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Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism
Feeding supplemental choline and Met during the periparturient period can have positive effects on cow performance; however, the mechanisms by which these nutrients affect performance and metabolism are unclear. The objective of this experiment was to determine if providing rumen-protected choline, rumen-protected Met, or both during the periparturient period modifies the choline metabolitic profile of plasma and milk, plasma AA, and hepatic mRNA expression of genes associated with choline, Met, and lipid metabolism. Cows (25 primiparous, 29 multiparous) were blocked by expected calving date and parity and randomly assigned to 1 of 4 treatments: control (no rumen-protected choline or rumen-protected Met); CHO (13 g/d choline ion); MET (9 g/d DL-methionine prepartum; 13.5 g/d DL-methionine, postpartum); or CHO + MET. Treatments were applied daily as a top dress from ~21 d prepartum through 35 d in milk (DIM). On the day of treatment enrollment (d −19 ± 2 relative to calving), blood samples were collected for covariate measurements. At 7 and 14 DIM, samples of blood and milk were collected for analysis of choline metabolites, including 16 species of phosphatidylcholine (PC) and 4 species of lysophosphatidylcholine (LPC). Blood was also analyzed for AA concentrations. Liver samples collected from multiparous cows on the day of treatment enrollment and at 7 DIM were used for gene expression analysis. There was no consistent effect of CHO or MET on milk or plasma free choline, betaine, sphingomyelin, or glycerophosphocholine. However, CHO increased milk secretion of total LPC irrespective of MET for multiparous cows and in absence of MET for primiparous cows. Furthermore, CHO increased or tended to increase milk secretion of LPC 16:0, LPC 18:1, and LPC 18:0 for primi- and multiparous cows, although the response varied with MET supplementation. Feeding CHO also increased plasma concentrations of LPC 16:0 and LPC 18:1 in absence of MET for multiparous cows. Although milk secretion of total PC was unaffected, CHO and MET increased secretion of 6 and 5 individual PC species for multiparous cows, respectively. Plasma concentrations of total PC and individual PC species were unaffected by CHO or MET for multiparous cows, but MET reduced total PC and 11 PC species during wk 2 postpartum for primiparous cows. Feeding MET consistently increased plasma Met concentrations for both primi- and multiparous cows. Additionally, MET decreased plasma serine concentrations during wk 2 postpartum and increased plasma phenylalanine in absence of CHO for multiparous cows. In absence of MET, CHO tended to increase hepatic mRNA levels of betaine-homocysteine methyltransferase and phosphate cytidylyltransferase 1 choline, α, but tended to decrease expression of 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and peroxisome proliferator activated receptor α irrespective of MET. Although shifts in the milk and plasma PC profile were subtle and inconsistent between primi- and multiparous cows, gene expression results suggest that supplemental choline plays a probable role in promoting the cytidine diphosphate-choline and betaine-homocysteine S-methyltransferase pathways. However, interactive effects suggest that this response depends on Met availability, which may explain the inconsistent results observed among studies when supplemental choline is fed.
). Choline deficiency impairs an animal's ability to export lipids from the liver because it is required for synthesis of phosphatidylcholine (PC), the major phospholipid that comprises very-low-density lipoproteins (
). Phosphatidylcholine is synthesized through one of 2 pathways: (1) the cytidine diphosphate (CDP)-choline pathway using choline derived from the diet; and (2) the phosphatidylethanolamine N-methyltransferase (PEMT) pathway whereby a series of 3 methylation reactions occur to convert phosphatidylethanolamine to PC (
), it is likely that in ruminants the majority of PC is synthesized via the PEMT pathway. Feeding rumen-protected choline (RPC) could lead to more PC synthesis via the CDP-choline pathway.
suggested that PC species that contained PUFA were derived from PEMT origin in rats. Thus, the profile of individual PC species in blood or milk that result when RPC is supplemented in periparturient cows could be indicative of a change in PC source.
Methionine is an AA that is used not only for protein synthesis, but also for synthesis of S-adenosylmethionine (SAM), one of the most important methyl donors in the body (
), which is why Met has been investigated for its potential role as a lipotrope. Additionally, SAM is also implicated in the regulation of gene expression via histone methylation reactions (
Choline and Met metabolism are integrated through their participation in one-carbon metabolism. Choline indirectly serves as a methyl donor in the one-carbon metabolic pathway via betaine, the product of choline oxidation. Betaine is required for the regeneration of Met from homocysteine (Hcy) via the betaine-homocysteine S-methyltransferase (BHMT) pathway (
). Alternatively, Hcy can also be converted to Met via the methyltetrahydrofolate pathway by methionine synthase, an enzyme that requires vitamin B12 to function (
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.
). Methionine also has the potential to indirectly influence PC synthesis via the PEMT pathway through SAM.
In an effort to derive nutritional strategies that improve cow health and performance during the periparturient period, attention has been given to nutrients involved in one-carbon metabolism such as choline and Met (
Methyl donor supplementation suppresses the progression of liver lipid accumulation while modifying the plasma triacylglycerol lipidome in periparturient Holstein dairy cows.
) due to their functions associated with lipid transport and metabolism, immunity, and gene expression. Previous investigations have shown that supplementing many of these nutrients during periods of negative energy balance that occur around the time of calving can modify hepatic expression of genes associated with choline, Met, and lipid metabolism (
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
Choline supply during negative nutrient balance alters hepatic cystathionine β-synthase, intermediates of the methionine cycle and transsulfuration pathway, and liver function in Holstein Cows.
Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
Methyl donor supplementation suppresses the progression of liver lipid accumulation while modifying the plasma triacylglycerol lipidome in periparturient Holstein dairy cows.
