The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells.

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2's high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2's role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.

Impact of a Saccharomyces cerevisiae fermentation product during an intestinal barrier challenge in lactating Holstein cows on ileal microbiota and markers of tissue structure and immunity.

Feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during periods of metabolic stress is beneficial to the health of dairy cows partially through its effect on the gut microbiota. Whether SCFP alters the ileal microbiota in lactating cows during intestinal challenges induced by feed restriction (FR) is not known. We used 16S rRNA sequencing to assess if feeding SCFP during FR to induce gut barrier dysfunction alters microbiota profiles in the ileum. The mRNA abundance of key genes associated with tissue structures and immunity was also detected. Multiparous cows (97.1 ±â€…7.6 days in milk (DIM); n = 7 per treatment) fed a control diet or the control plus 19 g/d NutriTek for 9 wk were subjected to an FR challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR. DNA extracted from ileal digesta was subjected to PacBio Full-Length 16S rRNA gene sequencing. High-quality amplicon sequence analyses were performed with Targeted Amplicon Diversity Analysis and MicrobiomeAnalyst. Functional analysis was performed and analyzed using PICRUSt and STAMP. Feeding SCFP did not (P > 0.05) alter dry matter intake, milk yield, or milk components during FR. In addition, SCFP supplementation tended (P = 0.07) to increase the relative abundance of Proteobacteria and Bifidobacterium animalis. Compared with controls, feeding SCFP increased the relative abundance of Lactobacillales (P = 0.03). Gluconokinase, oligosaccharide reducing-end xylanase, and 3-hydroxy acid dehydrogenase were among the enzymes overrepresented (P < 0.05) in response to feeding SCFP. Cows fed SCFP had a lower representation of adenosylcobalamin biosynthesis I (early cobalt insertion) and pyrimidine deoxyribonucleotides de novo biosynthesis III (P < 0.05). Subsets of the Firmicutes genus, Bacteroidota phylum, and Treponema genus were correlated with the mRNA abundance of genes associated with ileal integrity (GCNT3, GALNT5, B3GNT3, FN1, ITGA2, LAMB2) and inflammation (AOX1, GPX8, CXCL12, CXCL14, CCL4, SAA3). Our data indicated that the moderate FR induced dysfunction of the ileal microbiome, but feeding SCFP increased the abundance of some beneficial gut probiotic bacteria and other species related to tissue structures and immunity.

Stressors, including limited access to feed, heat stress, transportation, and disease are factors that reduce integrity of the gut epithelial barrier in livestock. Feeding Saccharomyces cerevisiae fermentation products (SCFP) mitigated immunological, aflatoxin, and subclinical mastitis challenges, heat stress, and grain-based subacute ruminal acidosis indicating it also could alleviate gut damage. Microbiota profiling of ileal epithelium using 16S rRNA sequencing and bioinformatics revealed that Lactobacillales and Animalis abundance was greater in cows fed SCFP versus controls during a 5-d feed restriction to induce intestinal dysfunction. Some genera of Firmicutes, Bacteroidota phylum, and Treponema genus were correlated with mRNA abundance of genes associated with integrity and inflammation of ileal epithelium. Thus, feeding SCFP can increase the abundance of beneficial bacteria during a gut challenge.

Alterations in ileal transcriptomics during an intestinal barrier challenge in lactating Holstein cows fed a Saccharomyces cerevisiae fermentation product identify potential regulatory processes.

