Postruminal choline supply during negative nutrient balance alters components of hepatic mTOR signaling and plasma amino acids in lactating Holstein cows.

Choline requirements for dairy cattle are unknown. However, enhanced postruminal supply of choline may increase flux through the methionine cycle to spare Met for other functions such as protein synthesis and phosphatidylcholine (PC) synthesis during periods of negative nutrient balance (NNB). The objective was to investigate the effects of postruminal choline supply during a feed restriction-induced NNB on hepatic abundance and phosphorylation of mTOR (mechanistic target of rapamycin)-related signaling proteins, hepatic lipidome and plasma AA. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 DIM) were used in a replicated 5 × 5 Latin square design with 4 d of treatment and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements to induce NNB) with abomasal infusion of water (R0) or restriction plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected via biopsy on d 5 after infusions ended and used for Western blot analysis to measure proteins involved in mTOR signaling and untargeted lipidomics. Blood was collected on d 1 to 5 for plasma AA analysis. Statistical contrasts for protein and AA data were A0 versus R0 (CONT1), R0 versus the average of choline dose (CONT2) and tests of linear and quadratic effects of choline dose. Analysis of lipidomic data were performed with the web-based metabolomic processing tool MetaboAnalyst 5.0. Ratios of p-RPS6KB1:tRPS6KB1, p-EEF2:tEEF2, and p-EIF2:tEIF2 were greater with R (CONT1). Among those, supply of choline led to decreases in p-EEF2:tEEF2 (CONT2), p-EIF2:tEIF2 and tended to decrease p-EIF4BP1:tEIF4BP1. However, the effect was quadratic only for p-EEF2:tEEF2 and p-EIF2A:tEIF2A, reaching a nadir at 6.25 to 12.5 g/d choline ion. The ratio of p-RPS6KB1:tRPS6KB1 was not affected by supply of choline and was close to 2-fold greater at 25 g/d choline versus A0. Plasma Met concentration decreased with R (CONT1), but increased linearly with choline. Restriction also increased plasma 3-methyl-histidine (CONT1). The partial least squares discriminant analysis model of liver lipids distinguished treatments, with 13.4% of lipids being modified by treatment. One-way ANOVA identified 109 lipids with a false discovery rate ≤0.05. The largest group identified was PC species; all 35 detected decreased with R versus A0, but there were few differences among choline treatments. Overall, data suggested that dephosphorylation of EEF2 and EIF2A due to enhanced choline supply potentially helped maintain or increase protein synthesis during NNB. While activation of mTOR was not altered by choline, this idea of increased protein synthesis is partly supported by the increased circulating Met. However, enhanced postruminal choline had limited effects on the species of lipid produced during a period of NNB.

Feeding rumen-protected lysine altered immune and metabolic biomarkers in dairy cows during the transition period.

This experiment was conducted to determine the effects of feeding rumen-protected lysine (RPL; AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America Inc.) from -26 ± 4.6 d prepartum (0.54% RPL of dietary dry matter intake) to 28 d postpartum (0.39% RPL of dietary dry matter intake) on immunometabolic status and liver composition in dairy cows. Seventy-five multiparous Holstein cows, blocked by parity, previous 305-d mature-equivalent milk production, expected calving date, and body condition score during the far-off dry period were assigned to 1 of 4 dietary treatments in a randomized, complete block design with a 2 × 2 factorial arrangement of treatments. Treatments prepartum consisted of total mixed ration top dressed with RPL (PRE-L) or without RPL (PRE-C), and postpartum treatments consisted of total mixed ration top dressed PRE-L prepartum and postpartum, PRE-L prepartum and PRE-C postpartum, PRE-C prepartum and PRE-L postpartum, and PRE-C prepartum and postpartum in 300 g of molasses. Blood samples were taken on -7 ± 0.5, 0 ± 0.5, 7 ± 0.9, 14 ± 0.9, and 28 ± 0.5 d relative to calving. Whole blood samples were taken on -14 ± 0.5, -7 ± 0.5, 7 ± 0.9, and 14 ± 0.9 d relative to calving for oxidative burst and phagocytic capacity of monocytes and neutrophils. Liver samples were collected via a biopsy on -12 ± 4.95 and 13 ± 2.62 d relative to calving and analyzed for liver composition (triacylglyceride and carnitine concentrations), mRNA expression of hepatic genes, and protein abundance. Protein abundance was calculated by normalizing intensity bands for a specific protein with glyceraldehyde-3-phosphate dehydrogenase. Concentrations of haptoglobin and glutathione peroxidase activity in plasma were lower at d 0 for cows in PRE-L (102 µg/mL and 339 nmol/min per mL, respectively) compared with cows in PRE-C (165 µg/mL and 405 nmol/min per mL, respectively). Oxidative burst capacity in monocytes tended to be greater on d 7 postpartum for cows in PRE-L (65.6%) than cows in PRE-C (57.5%). Additionally, feeding RPL altered the mRNA expression in liver tissue prepartum [decreased INSR (insulin receptor), CPT1A (carnitine palmitoyltransferase 1A), and IL1B (interleukin 1 ß)] and postpartum [increased IL8 (interleukin 8), EHMT2 (euchromatic histone lysine methyltransferase 2), TSPO (translocator protein), and SLC3A2 (solute carrier family 3 member 2); and decreased SLC7A1 (solute carrier family 7 member 1), SOD1 (superoxide dismutase 1), and SAA3 (serum amyloid A 3)] compared with cows not consuming RPL]. Additionally, cows in the PRE-C prepartum and PRE-L postpartum treatment tended to have greater protein abundance of mTOR postpartum compared with the PRE-C prepartum and postpartum treatment. Protein abundance of SLC7A7 (solute carrier family 7 member 7) pre- and postpartum tended to be greater and BBOX1 (gamma-butyrobetaine dioxygenase 1) tended to be less when RPL was consumed prepartum. In conclusion, cows that consumed RPL during the transition period had molecular changes related to liver composition, enhanced liver function indicated by greater total protein and albumin concentrations in plasma, and improved immune status indicated by decreased haptoglobin, glutathione peroxidase activity, and immune related mRNA expression.