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
) investigated specific effects associated with supplementation of choline, Met, or both during the periparturient period. Recent attention has also been given to the effects of methyl donors on the blood choline metabolite profile in lactating cows (
Short communication: Effects of dietary deoiled soy lecithin supplementation on circulating choline and choline metabolites, and the plasma phospholipid profile in Holstein cows fed palm fat.
) in an effort to elucidate mechanisms by which these nutrients affect metabolism and performance of postpartum cows. We hypothesized that choline and Met supplementation would modulate methyl group metabolism via (1) the CDP-choline pathway using choline derived from the diet; and (2) the PEMT pathway.
The objective of this study was to determine if providing supplemental choline, Met, or both during the periparturient period modifies plasma AA concentrations, the choline metabolic profile of plasma and milk, as well as hepatic expression of genes associated with choline, Met, and lipid metabolism.
MATERIALS AND METHODS
All procedures that utilized animals were approved by the University of Maryland, College Park, Institutional Animal Care and Use Committee (Protocol #857801–6).
Animals and Study Design
Between March and December of 2017, 25 primiparous and 29 multiparous Holstein cows from the Central Maryland Research and Education Center (Clarksville, MD) were utilized in a randomized block design experiment with a 2 × 2 factorial treatment structure. The 2 factors were 0 or 60 g/d RPC (ReaShure, Balchem Corporation) and 0 or 12 g/d RPM (Smartamine M; Adisseo USA, Inc.) prepartum and 18 g/d RPM postpartum. Thus, the 4 resulting treatments included: (1) no RPC or RPM (control; CON); (2) 60 g/d RPC (CHO); (3) 12 g/d RPM prepartum and 18 g/d RPM postpartum (MET); and (4) a combination of CHO and MET treatments (CHO + MET; 60 g/d RPC + 12 g/d RPM prepartum and 18 g/d RPM postpartum). Although not reported in our previous paper on production responses (
), the number of animals included in the study were based on a power analysis to provide an 80% chance of detecting a 2 kg/d difference in milk production across the main effects of CHO and MET within parity at P ≤ 0.05. Before the start of the experiment, cows were blocked by age (primiparous vs. multiparous) and expected calving date and randomly assigned to treatment. Treatments were applied daily as a top dress from 21 d before expected calving through 35 DIM. Because treatments were administered daily by project personnel, treatment assignments were not blinded. Additional details regarding basal pre- and postpartum diets and animal care and housing were described by
Blood was collected via coccygeal venipuncture before feeding on approximately −21, 7, and 14 d relative to calving. Because some cows calved early or late, prepartum samples were actually collected on −19 ± 2 d relative to calving. Samples were collected into one 10-mL evacuated tube containing potassium EDTA and one 10-mL evacuated tube containing sodium heparin. Tubes were immediately placed on ice and then centrifuged at 2,000 × g for 15 min at 4°C. Plasma aliquots were stored at −80°C until analysis.
Choline metabolite concentrations of plasma and milk (7 and 14 d postpartum) were determined by hydrophilic interaction liquid chromatography tandem MS (
). Metabolites examined included betaine, free choline, glycerophosphocholine (GPC), sphingomyelin (SM), 16 species of PC, and 4 species of lysophosphatidylcholine (LPC). Amino acid concentrations of plasma were determined using the methods described by
. Briefly, plasma samples (50 µL) were deproteinized with 500 µL of cold acetonitrile and evaporated to dryness under N2. Amino acids and organic acids were then converted to their respective t-butyldimethylsilyl derivatives by heating at 90°C for 1 h. Metabolites were separated by GC (HP-5ms, 30 m × 0.25 mm × 0.25 μm, Agilent Technologies) before selected ion monitoring of specific ion fragments with MS under electron ionization.
Approximately 150 mg of liver tissue was collected from each animal via percutaneous liver biopsies at approximately −21 d (actual prepartum samples were obtained on −19 ± 2 d) and 7 d relative to calving using a 14-gauge biopsy needle (14-gauge × 15 cm; Tru-Cut, Merit Medical). Further details regarding the liver biopsy procedure are described by
. Tissue was immediately snap frozen in liquid nitrogen and stored at −80°C. Because 2 multiparous cows in block 6 and 1 multiparous cow in block 3 were removed early from the experiment due to health problems (
), gene expression analysis was completed on liver samples obtained from the 5 completed blocks of multiparous cows (n = 20, 5 cows per treatment).
Hepatic mRNA levels of genes related to choline and Met metabolism (BHMT; PEMT; phosphate cytidylyltransferase 1A, PCYT1A; glutathione synthetase, GSS; 5-methyltetrahydrofolate-homocysteine methyltransferase, MTR) and lipid metabolism (carnitine palmitoyltransferase 1A; diacylglycerol O-acyltransferase 1, DGAT1; 3-hydroxy-3-methylglutaryl-CoA synthase 2, HMGCS2; microsomal triglyceride transfer protein, MTTP; peroxisome proliferator activated receptor α, PPARα) were determined via real-time quantitative PCR. Primers were designed (Table 1) using the National Center for Biotechnology Information primer BLAST Software (2012). Primers needed to (1) span an intron, (2) target a region as close to the 3′ end of the sequence as possible, (3) amplify all splice variants, (4) have an annealing temperature of 58 to 60°C, (5) have a guanine:cytosine ratio of 40 to 60%, (6) be between 18 and 30 nucleotides in length, (6) generate a PCR product that was 100 to 250 nucleotides in length, and (7) specifically target the gene of interest. Primers (25 nmol DNA oligos; standard desalting purification) were obtained from Integrated DNA Technologies and reconstituted in ultrapure water. Amplification efficiencies were determined for each primer pair by performing reverse-transcription quantitative PCR (QuantiTect SYBR Green PCR Kit, Qiagen Inc.) using 2-fold serial dilutions of pooled cDNA (1 µg). Efficiency was calculated using the equation [(10(−1/slope)) − 1], where slope is equal to the slope of the regression of the cycle threshold value on the log10 (copy number). Efficiencies ranged from 0.96 to 1.08 for all primers tested. Primer specificity was verified by dissociation curve analysis, agarose gel electrophoresis, and sequencing of PCR products.
