = 21, fed the basal diet + Smartamine ® M at a rate of 0.08% (w/w) of the dry matter and 60 g/d Reashure ® ), and CON (n = 20, no supplementation, fed the close-up and fresh cow diets). Follicular development was monitored via ultrasound every 2 d starting at 7 DIM until ovulation (n = 37) or aspiration (n = 40) of the first postpartum dominant follicle (DF). Follicular fluid from 40 cows was aspirated and cells were retrieved immediately by centrifugation. Gene expression of TLR4, TNF, IL1-β, IL8, IL6, LHCGR, STAR, 3β-HSD, P450scc, CYP19A1, IRS1, IGF, MAT1A, and SAHH, was measured in the follicular cells of the first DF. Cows in CON had higher TNF, TLR4, and IL1-β mRNA expression (11.70 ± 4.6, 21.29 ± 10.4, 6.28 ± 1.4, respectively) than CHO (2.77 ± 0.9, 2.16 ± 0.9, 2.29 ± 0.7, respectively), and MIX (2.23 ± 0.7, 1.46 ± 0.6, 2.92 ± 0.8, respectively). Cows in CON had higher IL1-β expression (6.27 ± 1.4) than cows in MET (3.28 ± 0.6). Expression of IL8 mRNA was lower for cows in CHO (0.98 ± 0.3) than cows in CON (4.90 ± 0.7), MET (6.10 ± 1.7), or MIX (5.05 ± 1.8). Treatments did not affect mRNA expression of LHCGR, STAR, P450scc, CYP19A, SAHH, MAT1A, or IL6 however, 3β-HSD expression was higher for cows in MET (1.46 ± 0.3) and MIX (1.25 ± 0.3) than CON (0.17 ± 0.04) and CHO (0.26 ± 0.1). Supplementation of methionine, choline, and both methionine and choline during the transition period did not affect days to first ovulation or number of cows that ovulated the first follicular wave. Plasma and follicular fluid estradiol and progesterone concentrations were not different among treatments. Methionine concentrations in the follicular fluid of the first postpartum DF was higher for cows in MET (18.2 ± 0.1 μM) than cows in CON (11.1 ± 0.9 μM). In conclusion, supplementing choline and methionine during the transition period changed mRNA expression in follicular cells and dietary methionine supplementation increased plasma and follicular fluid concentrations of methionine of the first postpartum DF in Holstein cows., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methionine, together with Lys, is the most limiting AA for milk production in dairy cows. Besides its crucial role in milk production, Met and its derivate metabolites (e.g., glutathione, taurine, polyamines) are well-known immunonutrients in nonruminants, helping support and boost immune function and activity. In the present study, the effects of Met or choline, as its precursor, were investigated using an ex vivo whole blood challenge. The study involved 33 multiparous Holstein cows (from a larger cohort with a factorial arrangement of treatments) assigned from d -21 to +30 relative to parturition to a basal control (CON) diet, CON plus rumen-protected Met (MET, Smartamine M, Adisseo NA, Alpharetta, GA) at a rate of 0.08% of dry matter, or CON plus rumen-protected choline (CHOL, ReaShure, Balchem Inc., New Hampton, NY) at 60 g/d. Blood was sampled on d -15, -7, 2, 7, and 20 for ex vivo lipopolysaccharide (LPS) challenge, and on d 1, 4, 14, and 28 relative to parturition for phagocytosis and oxidative burst assays. The MET cows had greater energy-corrected milk production and milk protein content. Overall, IL-6 response to LPS increased around parturition, whereas IL-1β remained constant, casting doubt on the existence of systemic immunosuppression in the peripartal period. Supplementation with MET dampened the postpartal blood response to LPS (lower IL-1β), while improving postpartum neutrophil and monocyte phagocytosis capacity and oxidative burst activity. In contrast, CHOL supplementation increased monocyte phagocytosis capacity. Overall, the data revealed a peripartal immune hyper-response, which appeared to have been mitigated by MET supplementation. Both MET and CHOL effectively improved immune function; however, MET affected the immune and antioxidant status before parturition, which might have been beneficial to prepare the cow to respond to metabolic challenges after parturition. These results provide insights on potential differences in the immunomodulatory action of methionine and choline in dairy cows. As such, the effects observed could have implications for ration formulation and dietary strategies., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 μM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-β-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless of treatment. The gradual upregulation over time of BHMT2 and SAHH coupled with the gradual upregulation of MAT1A and the DNA (cytosine-5-)-methyltransferases (DNMT1, DNMT3A, DNMT3B) in MET and CON calves was indicative of adaptations potentially driven by differences in intake of milk replacer and starter feed as calves grew. In that context, the ∼2.5-fold increase in abundance of DNMT3B at d 50 versus 4 in MET and CON indicate that DNA methylation might be an important component of the physiologic adaptations of calf liver. The data indicate that calves from MET-supplemented cows underwent alterations in Met, choline, and homocysteine metabolism partly to synthesize taurine and glutathione, which would be advantageous for controlling metabolic-related stress. Whether the effects in MET calves were directly related to increased Met supply in utero remains to be determined., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 L )/kg of DM] with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NE L /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 L /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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 L /kg of DM) with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NE L /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 L /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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The availability of Met in metabolizable protein (MP) of a wide range of diets for dairy cows is low. During late pregnancy and early lactation, in particular, suboptimal Met in MP limits its use for mammary and liver metabolism and also for the synthesis of S-adenosylmethionine, which is essential for many biological processes, including DNA methylation. The latter is an epigenetic modification involved in the regulation of gene expression, hence, tissue function. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo NA, Alpharetta, GA; n=12), or CON plus Smartamine M (SM; Adisseo NA; n=13). The total mixed ration dry matter for the close-up and lactation diets was measured weekly, then the Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 (MS) or 0.07% (SM) on a dry matter basis. Liver tissue was collected on -10, 7, and 21 d for global DNA and peroxisome proliferator-activated receptor alpha (PPARα) promoter region-specific methylation. Several PPARα target and putative target genes associated with carnitine synthesis and uptake, fatty acid metabolism, hepatokines, and carbohydrate metabolism were also studied. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrast CON versus SM + MS. Global hepatic DNA methylation on d 21 postpartum was lower in Met-supplemented cows than CON. However, of 2 primers used encompassing 4 to 12 CpG sites in the promoter region of bovine PPARA, greater methylation occurred in the region encompassing -1,538 to -1,418 from the transcription start site in cows supplemented with Met. Overall expression of PPARA was greater in Met-supplemented cows than CON. Concomitantly, PPARA-target genes, such as ANGPTL4, FGF21, and PCK1, were also upregulated overall by Met supplementation. The upregulation of PPARα target genes indicates that supplemental Met, likely through the synthesis of S-adenosylmethionine, activated PPARA-regulated signaling pathways. Upregulation of hepatic PPARA has been associated with improved lipid metabolism and immune function, both of which were reported in companion publications from this study. In turn, those positive effects resulted in improved postpartal health and performance. Further research is needed to study more closely the mechanistic connections between global DNA and promoter region-specific PPARA methylation with PPARA expression and functional outcomes in liver., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)