Prepartum dietary energy intake alters hepatic expression of genes related to peroxisome proliferator-activated receptor and inflammation in peripartal dairy cows.

We determined the effect of prepartum plane of energy intake on liver function and metabolism pre- and postpartum by combining in vivo and in vitro data with mRNA expression data. A subset of multiparous prepartal Holsteins (n = 18) from a previously conducted experiment consumed 1 of 3 amounts of dietary energy intake, relative to their requirements. A diet formulated to allow consumption of ≥150% of net energy requirements during the far-off dry period and the close-up dry period was fed for ad libitum intake (150E) or in restricted amounts so that cows consumed 80% of requirements for energy (80E). A second diet was formulated to include wheat straw (26.1% of dry matter) to limit energy intake to 100% of NRC (2001) requirements for energy when fed ad libitum during the far-off period (100E). In the close-up period, 100E was fed the 150E diet for ad libitum intake. Expression of mRNA for genes related to fatty acid oxidation (PPARA, CPT1A, ACOX1) was greater for 100E cows than 150E cows on d 14 postpartum. These expression patterns were related to in vitro data for conversion of palmitate to CO2, acid-soluble products, and esterified products by liver slices. Abundance of mRNA for PC displayed a sharp peak for all groups on d 1 postpartum, but serum glucose did not reflect this peak. The mRNA expression of SREBF1 was greater for 150E and 100E cows prepartum compared with 80E, and was positively related to rate of palmitate esterification postpartum. Expression of NR1H3 (LXRA) mRNA was greater for 100E cows on d 14 postpartum compared with 150E cows, which corresponded to expression of PPARA. An inflammatory response occurred in the liver around the time of parturition for 150E cows, as expression of IL1B was elevated both pre- and postpartum compared with 100E cows. The spike in IL1B expression for 150E cows on d 14 postpartum corresponded to the peak concentration of total lipids in liver tissue for all groups in this experiment. Overconsumption of energy prepartum was detrimental to the expression of important genes related to PPAR and liver function, especially postpartum. Furthermore, results provide evidence for inflammation related to accumulation of lipids in liver and overnutrition prepartum.

Residual feed intake in peripartal dairy cows is associated with differences in milk fat yield, ruminal bacteria, biopolymer hydrolyzing enzymes, and circulating biomarkers of immunometabolism.

Residual feed intake (RFI) measures feed efficiency independent of milk production level, and is typically calculated using data past peak lactation. In the current study, we retrospectively classified multiparous Holstein cows (n = 320) from 5 of our published studies into most feed-efficient (M-eff) or least feed-efficient (L-eff) groups using performance data collected during the peripartal period. Objectives were to assess differences in profiles of plasma biomarkers of immunometabolism, relative abundance of key ruminal bacteria, and activities of digestive enzymes in ruminal digesta between M-eff and L-eff cows. Individual data from cows with ad libitum access to a total mixed ration from d -28 to d +28 relative to calving were used. A linear regression model including dry matter intake (DMI), energy-corrected milk (ECM), changes in body weight (BW), and metabolic BW was used to classify cows based on RFI divergence into L-eff (n = 158) and M-eff (n = 162). Plasma collected from the coccygeal vessel at various times around parturition (L-eff = 60 cows; M-eff = 47 cows) was used for analyses of 30 biomarkers of immunometabolism. Ruminal digesta collected via esophageal tube (L-eff = 19 cows; M-eff = 29 cows) was used for DNA extraction and assessment of relative abundance (%) of 17 major bacteria using real-time PCR, as well as activity of cellulase, amylase, xylanase, and protease. The UNIVARIATE procedure of SAS 9.4 (SAS Institute Inc.) was used for analyses of RFI coefficients. The MIXED procedure of SAS was used for repeated measures analysis of performance, milk yield and composition, plasma immunometabolic biomarkers, ruminal bacteria, and enzyme activities. The M-eff cows consumed less DMI during the peripartal period compared with L-eff cows. In the larger cohort of cows, despite greater overall BW for M-eff cows especially in the prepartum (788 vs. 764 kg), no difference in body condition score was detected due to RFI or the interaction of RFI × time. Milk fat content (4.14 vs. 3.75 ± 0.06%) and milk fat yield (1.75 vs. 1.62 ± 0.04 kg) were greater in M-eff cows. Although cumulative ECM yield did not differ due to RFI (1,138 vs. 1,091 ± 21 kg), an RFI × time interaction due to greater ECM yield was found in M-eff cows. Among plasma biomarkers studied, concentrations of nonesterified fatty acids, ß-hydroxybutyrate, bilirubin, ceruloplasmin, haptoglobin, myeloperoxidase, and reactive oxygen metabolites were overall greater, and glucose, paraoxonase, and IL-6 were lower in M-eff compared with L-eff cows. Among bacteria studied, abundance of Ruminobacter amylophilus and Prevotella ruminicola were more than 2-fold greater in M-eff cows. Despite lower ruminal activity of amylase in M-eff cows in the prepartum, regardless of RFI, we observed a marked linear increase after calving in amylase, cellulase, and xylanase activities. Protease activity did not differ due to RFI, time, or RFI × time. Despite greater concentrations of biomarkers reflective of negative energy balance and inflammation, higher feed efficiency measured as RFI in peripartal dairy cows might be associated with shifts in ruminal bacteria and amylase enzyme activity. Further studies could help address such factors, including the roles of the liver and the mammary gland.