Table 1Forward and reverse primers used for real-time quantitative PCR
Liver total RNA was extracted using the RNeasy Lipid Tissue Mini Kit with on-column DNase digestion (Qiagen Inc.). Approximately 20 to 30 mg of liver tissue was weighed and kept frozen in liquid nitrogen until homogenization in 0.5 mL of QIAzol Lysis Reagent (Qiagen Inc.). The remainder of the RNA extraction protocol was carried out according to manufacturer instructions. After extraction, RNA was stored at −80°C. The concentration of RNA in each sample was determined using a commercially available kit (Quant-iT RiboGreen RNA Assay Kit, Catalog #R11490, ThermoFisher Scientific) and 1 µg of RNA was used for cDNA synthesis (QuantiTect Reverse Transcription Kit; Qiagen Inc.). For the reverse transcription reactions, a reaction of a pool of total RNA without reverse transcriptase was conducted as a control for genomic DNA contamination. Complementary DNA was not diluted before PCR analysis and was stored at −20°C.
The PCR reactions were carried out using a commercially available kit according to the manufacturer instructions (QuantiTect SYBR Green PCR Kit; Qiagen Inc.). The PCR reactions were performed in 96-well plates (VWR International, LLC) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) for 40 cycles using the following program: 95°C for 15 min (activation), 94°C for 15s (denaturation), 60°C for 30s (annealing), and 72°C for 30s (extension). Dissociation curve analysis ensured amplification of a single PCR product and absence from the no reverse-transcription and water controls. Data were normalized to phosphoglycerate kinase 1 and analyzed by the 2−ΔΔCt method.
Statistical Analysis
Because production responses to treatment appeared to be different between primi- and multiparous cows (
), AA and choline metabolitic data were analyzed for primi- and multiparous cows separately. Postpartum blood AA and choline metabolite concentrations were analyzed using a repeated-measures mixed model (SAS Institute, version 9.4) that included the random effect of cow nested within block and fixed effects of wk relative to calving (1 or 2), the main effects of CHO and MET, and all 2- and 3-way interactions. Prepartum metabolite concentration, determined from samples collected in the morning before first treatment application (−19 ± 2 d relative to calving), was also included in the model as a covariate. Week relative to calving served as the repeated factor and a total of 8 covariance structures were tested, and the one that resulted in the lowest Akaike information criterion was selected for each variable. Milk choline metabolite yields were analyzed using a similar model that did not include a prepartum covariate measurement.
Hepatic mRNA level fold-changes (relative to postpartum CON) were analyzed in a mixed model that included the main effects of CHO and MET, their interaction, and the random effect of cow. Prepartum gene expression fold-change (relative to postpartum CON) was included in the model as a covariate. Thus, postpartum gene expression results are expressed as covariate-adjusted fold-changes relative to CON. Cook's distance was used to detect potential outliers using a threshold of 4 divided by number of observations (4/n). If a cow was identified as a potential outlier for more than 4 of the 10 genes of interest, she was removed from the analysis. Based on this method, results from 2 cows (one from CON and one from CHO) were considered outliers and removed from the final analysis. Statistical significance was declared at P ≤ 0.05, and tendencies were considered at P < 0.10.
RESULTS
Milk Choline Metabolites
Multiparous Cows.
Milk secretion of free choline, GPC, SM, and total PC was unaffected by CHO and MET (Table 2). However, CHO decreased betaine secretion during wk 1 postpartum (4,940 vs. 2,830 µmol/d) but not wk 2 (CHO × wk: P = 0.04). In contrast, feeding CHO increased total LPC secretion (P = 0.05). Aligning with this observation, CHO increased secretion of LPC 18:0 (P < 0.01), tended to increase secretion of LPC 16:0 (P = 0.08), and increased secretion of LPC 18:1 when fed without MET (CHO × MET: P = 0.03; Table 3).
Table 2Secretion rates (μmol/d) of major choline metabolites in milk for multiparous cows (n = 27) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
2 GPC = glycerophosphocholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; PC = phosphatidylcholine.
Table 3Secretion rates (μmol/d) of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) species in milk for multiparous cows (n = 27) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
a,b Means with differing superscripts are significantly different (P < 0.05).
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
Although secretion of total PC was unaffected, secretion of several individual PC species were elevated by CHO or MET. Feeding CHO increased secretion of PC 16:0/20:5 + 16:1/20:4, PC 18:1/22:6, and PC 18:0/22:6 + 18:1/22:5 and tended to increase secretion of PC 16:0/18:1 (P < 0.01, P = 0.03, P = 0.02, and P = 0.08, respectively; Table 3). Furthermore, CHO increased secretion of PC 18:1/20:4 + 18:0/20:5 + 16:0/22:5, PC 18:0/20:4, and PC 18:0/22:5 during wk 2 but not wk 1 postpartum (CHO × wk: P = 0.05, P = 0.01, and P = 0.05, respectively). Feeding MET increased secretion of PC 18:1/20:4 + 18:0/20:5 + 16:0/22:5, PC 18:0/20:4, PC 18:0/20:3, and PC 18:0/22:5 during wk 1 postpartum but not wk 2 (MET × wk: P = 0.03, P < 0.01, P = 0.03, and P < 0.01, respectively). In addition, feeding CHO without MET increased secretion of PC 18:0/22:6 + 18:1/22:5 and PC 18:0/18:1 during wk 2 postpartum but not wk 1, whereas MET increased their secretion irrespective of wk or CHO (CHO × MET × wk: both P = 0.03). In contrast, feeding CHO with MET increased secretion of PC 18:0/18:1 during wk 1 but not wk 2 postpartum, but CHO fed without MET increased its secretion during wk 2 postpartum (CHO × MET × wk: P = 0.04).