Effect of stage of lactation and dietary starch content on endocrine-metabolic status, blood amino acid concentrations, milk yield, and composition in Holstein dairy cows.

Milk yield and composition are modified by level and chemical characteristics of dietary energy and protein. Those factors determine nutrient availability from a given diet, and once absorbed, they interact with the endocrine system and together determine availability of metabolites to the mammary gland. Four multiparous dairy cows in early lactation and subsequently in late lactation were fed 2 diets for 28 d in a changeover design that provided, within the same stage of lactation, similar amounts of rumen fermentable feed with either high (HS) or low starch (LS). All diets had similar dietary crude protein (15.5% dry matter) and rumen-undegradable protein (∼40% of crude protein) content. Profiles of AA were calculated to be similar to that of casein. On d 28, [1-13C] Leu was infused into one jugular vein with blood samples taken at 0, 2, 4, 6, and 8 h, and cows milked at 0, 2, 4, 5, 6, 7, and 8 h from start of infusion. Isotopic enrichments of plasma Leu, keto-isocaproic acid, and milk casein were determined for calculation of Leu kinetics. Data were subjected to ANOVA using the MIXED procedure of SAS (SAS Institute Inc.), with time as repeated factor and cow as the random effect. Dry matter intake within each stage of lactation was similar between groups. Feeding LS resulted in lower blood glucose and greater ratio of bovine somatotropin to insulin. This response was associated with greater blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, which might have contributed to greater milk fat content in LS-fed cows. Except for His, average concentrations of all AA in blood were higher in late than early lactation. Diet did not alter average plasma concentrations of AA. However, for most of the essential AA (particularly branched-chain), the HS diet led to a marked decrease in concentrations after the forage meal, resulting in significant differences between dietary groups in early lactation. In early-lactating cows fed HS, a greater reduction in plasma concentrations at 8 h relative to pre-feeding values (time zero) was observed for Met, Lys, and His, resulting in decreases of 27.9%, 33.6%, and 38.5%, respectively. A higher bovine somatotropin/insulin ratio in early lactation and in cows fed LS could possibly have led to greater breakdown and, consequently, higher AA flux from peripheral tissues. In LS-fed cows, higher mobilization of body fat and protein was confirmed by the greater body weight loss in both stages of lactation. Higher irreversible loss of [1-13C] Leu in early lactation suggested lower protein retention in peripheral tissues during early compared with late lactation. Milk yield, protein output, and composition were similar between groups at both stages of lactation, whereas milk coagulation was faster (lower curd firming rate) and with higher curd firmness in response to feeding HS in late lactation. Overall, data indicated that rate of carbohydrate fermentability in the rumen can modify the availability of metabolites to the mammary gland and consequently modify milk protein coagulation.

Rumen-protected methionine during heat stress alters mTOR, insulin signaling, and 1-carbon metabolism protein abundance in liver, and whole-blood transsulfuration pathway genes in Holstein cows.

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione ß-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

Methionine supplementation during a hydrogen peroxide challenge alters components of insulin signaling and antioxidant proteins in subcutaneous adipose explants from dairy cows.

Enhanced postruminal supply of methionine (Met) during the peripartal period alters protein abundance of insulin, AA, and antioxidant signaling pathways in subcutaneous adipose tissue (SAT). Whether SAT is directly responsive to supply of Met and can induce molecular alterations is unknown. Our objective was to examine whether enhanced Met supply during an oxidative stress challenge in vitro alters insulin, AA, inflammation, and antioxidant signaling-related protein networks. Four late-lactation Holstein cows (average 27.0 kg of milk per day) were used for SAT collection. Tissue was incubated in duplicate for 4 h in a humidified incubator with 5% CO2 at 37°C according to the following experimental design: control medium with an "ideal" profile of essential AA (CTR; Lys:Met 2.9:1), CTR plus 100 µM H2O2 (HP), or CTR with greater Met supply plus 100 µM H2O2 (HPMET; Lys:Met 2.5:1). Molecular targets associated with insulin signaling, lipolysis, antioxidant nuclear factor, erythroid 2 like 2 (NFE2L2), inflammation, and AA metabolism were determined through reverse-transcription quantitative PCR and western blotting. Data were analyzed using the MIXED procedure of SAS 9.4 (SAS Institute Inc.). Among proteins associated with insulin signaling, compared with CTR, HP led to lower abundance of phosphorylated AKT serine/threonine kinase (p-AKT) and solute carrier family 2 member 4 (SLC2A4; insulin-induced glucose transporter). Although incubation with HPMET restored abundance of SLC2A4 to levels in the CTR and upregulated abundance of fatty acid synthase (FASN) and phosphorylated 5'-prime-AMP-activated protein kinase (p-AMPK), it did not alter p-AKT, which remained similar to HP. Among proteins associated with AA signaling, compared with CTR, challenge with HP led to lower abundance of phosphorylated mechanistic target of rapamycin (p-MTOR), and HPMET did not restore abundance to CTR levels. Among inflammation-related targets studied, incubation with HPMET led to greater protein abundance of nuclear factor kappa B subunit p65 (NFKB-RELA). The response in NFKB observed with HPMET was associated with a marked upregulation of the antioxidant transcription regulator NFE2L2 and the antioxidant enzyme glutathione peroxidase 1 (GPX1). No effects of treatment were detected for mRNA abundance of proinflammatory cytokines or antioxidant enzymes, underscoring the importance of post-transcriptional regulation. Overall, data indicated that short-term challenge with H2O2 was particularly effective in reducing insulin and AA signaling. Although a greater supply of Met had little effect on those pathways, it seemed to restore the protein abundance of the insulin-induced glucose transporter. Overall, the concomitant upregulation of key inflammation and antioxidant signaling proteins when a greater level of Met was supplemented to oxidant-challenged SAT highlighted the potential role of this AA in regulating the inflammatory response and oxidant status. Further studies should be conducted to assess the role of postruminal supply of Met and other AA in the regulation of immune, antioxidant, and metabolic systems in peripartal cow adipose tissue.

Hepatic transcriptome perturbations in dairy cows fed different forage resources.

BACKGROUND: Forage plays critical roles in milk performance of dairy. However, domestic high-quality forage such as alfalfa hay is far from being sufficient in China. Thus, more than 1 million tons of alfalfa hay were imported in China annually in recent years. At the same time, more than 10 million tons of corn stover are generated annually in China. Thus, taking full advantage of corn stover to meet the demand of forage and reduce dependence on imported alfalfa hay has been a strategic policy for the Chinese dairy industry. Changes in liver metabolism under different forage resources are not well known. Thus, the objective of the present study was to investigate the effect of different forage resources on liver metabolism using RNAseq and bioinformatics analyses. RESULTS: The results of this study showed that the cows fed a diet with corn stover (CS) as the main forage had lower milk yield, DMI, milk protein content and yield, milk fat yield, and lactose yield than cows fed a mixed forage (MF) diet (P <  0.01). KEGG analysis for differently expressed genes (DEG) in liver (81 up-regulated and 423 down-DEG, Padj ≤0.05) showed that pathways associated with glycan biosynthesis and metabolism and amino acid metabolism was inhibited by the CS diet. In addition, results from DAVID and ClueGO indicated that biological processes related to cell-cell adhesion, multicellular organism growth, and amino acid and protein metabolism also were downregulated by feeding CS. Co-expression network analysis indicated that FAM210A, SLC26A6, FBXW5, EIF6, ZSCAN10, FPGS, and ARMCX2 played critical roles in the network. Bioinformatics analysis showed that genes within the co-expression network were enriched to "pyruvate metabolic process", "complement activation, classical pathway", and "retrograde transport, endosome to Golgi". CONCLUSIONS: Results of the present study indicated that feeding a low-quality forage diet inhibits important biological functions of the liver at least in part due to a reduction in DMI. In addition, the results of the present study provide an insight into the metabolic response in the liver to different-quality forage resources. As such, the data can help develop favorable strategies to improve the utilization of corn stover in China.

Energy, nitrogen partitioning, and methane emissions in dairy goats differ when an isoenergetic and isoproteic diet contained orange leaves and rice straw crop residues.

The aim of this study was to evaluate the effects of incorporating rice straw and orange leaves into the diets for goats. Ten Murciano-Granadina goats at mid lactation weighing 45 ± 0.3 kg were used in a crossover design. Two isoproteic and isoenergetic diets (180 g/kg DM and 17 MJ/kg DM, respectively) with alfalfa hay as forage source (33% of DM) were fed. A control diet (CON) incorporated barley as energy source and soy hulls as fiber component. The experimental diet (ORG) replaced barley and soy hulls with orange leaves (19% on DM basis), rice straw (12%, on DM basis) and soya oil (2%). Peas and horsebeans were the protein source in both diets. Each goat received the 2 treatments in 2 periods. Goats were fed the experimental diets and after 14 d on their respective treatments moved to individual metabolism cages for another 7 d. Subsequently, feed intake, total fecal and urine output and milk yield were recorded daily over the first 5 d. During the next 2 d ruminal fluid and blood samples were collected, and then individual gas-exchange measurements were recorded by a mobile open-circuit indirect calorimetry system using a head box. No differences in dry matter intake were detected, and apparent total-tract digestibility was greater in CON than ORG. Efficiency of metabolizable energy intake for milk and maintenance also was lower in response to ORG (0.65 vs. 0.63), with energy balance being negative (-12 kJ/kg of BW0.75) due to mobilization of fat (-16 g/animal vs. 68 g/animal for ORG and CON, respectively). Although actual milk yield was lower in goats fed ORG (2.32 vs. 2.06 kg/d, respectively), energy-corrected milk did not differ (2.81 kg/d on average). In terms of milk quality, milk fat content, and concentrations of monounsaturated (18.54 vs. 11.55 g/100 g milk fat) and polyunsaturated fatty acids (5.75 vs. 3.99 g/100 g milk fat) were greater in goats fed ORG. Based on various indices, the milk produced by ORG would be less atherogenic and thrombogenic than CON milk. Compared with CON, enteric CH4 emission was lower due to feeding ORG (reduction of 38 g CH4/kg milk fat). Data suggest that greater fat mobilization in goats fed ORG might have been due to the apparent lack of synchrony between degradable protein and carbohydrate and the lipogenic nutrients associated with the lower cereal content of the ORG diet. Thus, goats fed ORG seemed to rely more on fat depots to help meet energy requirements and reach optimal performance. As such, the lower content of glucogenic nutrients in ORG did not favor body fat deposition and partitioning of ME into body tissue. Overall, responses in terms of CH4 emissions and milk quality suggest that inclusion of rice straw and orange leaves in diets for small ruminants could be a valuable alternative to reuse, recycle and revalue agricultural by-products.

Alterations in immune and antioxidant gene networks by gamma-d-glutamyl-meso-diaminopimelic acid in bovine mammary epithelial cells are attenuated by in vitro supply of methionine and arginine.