Primiparous Cows.
Milk secretion of GPC, SM, and total PC was unaffected by CHO or MET. Feeding CHO tended to increase milk secretion of betaine (P = 0.09) and free choline (P = 0.06; Table 4). However, the effect of CHO on total LPC secretion was dependent on MET (CHO × MET: P = 0.01) as CHO increased total LPC secretion when fed without MET. Aligning with this result, CHO increased secretion of LPC 18:0 and LPC 16:0 (both P = 0.02; Table 5) when fed without MET. However, CHO tended to increase secretion of LPC 18:1 irrespective of MET (P = 0.08). In contrast, feeding MET with CHO suppressed the increase in secretion of LPC 18:2 during wk 1 but not wk 2 postpartum (CHO × MET × wk interaction: P = 0.05). The lack of CHO or MET effects on total PC secretion largely carried through to individual PC species (Table 5). However, CHO reduced secretion of PC 16:0/16:1 (P = 0.04) but increased secretion of PC 16:0/20:5 + 16:1/20:4 during wk 1 postpartum but not wk 2 (CHO × wk: P = 0.03). Additionally, MET increased secretion of PC 18:0/22:6 + 18:1/22:5 (P = 0.05) and tended to increase secretion of PC 18:0/22:5 (P = 0.06).
Table 4Secretion rates (μmol/d) of major choline metabolites in milk for primiparous cows (n = 24) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
Means with differing superscripts are significantly different (P < 0.05).
8.58
72.9
90.6
5.07
0.14
0.47
0.01
<0.01
Total PC
2,608
2,408
2,966
2,367
314
2,641
2,532
197
0.20
0.61
0.52
0.65
a,b Means with differing superscripts are significantly different (P < 0.05).
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
2 GPC = glycerophosphocholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; PC = phosphatidylcholine.
Table 5Secretion rates (μmol/d) of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) species in milk for primiparous cows (n = 24) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
a,b Means with differing superscripts are significantly different (P < 0.05).
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Milk samples were obtained during wk 1 and 2 postpartum.
There were no effects of CHO or MET on plasma betaine, free choline, GPC, or total PC concentrations (Table 6). However, CHO decreased plasma SM concentrations (P = 0.04) and MET tended to decrease total LPC concentrations (P = 0.09). In line with the latter observation, MET reduced plasma concentrations of LPC 18:0 (P = 0.05; Table 5). In addition, feeding CHO increased plasma concentrations of LPC 16:0 and LPC 18:1, but this effect was dependent upon MET (CHO × MET: P = 0.05 and P = 0.02, respectively). Plasma total PC concentrations were unaffected and individual PC species were largely unaffected by CHO or MET (Table 7). However, CHO increased concentration of PC 18:0/18:1 during wk 1 postpartum (CHO × wk: P = 0.05) and tended to increase plasma concentrations of PC 16:0/20:4 when fed without MET (CHO × MET: P = 0.09). Furthermore, feeding CHO and MET together tended to reduce PC 16:0/18:2 concentrations (CHO × MET: P = 0.09).
Table 6Postpartum plasma choline metabolite concentrations (μM) for multiparous cows (n = 27) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
GPC = glycerophosphocholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; PC = phosphatidylcholine.
Treatment
SEM
Week postpartum
SEM
P-value
CON
CHO
MET
CHO + MET
1
2
CHO
MET
MET × CHO
Week
Betaine
49.9
42.6
42.7
41.1
7.56
48.5
39.7
3.16
0.58
0.49
0.64
<0.01
Free choline
4.21
4.59
4.07
3.75
0.44
4.52
3.79
0.22
0.95
0.15
0.29
0.02
GPC
11.6
18.4
11.1
13.1
4.49
8.6
18.5
3.93
0.29
0.45
0.53
0.03
SM
338
259
340
250
37.9
275
319
18
0.04
0.92
0.85
0.01
Total LPC
250
296
238
202
35.2
197
296
30.2
0.88
0.09
0.16
0.01
Total PC
2,406
2,583
2,528
2,094
370
2,104
2,701
230
0.74
0.57
0.34
0.03
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
2 GPC = glycerophosphocholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; PC = phosphatidylcholine.
Table 7Postpartum plasma lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) species concentrations (μM) for multiparous cows (n = 27) fed the control (CON) diet or the control diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d rumen-protected choline (RPC; ReaShure, Balchem Corp.; CHO), 12 g/d rumen-protected Met (RPM; Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
a,b Means with differing superscripts are significantly different (P < 0.05).
1 Prepartum, cows were supplemented with 60 g/d rumen-protected choline (RPC; ReaShure, Balchem Corp.; CHO), 12 g/d rumen-protected Met (RPM; Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
Plasma concentrations of betaine, GPC, and SM were unaffected by CHO or MET but MET tended to increase plasma free choline (P = 0.09; Table 8). Total LPC and individual LPC species concentrations were also unaffected by CHO or MET (Table 8, Table 9, respectively). There was no effect of CHO on total PC concentrations and the effect on individual PC species was minimal as only PC 18:0/18:1 tended to be reduced by CHO (P = 0.06). Feeding MET decreased total PC concentrations at wk 2 postpartum (MET × wk: P = 0.01; Table 9). Aligning with this observation, MET reduced concentrations of several PUFA-PC species (including PC 16:0/20:4, PC 18:1/20:4 + 18:0/20:5 + 16:0/22:5, PC 18:0/20:4, PC 18:0/20:3, and PC 18:1/22:6) during wk 2 postpartum (MET × wk: P = 0.02, P = 0.02, P = 0.04, P = 0.04, and P = 0.05, respectively). Similarly, MET decreased concentrations of all SFA- and UFA-PC species during wk 2 postpartum (MET × wk: all P < 0.01; Table 9). In contrast, MET fed without CHO increased concentrations of PC 16:0/20:3 during wk 1 postpartum, whereas other treatments failed to attain similar levels until wk 2 postpartum (CHO × MET × wk: P = 0.03).