Nucleotide-binding oligomerization domain (NOD)-like receptor 1 (NOD1) is a cytosolic pattern recognition receptor with a crucial role in the innate immune response of cells triggered by the presence of compounds such as gamma-d-glutamyl-meso-diaminopimelic acid (iE-DAP) present in the peptidoglycan of all gram-negative and certain gram-positive bacteria. Methionine (Met) and arginine (Arg) are functional AA with immunomodulatory properties. In the present study, we aimed to assess the effect of increased Met and Arg supply on mRNA abundance of genes associated with innate immune response, antioxidant function, and AA metabolism during iE-DAP challenge in bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 per treatment) were precultured in modified medium for 12 h with the following AA formulations: ideal profile of AA (control), increased Met supply (incMet), increased Arg supply (incArg), or increased supply of Met plus Arg (incMetArg). Subsequently, cells were challenged with or without iE-DAP (10 µg/mL) for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4. Greater mRNA abundance of NOD1, the antioxidant enzyme SOD1, and AA transporters (SLC7A1 and SLC3A2) was observed in the incMet cells after iE-DAP stimulation. Although increased Met alone had no effect, incMetArg led to greater abundance of the inflammatory cytokine IL-6, and the antioxidant enzyme GPX1 after iE-DAP stimulation. The increased Arg alone downregulated NOD1 after iE-DAP stimulation, coupled with a downregulation in the AA transporters mRNA abundance (SLC7A1, SLC7A5, SLC3A2, and SLC38A9), and upregulation in GSS and KEAP1 mRNA abundance. Overall, the data indicated that increased supply of both Met and Arg in the culture medium were more effective in modulating the innate immune response and antioxidant capacity of BMEC during in vitro iE-DAP stimulation.

One-carbon, carnitine, and glutathione metabolism-related biomarkers in peripartal Holstein cows are altered by prepartal body condition.

We investigated how prepartal body condition score (BCS) alters key hepatic enzymes associated with 1-carbon, carnitine, and glutathione metabolism and the related biomarkers in liver tissue and plasma of periparturient dairy cows. Twenty-six multiparous Holstein dairy cows were retrospectively selected according to BCS at 4 wk prepartum and divided into high (HighBCS, BCS ≥ 3.50) and normal (NormBCS, BCS ≤ 3.25) BCS groups (n = 13 each). Blood plasma samples were obtained at -30, -10, 7, 15, and 30 d relative to calving. Liver tissue biopsies were performed at -15, 7, and 30 d relative to calving, and samples were used to assess protein abundance via Western blot assay. Cows in the HighBCS group lost ∼1 unit of BCS between -4 and 4 wk around calving, while NormBCS cows lost ∼0.5 unit in the same period. Prepartal dry matter intake (DMI, kg/d) did not differ between groups. Compared with NormBCS cows, HighBCS cows had higher postpartal DMI and milk yield (+5.34 kg/d). In addition, greater overall plasma concentrations of fatty acids and activity of the neutrophil-enriched enzyme myeloperoxidase were observed in HighBCS compared with NormBCS cows. Despite similar reactive oxygen metabolite concentrations in both groups at 30 d, HighBCS cows had lower overall concentrations of ß-carotene and tocopherol, explaining the lower (BCS × Time) antioxidant capacity (ferric reducing ability of plasma). The HighBCS cows also had greater liver malondialdehyde concentrations and superoxide dismutase activity at 30 d. Overall, compared with NormBCS cows, HighBCS cows had lower hepatic protein abundance of the 1-carbon metabolism enzymes cystathionine-ß-synthase, betaine-homocysteine methyltransferase, and methionine adenosyltransferase 1 A (MAT1A), as well as the glutathione metabolism-related enzymes glutathione S-transferase α 4 and glutathione peroxidase 3 (GPX3). A lower protein abundance of glutathione S-transferase mu 1 (GSTM1) at -15 and 7 d was also observed. Regardless of BCS, cows had increased abundance of GSTM1 and GPX3 between -15 and 7 d around calving. A marked decrease of gamma-butyrobetaine dioxygenase 1 from -10 to 7 d in HighBCS compared with NormBCS cows suggested a decrease in de novo carnitine synthesis that was partly explained by the lower abundance of MAT1A. Overall, data suggest biologic links between BCS before calving, milk yield, immune response, and hepatic reactions encompassing 1-carbon metabolism, carnitine, and antioxidant synthesis.

Maternal supplementation with cobalt sources, folic acid, and rumen-protected methionine and its effects on molecular and functional correlates of the immune system in neonatal Holstein calves.

Calves born to multiparous Holstein cows fed during the last 30 d of pregnancy 2 different cobalt sources [cobalt glucoheptonate (CoPro) or cobalt pectin (CoPectin)], folic acid (FOA), and rumen-protected methionine (RPM) were used to study neonatal immune responses after ex vivo lipopolysaccharide (LPS) challenge. Groups were (n = 12 calves/group) CoPro, FOA+CoPro, FOA+CoPectin, and FOA+CoPectin+RPM. Calves were weighed at birth and blood collected at birth (before colostrum), 21 d of age, and 42 d of age (at weaning). Growth performance was recorded once a week during the first 6 wk of age. Energy metabolism, inflammation, and antioxidant status were assessed at birth through various plasma biomarkers. Whole blood was challenged with 3 µg/mL of LPS or used for phagocytosis and oxidative burst assays. Target genes evaluated by real-time quantitative PCR in whole blood samples were associated with immune response, antioxidant function, and 1-carbon metabolism. The response in mRNA abundance in LPS challenged versus nonchallenged samples was assessed via Δ = LPS challenged - LPS nonchallenged samples. Phagocytosis capacity and oxidative burst activity were measured in neutrophils and monocytes, with data reported as ratio (percentage) of CD14 to CH138A-positive cells. Data including all time points were subjected to ANOVA using PROC MIXED in SAS 9.4 (SAS Institute Inc.), with Treatment, Sex, Age, and Treatment × Age as fixed effects. A 1-way ANOVA was used to determine differences at birth, with Treatment and Sex as fixed effects. Calf birth body weight and other growth parameters did not differ between groups. At birth, plasma haptoglobin concentration was lower in FOA+CoPro compared with CoPro calves. We detected no effect for other plasma biomarkers or immune function due to maternal treatments at birth. Compared with CoPro, in response to LPS challenge, whole blood from FOA+CoPectin and FOA+CoPectin+RPM calves had greater mRNA abundance of intercellular adhesion molecule 1 (ICAM1). No effect for other genes was detectable. Regardless of maternal treatments, sex-specific responses were observed due to greater plasma concentrations of haptoglobin, paraoxonase, total reactive oxygen metabolites, nitrite, and ß-carotene in female versus male calves at birth. In contrast, whole blood from male calves had greater mRNA abundance of IRAK1, CADM1, and ITGAM in response to LPS challenge at birth. The longitudinal analysis of d 0, 21, and 42 data revealed greater bactericidal permeability-increasing protein (BPI) mRNA abundance in whole blood from FOA+CoPectin versus FOA+CoPro calves, coupled with greater abundance in FOA+CoPro compared with CoPro calves. Regardless of maternal treatments, most genes related to cytokines and cytokine receptors (IL1B, IL10, TNF, IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (ICAM1, ITGAM), antimicrobial function (MPO), and antioxidant function (GPX1) were downregulated over time. Phagocytosis capacity and oxidative burst activity in both neutrophils and monocytes did not differ due to maternal treatment. Regardless of maternal treatments, we observed an increase in the percentage of neutrophils capable of phagocytosis and oxidative burst activity over time. Overall, these preliminary assessments suggested that maternal supplementation with FOA and Co combined with RPM had effects on a few plasma biomarkers of inflammation at birth and molecular responses associated with inflammatory mechanisms during the neonatal period.

Phosphatase and tensin homolog (PTEN) suppresses triacylglycerol accumulation and monounsaturated fatty acid synthesis in goat mammary epithelial cells.

Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor in nonruminants and regulates various cellular processes including growth through dephosphorylation of phosphoinositide substrates. Although studies with bovine mammary tissue suggested a role for PTEN during lactation, its potential role in lipid metabolism remains unknown. Objectives of the present study were to determine PTEN abundance in goat mammary tissue at 2 stages of lactation (n = 6 Xinong Saanen dairy goats per stage), and to use gene-silencing and adenoviral transfections in vitro with isolated goat mammary epithelial cells (GMEC) to evaluate the role of PTEN abundance of lipid metabolism-related genes. Abundance of PTEN decreased by 51.5% at peak lactation compared with the dry period. The PTEN was overexpressed in isolated GMEC through adenoviral transfection using an adenovirus system with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and silenced via targeted small interfering RNA (siRNA) transfection with a scrambled small interfering RNA as a negative control. Cell culture was performed for 48 h before RNA extraction, triacylglycerol (TAG) analysis, and fatty acid analysis. Overexpression of PTEN downregulated abundance of acetyl-coenzyme A carboxylase α (ACACA), fatty acid synthase (FASN), sterol regulatory element binding transcription factor1 (SREBF1), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acytransferase 1 (DGAT1), 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) coupled with an increase in patatin-like-phospholipase domain containing 2 (PNPLA2), hormone-sensitive lipase (LIPE), and carnitine palmitoyltransferase 1 ß (CPT1B). Furthermore, overexpressing PTEN in vitro resulted in a significant decrease in TAG concentration and concentration of C16:1. In contrast, interference of PTEN led to an opposite effect on lipid metabolism in GMEC. These changes suggested a shift from lipogenesis and esterification to lipolysis and fatty acid oxidation. Collectively, PTEN seems to play a role in monounsaturated fatty acids synthesis and lipid accumulation in GMEC.

Maternal body condition influences neonatal calf whole-blood innate immune molecular responses to ex vivo lipopolysaccharide challenge.

Managing body condition in dairy cows during the close-up period could alter the availability of nutrients to the fetus during the final growth stages in utero. We investigated how maternal body condition score (BCS) in late pregnancy affected calf whole-blood mRNA abundance and IL-1ß concentrations after ex vivo lipopolysaccharide (LPS) challenge. Thirty-eight multiparous Holstein cows and their calves from a larger cohort were retrospectively grouped by prepartal BCS as normal BCS (≤3.25; n = 22; NormBCS) and high BCS (≥3.75; n = 16; HighBCS). Calf blood samples collected at birth (before receiving colostrum, d 0) and at ages 21 and 42 d (at weaning) were used for ex vivo whole-blood challenge with 3 µg/mL of LPS before mRNA isolation. Target genes evaluated by real-time quantitative PCR were associated with immune response, antioxidant function, and 1-carbon metabolism. Plasma IL-1ß concentrations were also measured. Responses in plasma IL-1ß and mRNA abundance were compared between LPS-challenged and nonchallenged samples. Statistical analyses were performed at all time points using a MIXED model in SAS 9.4. Neither birth body weight (NormBCS = 43.8 ± 1.01 kg; HighBCS = 43.9 ± 1.2 kg) nor colostrum IgG concentration (NormBCS = 70 ± 5.4 mg/mL; HighBCS = 62 ± 6.5 mg/mL) differed between groups. At birth, whole blood from calves born to HighBCS cows had greater mRNA abundance of IL1B, NFKB1, and GSR and lower GPX1 and CBS abundance after LPS challenge. The longitudinal analysis of d 0, 21, and 42 data revealed a BCS × age effect for SOD2 and NOS2 due to lower mRNA abundance at 42 d in the HighBCS calves. Regardless of maternal BCS, mRNA abundance decreased over time for genes encoding cytokines (IL1B, IL6, IL10, TNF), cytokine receptors (IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (CADM1, ICAM1, ITGAM), and antimicrobial function (MPO). Concentration of IL-1ß after LPS challenge was also markedly lower at 21 d regardless of maternal BCS. Overall, results suggested that maternal BCS in late prepartum influences the calf immune system response to an inflammation challenge after birth. Although few genes among those studied were altered due to maternal BCS, the fact that genes related to oxidative stress and 1-carbon metabolism responded to LPS challenge in HighBCS calves underscores the potential role of methyl donors (e.g., methionine, choline, and folic acid) in the early-life innate immune response.

Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance.

Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chol)-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 ± 5 d postpartum, 34.63 ± 2.73 kg/d of milk production; mean ± SD). The PMN were incubated for 2 h at thermal-neutral (37°C; TN) or heat stress (42°C; HS) temperatures with 3 levels of Chol (0, 400, or 800 µg/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1ß, IL-6, and tumor necrosis factor-α were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42°C (HS) relative to 37°C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 µg of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 µg/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 µg of Chol/mL compared with PMN receiving 0 µg of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys:Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 µg/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.

Inclusion of lemon leaves and rice straw into compound feed and its effect on nutrient balance, milk yield, and methane emissions in dairy goats.

The objective of this experiment was to study the effects of incorporating lemon leaves and rice straw into the compound feed of diets for dairy goats. Ten Murciano-Granadina dairy goats (n = 5 per group) in mid-lactation were used in a crossover design experiment (2 treatments across 2 periods). Goats were fed a mixed ration with barley grain (control, CON) or CON plus lemon leaves [189 g/kg of dry matter (DM)] and rice straw (120 g/kg of DM) in place of barley grain (LRS). Soybean oil (19 g/kg of DM) was added to the LRS diet to make it isoenergetic (17 MJ of gross energy/kg of DM) relative to CON. After 14 d on their respective treatments, goats were allocated to individual metabolism cages for another 7 d. Subsequently, feed intake, total fecal and urine output, and milk yield were recorded daily over the first 5 d. During the last 2 d, ruminal fluid and blood samples were collected, along with individual gas exchange measurements recorded by a mobile open-circuit indirect calorimetry system using a head box. No differences in DM intake were detected, and ME intake in LRS was lower than in CON (1,095 vs. 1,180 kJ/kg of metabolic body weight). No differences were observed in milk production, but milk fat content was greater in LRS (6.4%) than in CON (5.6%). Greater concentrations of monounsaturated (14.94 vs. 11.96 g/100 g of milk fat) and polyunsaturated fatty acids (4.53 vs. 4.03 g/100 g of milk fat) were detected in the milk of goats fed LRS compared with CON. Atherogenicity (2.68 vs.1.91) and thrombogenic (4.58 vs. 2.81) indices were lower with LRS compared with CON. Enteric CH4 emission was lower in LRS (24.3 g/d) compared with CON (31.1 g/d), probably due to the greater lipid content and unsaturated fatty acid profile of lemon leaves and the soybean oil added in the LRS diet. Overall, data suggest that incorporating lemon leaves and rice straw into lactating goat diets is effective in reducing CH4 emissions while allowing improvements in milk fat production and milk thrombogenic index without affecting production performance. Thus, their inclusion in compound feeds fed to small ruminants appears warranted and would have multiple positive effects, as on efficiency of nutrient use, human health, and the environment.

Short communication: The antilipogenic effect of trans-10,cis-12 conjugated linoleic acid in bovine mammary epithelial cells is associated with proteasome activity and ATP production.

Proteasomes play a widespread role in the control of protein abundance via degrading ubiquitinated proteins. Activity of proteasomes is regulated by constitutive ATPases that respond to intracellular concentrations of ATP. Although recent data suggest a role of proteasomes in fatty acid metabolism, whether lipogenic activity in mammary cells is responsive to ATP concentrations and proteasome activity is unknown. To investigate whether proteasomes play a role in milk fat depression induced by trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA), a bovine mammary epithelial cell line was treated with t10,c12 CLA for 24 h before analysis of lipogenic protein abundance. Western blot analysis of inactive sterol response element-binding protein-1 (pSREBP1) and active (nSREBP1) fragments indicated a decrease in abundance induced by exogenous t10,c12 CLA. At 150 nM t10,c12 CLA, abundance of both pSREBP1 and nSREBP1 was lowest, and decreased from basal levels by 16 and 64%, respectively. Exogenous t10,c12 CLA had no effect on abundance of peroxisome proliferator-activated receptor-gamma (PPARγ), but at 150 and 300 nM it decreased abundance of SREBF chaperone (SCAP). Inhibition of proteasome activity via incubation with MG-132 (a proteasome inhibitor) alone had no effect on pSREBP1, nSREBP1, PPARγ, or SCAP abundance. However, when cells were pre-incubated with MG-132, treatment with t10,c12 CLA reduced pSREBP1 (∼27%) and nSREBP1 (∼41%) abundance without affecting PPARγ or SCAP. Compared with the control, exogenous t10,c12 CLA increased ATP concentrations, and MG-132 alone had no effect. However, ATP concentration decreased markedly in cells incubated with both MG-132 and t10,c12 CLA. Combined with the alteration of SCAP and nSREBP1, the increase of ATP concentrations with t10,c12 CLA suggested that this fatty acid influenced the function of the SREBP1-SCAP complex through altering proteasome activity. Collectively, the current data highlight a role of proteasomes and intracellular ATP concentrations in the antilipogenic effect induced by t10,c12 CLA that leads to milk fat depression.

Short communication: A decrease in diameter of milk fat globules accompanies milk fat depression induced by conjugated linoleic acid supplementation in lactating dairy cows.