Table 8Postpartum plasma choline metabolite concentrations (μM) for primiparous cows (n = 24) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
2 GPC = glycerophosphocholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; PC = phosphatidylcholine.
Table 9Postpartum plasma lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) species concentrations (μM) for primiparous cows (n = 24) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
Feeding MET increased plasma Met concentrations (P < 0.01) but reduced Ser concentrations during wk 2 postpartum (MET × wk: P = 0.03; Table 10). Feeding MET without CHO increased Phe concentrations and tended to increase Ile, Leu, and total branched-chain AA concentrations (CHO × MET: P = 0.04, P = 0.06, P = 0.09, and P = 0.08, respecitvely). There were few effects of CHO on plasma AA, but feeding CHO without MET decreased glutamate concentrations during wk 1 but not wk 2 postpartum (CHO × MET × wk: P = 0.03).
Table 10Postpartum plasma amino acid concentrations (μM) for multiparous cows (n = 27) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
a,b Means with differing superscripts are significantly different (P < 0.05).
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
Results for plasma AA in primiparous cows are shown in Table 11. Feeding MET increased plasma Met concentrations (P < 0.01) but tended to reduce Gly concentrations (P = 0.08). Feeding MET reduced Ser concentrations (P = 0.01), but this effect tended to be intensified when CHO was fed with MET (CHO × MET: P = 0.08). The effect of CHO on plasma AA were minimal, although CHO increased Gln and tended to increase Gly concentrations during wk 1 postpartum (CHO × wk: P = 0.04 and P = 0.07, respectively).
Table 11Postpartum plasma amino acid concentrations (μM) for primiparous cows (n = 24) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Blood samples were obtained during wk 1 and 2 postpartum.
Relative mRNA levels of hepatic genes associated with choline, Met, and fatty acid metabolism for a subset of multiparous cows (n = 20) is shown in Table 12. Of the one-carbon metabolism genes examined, only BHMT and PCYT1A were affected by CHO or MET. Feeding MET reduced mRNA levels of PCYT1A (P < 0.01). Additionally, CHO tended to increase mRNA levels of BHMT and PCYT1A when fed without MET (Figure 1, CHO × MET: P = 0.09 and P = 0.10, respectively). The mRNA levels of HMGCS2 and PPARα tended to be reduced by CHO (P = 0.10 and P = 0.08). Feeding MET tended to reduce mRNA expression of DGAT1 (P = 0.08).
Table 12Postpartum covariate-adjusted gene expression fold-changes relative to control (CON) for multiparous cows (n = 20) fed the CON diet or the CON diet plus rumen-protected choline (RPC), rumen-protected Met (RPM), or both
Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Prepartum (−21 d relative to calving) gene expression fold-change was used as a covariate in the statistical model.
1 Prepartum, cows were supplemented with 60 g/d RPC (ReaShure, Balchem Corp.; CHO), 12 g/d RPM (Smartamine M, Adisseo USA Inc.; MET), or both (CHO + MET). Postpartum, cows were supplemented with 60 g/d RPC, 18 g/d RPM, or both. Cows were monitored and treatments were applied from 21 d before expected calving through 35 DIM. Prepartum (−21 d relative to calving) gene expression fold-change was used as a covariate in the statistical model.
Figure 1Differences in postpartum hepatic mRNA expression of the genes BHMT and PCYT1A that are associated with Met and choline metabolism, respectively, in multiparous cows (n = 20) fed the control (CON) diet or the CON diet plus rumen-protected choline (RPC; 60 g/d; CHO), rumen-protected Met (RPM; 12 g/d prepartum, 18 g/d postpartum; MET), or both (CHO + MET) from samples taken ~7 d after calving. Data are expressed as covariate-adjusted fold-changes relative to the CON treatment. For both genes, MET × CHO tended to be significant (P = 0.09 and P = 0.10, respectively). For PCYT1A, the effect of MET was significant (P < 0.01).
Our results suggest that supplemental choline and Met both seem to affect one-carbon metabolic pathways during the periparturient period but that responses are inconsistent between primi- and multiparous cows and appear to be dependent on Met availability. Although we did not examine metabolite flux through pathways associated with choline and Met metabolism, changes in metabolite secretion in milk and concentrations in plasma are indicative of shifts in these pathways. Alterations in hepatic expression of genes associated with one-carbon and lipid metabolism in multiparous cows further support this idea. However, the tendency for interactive effects on hepatic gene expression suggest that supplemental choline may play a role in promoting the CDP-choline and BHMT pathways but that the response is dependent on Met availability. Interactive effects on milk and plasma metabolites provide additional evidence of this.
Choline Metabolites
Baseline milk secretion and plasma concentrations of free choline, betaine, SM, total LPC, and total PC were comparable between our study and that of previous studies (
Short communication: Effects of dietary deoiled soy lecithin supplementation on circulating choline and choline metabolites, and the plasma phospholipid profile in Holstein cows fed palm fat.
indicated an increase in milk free choline and betaine secretion when RPC was fed to lactating cows. In our study, CHO decreased milk secretion of betaine during the first wk postpartum in multiparous cows, which coincides with our observation for the tendency of CHO to increase hepatic BHMT expression for multiparous cows when fed without MET. This increased use of betaine by the liver likely reduced betaine availability to the mammary gland. In addition to putative increases in dietary choline oxidation to betaine, the overall increase in milk secretion of LPC and several LPC species for cows fed CHO also suggest that CHO increased choline metabolite production via the CDP-choline or PEMT pathways.
Feeding CHO tended to increase milk secretion of free choline and betaine for primiparous cows in our study, which is contrary to our observations for multiparous cows but similar to the findings reported by
. This increase in free choline could suggest an adequate choline supply and increased ability to support the BHMT pathway for Met synthesis. However, in their study of choline and Met kinetics in lactating goats,
indicated that very little choline is directed toward Met synthesis, which would explain why there was no effect of CHO on plasma Met concentrations in our study. Varied responses to CHO between primi- and multiparous cows for milk secretion of choline metabolites in our study are likely due, in part, to differences in physiological state and production level, which presumably affect the demand for methyl donors. Younger cows seem to have lower choline and Met requirements than older cows (
). Therefore, it is possible that the methyl donor balance of the primiparous cows in our study was more similar to that of the mid-lactation cows studied by
, which may help explain the agreement of responses to CHO between the primiparous, but not multiparous, cows in our study and the cows in the study conducted by
. Although CHO did not affect plasma betaine or free choline concentrations in primiparous cows, MET tended to increase plasma free choline concentrations, which suggests either a preferential increase in the use of Met as a methyl donor over choline or that Met was used to synthesize additional choline (
reported no effect of MET on plasma free choline concentrations in multiparous, periparturient cows, which agrees with our observations for multiparous cows.
Plasma concentration and milk secretion of SM was unaffected by treatment in primiparous cows, which agrees with data reported by
. Milk secretion of SM was also unaffected by treatment in multiparous cows. However, the decrease in plasma SM concentrations for multiparous cows fed CHO was somewhat surprising, given that previous work by
showed no effect of postruminal choline supply on milk or plasma SM. Sphingomyelin is produced from PC and is involved with cell signaling associated with inflammation (
), so a decrease in SM could suggest a reduced inflammatory response. This presents a mechanism by which choline may exert favorable effects with regard to minimizing inflammation during the periparturient period. Sphingomyelin has also been shown to be inversely associated with insulin sensitivity in monogastrics (
), which would suggest greater insulin sensitivity for multiparous cows fed CHO in our study. However, additional research is needed to determine what, if any, effects dietary choline has on insulin sensitivity in dairy cows.
indicated no change in total LPC secretion in milk when supplemental choline was delivered via abomasal infusion or as RPC. The increased milk secretion of LPC in our study combined with the trend for reduced betaine secretion, is supportive of increased use of choline for PC synthesis. Because GPC secretion was also unaffected, CHO may have diverted additional PC toward LPC production, rather than to GPC. Our observations for primiparous cows were similar, although this effect seemed to depend on Met availability. This was largely driven by changes in LPC 16:0 and LPC 18:0, where CHO increased their secretion only when fed without MET. Previous studies have shown increases in total plasma LPC (
Short communication: Effects of dietary deoiled soy lecithin supplementation on circulating choline and choline metabolites, and the plasma phospholipid profile in Holstein cows fed palm fat.
). Although we did not observe an increase in plasma total LPC concentrations in response to CHO, our results did indicate increases in concentrations of LPC 16:0 and 18:1 when CHO was fed without MET for multiparous cows. The fact that MET negated this increase seems indicative of a shift in priority when sufficient Met was available. The tendency for MET to decrease total plasma LPC concentrations in multiparous cows further supports this idea. Assuming that Met supply was sufficient for primiparous cows in our study, this observation would coincide with the lack of effect of CHO or MET on plasma total LPC and LPC 16:0, 18:1, and 18:0 in primiparous cows. The observation for plasma LPC 18:1 concentration aligns with our observation for milk secretion of LPC 18:1 in multiparous cows, in which MET minimized the CHO-induced increase. Research in nonruminants has indicated that LPC has immunomodulatory effects (
), which provides a mechanism through which dietary choline or Met could interact with the immune system. Because MET seemed to mute the CHO-induced increases of these LPC species, it is possible that MET may have provided an alternative mechanism for activating the immune system, such as through glutathione (
). In addition to its effects on the immune system, LPC is also associated with energy metabolism. Both LPC 16:0 and LPC 18:1 are shown to be reduced during weight loss in obese humans (
). The elevation of these LPC species in multiparous cows fed CHO without MET could indicate that these cows were mobilizing less body fat. This idea is supported by the numerically greater body condition score, greater body weight, and lower plasma fatty acids observed for the multiparous cows fed this treatment during the postpartum period (
). The increase in LPC secretion in milk and concentration in plasma for cows fed CHO suggests an increase in PC conversion to LPC, which is supportive of a sufficient PC supply. Overall, these findings demonstrate that supplemental choline can affect LPC synthesis, which has the potential to modulate the immune system as well as energy metabolism, but that these changes depend on the Met availability to the animal.