Milk fat is secreted from the mammary gland in the form of milk fat globules (MFG). Although milk fat depression has been studied since the beginning of the last century, the extent to which this phenomenon alters MFG synthesis is not fully understood. The aim of this study was to evaluate the effect of conjugated linoleic acid (CLA) on the size and distribution of MFG during milk fat depression in dairy cows. Twelve Holstein cows in mid lactation (145 ± 31 d in milk, 583 ± 34.6 kg of body weight, and 27.2 ± 2.4 kg of milk/d) were randomly assigned to a control diet or control plus Ca-protected CLA at 15 g/kg of dry matter for a 6-d period. The average diameter and particle size distribution of MFG were measured using a Mastersizer 3000 laser particle size analyzer (Malvern Instruments Ltd., Malvern, UK). Feeding CLA did not affect dry matter intake (16.2 ± 0.4 kg/d), milk production (28.4 ± 0.4 kg/d), milk protein, or lactose, but it decreased milk fat content (3.46 vs. 2.52%). In addition, surface area-related mean diameter of fat globules in cows fed CLA was lower compared with controls (3.02 vs. 3.45 µm). The percentage of large fat globules decreased and that of small fat globules increased in response to CLA. Overall, the data suggest that the milk fat depression induced by CLA is accompanied by a decrease in average diameter of MFG.

Short communication: Inflammation, migration, and cell-cell interaction-related gene network expression in leukocytes is enhanced in Simmental compared with Holstein dairy cows after calving.

The aim of this study was to investigate changes in the abundance of genes involved in leukocyte function between cows highly specialized for milk production (Holstein, n = 12) and cows selected for meat and milk (Simmental, n = 13). Blood was collected on d 3 after calving in PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland) to measure mRNA abundance of 33 genes. Normalized gene abundance data were subjected to MIXED model ANOVA using SAS (SAS Institute Inc. Cary, NC). Simmental cows had greater transcript abundance of proinflammatory cytokines and receptor genes (IL1B, TNF, IL1R, TNFRSF1A), cell migration- and adhesion-related genes (CX3CR1, ITGB2, CD44, LGALS8), and the antimicrobial IDO1 gene. In contrast, compared with Holstein cows, Simmental cows had lower abundance of the toll-like receptor (TLR) recognition-related gene TLR2, the antimicrobial-related gene LTF, and S100A8, which is involved in cell maturation, regulation of inflammatory processes, and immune response. These results revealed that breed plays an important role in the modulation of genes involved in immune adaptation and inflammatory response, and the immune system of Simmental cows could potentially have a more acute response in early lactation. In turn, this might be beneficial for mounting a more efficient response after calving and allow for a smoother homeorhetic adaptation to lactation.

Hepatic one-carbon metabolism enzyme activities and intermediate metabolites are altered by prepartum body condition score and plane of nutrition in grazing Holstein dairy cows.

Precalving feeding level and body condition score (BCS) alter postcalving energy balance and oxidant status of dairy cows. We hypothesized that the reported benefits of a controlled restriction precalving depend on precalving BCS. The objective was to identify alterations in activity and intermediates of the hepatic one-carbon metabolism, transsulfuration, and tricarboxylic acid pathways. Twenty-eight pregnant and nonlactating grazing dairy cows of mixed age and breed (Friesian, Friesian × Jersey) were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial design: 2 prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); 10-point scale], by managing cows in late lactation to achieve the 2 groups at dry-off, and 2 levels of energy intake during the 3 wk preceding calving (75 or 125% of estimated requirements), obtained via allowance (m2/cow) of fresh pasture composed of mostly perennial ryegrass and white cover. Average (± standard deviation) age was 6 ± 2, 6 ± 3, 5 ± 1, and 7 ± 3 yr for BCS4 fed 75 and 125%, and BCS5 fed 75 and 125%, respectively. Breed distribution (average ± standard deviation) for the 4 groups was 79 ± 21, 92 ± 11, 87 ± 31, and 74 ± 23% Friesian, and 17 ± 20, 8 ± 11, 13 ± 31, and 25 ± 23% Jersey. Liver tissue was collected by biopsy at -7, 7, and 28 d relative to calving. Tissue was used for 14C radio-labeling assays to measure betaine-homocysteine S-methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine-ß-synthase (CBS) activity. Liver metabolomics was undertaken using a targeted liquid chromatography with tandem mass spectrometry-based profiling approach. After initial liquid chromatography separation, mass spectra were acquired under both positive and negative ionization, whereas multiple reaction monitoring was used to measure target compound signal response (peak area count). Enzyme activity and metabolite peak area count were normalized with the homogenate protein concentration. Repeated measures analysis of variance via PROC MIXED in SAS (SAS Institute Inc., Cary, NC), with BCS, feeding, and time as fixed effects, and cow as random effect was used. All enzyme activities were affected by time, with betaine-homocysteine S-methyltransferase activity peaking at 7 d, whereas CBS and MTR activity decreased postpartum. Overall, thin cows had greater MTR activity, whereas cows fed 125% requirements had greater CBS activity. An interaction was detected between BCS and feeding for CBS activity, as thin cows fed 125% of requirements had greater overall activity. Compared with liver from BCS4 cows, BCS5 cows had overall greater betaine, glycine, butyrobetaine/acetylcholine, serine, and taurine concentrations. The same metabolites, plus choline and N-N-dimethylglycine, were overall greater in liver of cows fed 75% compared with those fed 125% of requirements. An interaction of BCS and feeding level was detected for the aforementioned metabolites plus methionine, cystathionine, cysteinesulfinate, and hypotaurine, due to greater overall concentrations in BCS5 cows fed 75% of requirements compared with other groups. Overall, differences in hepatic enzyme activity and intermediate metabolites suggest that both BCS and feeding level can alter the internal antioxidant system (e.g., glutathione and taurine) throughout the periparturient period. Further studies are needed to better understand potential mechanisms involved.