Despite a lack of treatment effect on total PC secretion in milk and concentration in plasma, the changes in secretion of several PC species for multiparous cows suggests that both CHO and MET affected PC synthesis pathways. Research in rodents indicated that SFA- and UFA-PC species are typically derived from the CDP-choline pathway, whereas PUFA-PC species are derived from the PEMT pathway (
). Although these findings can provide valuable insight for interpreting our results, caution should be exercised when extrapolating these findings to ruminant animals, which typically have a relatively limited access to dietary choline in the small intestine. The fact that CHO and MET both enhanced secretion of several PUFA-PC species suggests that both promoted the PEMT pathway in multiparous cows. However, CHO only increased secretion of these PC species during wk 2, suggesting that choline can modulate the PEMT pathway but that this adaptation takes time. By contrast, the apparent increase in the PEMT pathway was not time-dependent for MET, as it increased PUFA-PC secretion, and likely flux through the PEMT pathway, irrespective of week. The PEMT pathway requires SAM to provide 3 methyl groups to produce PC from phosphatidylethanolamine (
). It is possible that there was insufficient Met available to support the PEMT pathway via SAM during wk 1 for cows fed CHO. It appears that feeding MET helped to overcome this challenge irrespective of CHO supplementation. Together, these data suggest that, while both CHO and MET have the ability to support PEMT pathway activities, MET may act earlier to enhance availability of PEMT-derived PC-species through the provision of additional SAM. The general lack of treatment effect on the secretion of individual PC species for primiparous cows suggests minimal change to the apparent balance between the PEMT and CDP-choline pathways (
). This finding agrees with the hypothesis that the primiparous cows in our study had a more positive choline balance and is supported by the increase in free choline and betaine secreted into milk when CHO was fed. If the primiparous cows had achieved a positive choline balance, it is likely that PC supply was already sufficient before CHO or MET supplementation and that there was little need to alter the pathways by which PC needs were being met.
There was minimal effect of CHO or MET on the plasma concentrations of individual PC species in multiparous cows, suggesting minimal shift in PC origin (
). However, in primiparous cows, MET reduced plasma concentrations of several species derived from both PEMT and CDP-choline pathways during wk 2, suggesting that MET decreased PC needs during this time. These results are somewhat contradictory to those presented by
, who showed an increase in plasma concentrations of several PC species of CDP-choline pathway origin when RPC was fed to feed-restricted dry cows. However, previous research in rats suggested that dietary Met alters plasma concentrations of various PC species, specifically by reducing those that contain linoleic acid and PC 16:0/20:4 (
), which would suggest a decrease in PC synthesized via the PEMT pathway. Indeed, we also observed a reduction in plasma concentration of several PEMT-PC species in primiparous cows fed MET during wk 2 postpartum, including PC 16:0/20:4. However, the biological significance of this change is unclear.
Research in humans suggests that the PC and LPC profiles differ among the lipoprotein fractions (
). Thus, the treatment-induced changes in plasma PC and LPC profiles for cows in our study could reflect alterations in the lipoprotein profile. However, we did not measure plasma lipoprotein fractions in our study. Although the measurement of lipoprotein fractions in bovine plasma is more complex than for humans (
Of the nonessential AA, only plasma Ser, glutamate, Gln, and Gly were affected by CHO or MET. Contrary to these observations, previous work with periparturient dairy cows by
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
indicated no effect of Met or choline on plasma Ser, glutamate, Gln, or Gly concentrations. Furthermore, they observed that Met increased plasma Ala, aspartate, Asn, Pro, and glutamate, which is also contrary to our results. It is curious that MET decreased plasma Ser for both primi- and multiparous cows in our study, given that
indicated that Met can be used to synthesize Ser to a limited extent in lactating goats and that Ser is required for the first step toward glutathione production from Hcy, as well as for regeneration of 5, 10-methyl-tetrahydrofolate from tetrahydrofolate in the methyltetrahydrofolate pathway (
), it is unlikely that this reduction is indicative of changes in glutathione production. For the same reasons, the decrease in glutamate concentration during wk 1 postpartum for multiparous cows fed CHO without MET are also probably not related to alterations in glutathione production. Instead, these observations are more likely indicative of increased protein synthesis. This aligns with the absence of a treatment effect on hepatic GSS expression and the increased milk protein content we observed for multiparous cows fed MET in our study (
Rumen-protected methionine fed to dairy cows: Bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations.
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
), MET increased plasma Met for both groups of cows, indicating that the Met fed as RPM was absorbed. However, effects of supplemental Met on plasma concentrations of other essential AA in previous studies has been variable. Previous research reported by
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
indicated that Met increased plasma concentrations of several other essential AA in periparturient cows, including Arg, Lys, and Trp. Contrary to these findings, the essential AA affected by MET for multiparous cows in our study were Ile, Leu, Met, and Phe. Previous work (
Rumen-protected methionine fed to dairy cows: Bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations.
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.
) showed that RPM decreased plasma concentrations of the branched-chain AA Leu, Ile, and Val. Interestingly, we observed the opposite, where MET tended to increase concentrations of the branched-chain AA Ile and Leu. Although changes in branched-chain AA concentrations can be indicative of changes in DMI, intake was similar across treatments in our study (
). Alternatively, this response could be explained by alterations in muscle AA metabolism because it occurred in absence of a change in DMI. A recent examination of muscle tissue from periparturient dairy cows fed RPM that demonstrated the ability of MET to affect key pathways related to AA and protein metabolism (
Alterations in skeletal muscle mRNA abundance in response to ethyl-cellulose rumen-protected methionine during the periparturient period in dairy cows.
). Thus, the increase in plasma Ile and Leu concentrations for multiparous cows in our study could help explain the increased milk protein concentration observed for multiparous cows fed MET (
The PCYT1A gene encodes phosphate cytidylyltransferase 1 choline α, which is the enzyme that catalyzes the rate-limiting step of the CDP-choline pathway (
). Our observations lend support to the hypothesis that supplemental choline increases PC synthesis via the CDP-choline pathway. This change coincided with the increase in milk secretion of several CDP-choline-derived PC species by CHO and is suggestive of the ability for supplemental choline to increase PC synthesis via this pathway in periparturient cows. Because this response seemed to be dependent on MET supplementation, it is possible that inclusion of MET redirected supplemental choline for another purpose.
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
also observed an increase in PCYT1A mRNA expression for periparturient cows fed choline, although they indicated that supplemental Met also increased PCYT1A mRNA expression. Contrary to our results, previous work by
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
indicated that supplemental choline did not affect expression of BHMT for periparturient cows. However, CHO tended to increase BHMT expression when fed without MET, suggesting an increase in Met recycling from Hcy via the BHMT pathway for these cows. This response seemed to be dependent on MET supplementation, which could be indicative of a reduced need to synthesize Met from Hcy via this pathway when Met supply is sufficient. In support of this hypothesis, recent in vitro work by
The effect of increasing concentrations of dl-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic genes controlling methionine regeneration and gluconeogenesis.
showed a reduction in BHMT mRNA expression with increasing concentrations of Met, indicating that when Met is in ample supply, less Met is regenerated from Hcy through the BHMT pathway.
Lack of an effect of CHO or MET on mRNA expression of PEMT, GSS, or MTR suggests that the effects of these nutrients on these pathways do not occur at the transcription level. This observation for PEMT expression aligns with the lack of effect of CHO or MET on the plasma concentrations PEMT-derived PC species in multiparous cows. However, this is contrary to previous work which indicated an increase in hepatic mRNA expression of PEMT in periparturient cows fed supplemental Met (
Smartamine M and MetaSmart supplementation during the peripartal period alter hepatic expression of gene networks in 1-carbon metabolism, inflammation, oxidative stress, and the growth hormone-insulin-like growth factor 1 axis pathways.
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
, who reported a decrease in MTR activity, but not MTR mRNA expression, for periparturient cows fed supplemental choline or Met. It is also possible that, with only 5 cows per treatment, we did not have sufficient statistical power in our study to detect changes in mRNA expression of these genes.
The mRNA levels of HMGCS2 and PPARα tended to be reduced by CHO (Table 12), which would suggest reductions in ketogenesis and hepatic fatty acid oxidation by cows fed choline. However, CHO did not affect postpartum plasma BHBA or nonesterified fatty acid concentrations for multiparous cows (
) mRNA expression when choline was fed to periparturient cows. Feeding MET tended to reduce mRNA expression of DGAT1 (Table 12), suggesting a reduction in liver triglyceride (TG) synthesis, although there was no effect of MET on liver TG content (
) suggests that choline has little effect on liver TG synthesis during the periparturient period. Our observations for hepatic gene expression agree with the lack of treatment effect on energy balance, plasma fatty acid concentrations, and liver TG content for multiparous cows in this study (
). However, it is worth noting that the multiparous cows in our study did not experience severe negative energy balance during the postpartum period (−3.1 Mcal/d) and that liver TG accumulation was relatively mild (4.9% DM), which may have affected gene expression responses.
Feeding CHO or MET did not affect mRNA levels of MTTP in this study. Microsomal triglyceride transfer protein is an enzyme involved in packaging TG into very low density lipoprotein in the liver (
). An increase in MTTP mRNA expression could indicate an increase in the rate of very low density lipoprotein formation, which is the proposed route by which choline could reduce hepatic TG concentration in dairy cows (
The effects of prepartum energy intake and peripartum rumen-protected choline supplementation on hepatic genes involved in glucose and lipid metabolism.
and suggest a limited effect of CHO on TG export for the cows in the current study.
CONCLUSIONS
With the exception of milk secretion of 3 LPC species, effects of choline and Met on apparent choline metabolism were not consistent for primi- and multiparous cows. Although there were subtle changes in the PC profile of milk and plasma in response to supplemental choline or Met, substantial, consistent changes did not suggest definitive shifts in the balance of PC synthesis via the PEMT or CDP-choline pathways. Supplemental choline alone tended to increase hepatic expression of genes associated with choline metabolism in multiparous cows, which suggests a probable role in promoting the CDP-choline and BHMT pathways. However, when supplemental Met was combined with supplemental choline, this response seemed to be negated, which suggests a dependency on Met availability to the cow. This is further supported by the differences in responses between primi- and multiparous cows, who likely differ in their choline and Met requirements. Additional work is required to explore how choline and Met affect one-carbon metabolism during the periparturient period and how these responses may differ with choline and methionine availability to the cow.
ACKNOWLEDGMENTS
Partial funding for this study was provided by Balchem Corporation (New Hampton, NY). The authors acknowledge the staff at the Central Maryland Research and Education Center Dairy Unit (Clarksville, MD) for their assistance with animal care and management. The authors are also grateful to Claudia Gomez and Emily Davis (University of Maryland, College Park) for assisting with sample and data collection. The authors have not stated any conflicts of interest.
REFERENCES
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Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices.
Rumen-protected methionine fed to dairy cows: Bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations.
The effects of prepartum energy intake and peripartum rumen-protected choline supplementation on hepatic genes involved in glucose and lipid metabolism.
Choline supply during negative nutrient balance alters hepatic cystathionine β-synthase, intermediates of the methionine cycle and transsulfuration pathway, and liver function in Holstein Cows.
Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.
Smartamine M and MetaSmart supplementation during the peripartal period alter hepatic expression of gene networks in 1-carbon metabolism, inflammation, oxidative stress, and the growth hormone-insulin-like growth factor 1 axis pathways.
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.
Alterations in skeletal muscle mRNA abundance in response to ethyl-cellulose rumen-protected methionine during the periparturient period in dairy cows.
Short communication: Effects of dietary deoiled soy lecithin supplementation on circulating choline and choline metabolites, and the plasma phospholipid profile in Holstein cows fed palm fat.
Methyl donor supplementation suppresses the progression of liver lipid accumulation while modifying the plasma triacylglycerol lipidome in periparturient Holstein dairy cows.
The effect of increasing concentrations of dl-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic genes controlling methionine regeneration and gluconeogenesis.
Hepatic activity and transcription of betaine-homocysteine methyltransferase, methionine synthase, and cystathionine synthase in periparturient dairy cows are altered to different extents by supply of methionine and choline.
Methionine and choline supply during the periparturient period alter plasma amino acid and one-carbon metabolism profiles to various extents: Potential role in hepatic metabolism and antioxidant status.
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