Stressors such as lack of access to feed, hot temperatures, transportation, and pen changes can cause impairment of ruminal and intestinal barrier function, also known as "leaky gut". Despite the known benefits of some nutritional approaches during periods of stress, little is understood regarding the underlying mechanisms, especially in dairy cows. We evaluated the effect of feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) on the ileal transcriptome in response to feed restriction (FR), an established model to induce intestinal barrier dysfunction. Multiparous cows [97.1 ±â€…7.6 days in milk (DIM); n = 5/group] fed a control diet or control plus 19 g/d SCFP for 9 wk were subjected to an FR challenge for 5 d during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR, and ileal scrapping RNA was used for RNAseq (NovaSeq 6000, 100 bp read length). Statistical analysis was performed in R and bioinformatics using the KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO databases. One thousand six hundred and ninety-six differentially expressed genes (DEG; FDR-adjusted P ≤ 0.10) were detected in SCFP vs. control, with 451 upregulated and 1,245 downregulated. "Mucin type O-glycan biosynthesis" was the top downregulated KEGG pathway due to downregulation of genes catalyzing glycosylation of mucins (GCNT3, GALNT5, B3GNT3, GALNT18, and GALNT14). An overall downregulation of cell and tissue structure genes (e.g., extracellular matrix proteins) associated with collagen (COL6A1, COL1A1, COL4A1, COL1A2, and COL6A2), laminin (LAMB2), and integrins (ITGA8, ITGA2, and ITGA5) also were detected with SCFP. A subset of DEG enriched in the GO term "extracellular exosome" and "extracellular space". Chemokines within "Cytokine-cytokine receptor interaction pathways" such as CCL16, CCL21, CCL14, CXCL12, and CXCL14 were downregulated by SCFP. The "Glutathione metabolism" pathway was upregulated by SCFP, including GSTA1 and RRM2B among the top upregulated genes, and GSTM1 and GPX8 as top downregulated genes. There were 9 homeobox transcription factors among the top 50 predicted transcription factors using the RNAseq DEG dataset, underscoring the importance of cell differentiation as a potential target of dietary SCFP. Taken together, SCFP downregulated immune-, ECM-, and mucin synthesis-related genes during FR. Homeobox transcription factors appear important for the transcriptional response of SCFP.

Stressors such as lack of access to feed, hot temperatures, transportation, and disease contribute to diminished gut epithelial barrier integrity in livestock. RNA-sequencing technology and bioinformatics were used to evaluate genome-wide mRNA abundance profiles in ileal tissue from dairy cows fed Saccharomyces cerevisiae fermentation product (SCFP) or an unsupplemented control diet during an intestinal challenge induced by feed restriction. Molecular responses were characterized according to metabolic pathways and other biological categories. Genes associated with "Mucin type O-glycan biosynthesis" and "Extracellular matrix-receptor interaction" were downregulated due to SCFP relative to controls. Alterations in cytokine and chemokine mRNA profiles induced by SCFP underscored differences in tissue immune response. Overall, SCFP altered the transcriptome of ileal tissue damaged by feed restriction.

Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide.

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to an inflammatory challenge using lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were two periods with two phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) cows were exposed to HS using electric heat blankets, whereas group 2 cows (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), whereas dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 µg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1, and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). Although LPS did not alter mRNA abundance of the antioxidant transcription factor NFE2L2 (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation in mammary tissue. Additionally, although HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

Heat stress (HS) is an environmental issue worldwide and occurs when animals experience a heat load that exceeds their thermoregulatory capacity. Milk protein synthesis and overall production often decrease when cows are exposed to HS conditions, in part due to lower feed intake and a limit in the mammary supply of amino acids. Increasing post-ruminal supply of methionine to late-lactation cows upregulated abundance of p-mTOR in mammary tissue, providing a link with the greater milk protein production. Exposure of cows to a HS challenge also increased abundance of p-mTOR, but did not alter milk protein suggesting this response might have been associated with synthesis of other proteins. Further work at a translational level is needed to understand potential mechanisms whereby methionine may modulate mammary metabolism during periods of HS.

Dietary Fiber as a Counterbalance to Age-Related Microglial Cell Dysfunction.

With increasing age, microglia shift toward a pro-inflammatory phenotype that may predispose individuals to neurodegenerative disease. Because fiber fermentation in the colon produces bioactive short-chain fatty acids (SCFAs; e.g., acetate, butyrate, and propionate) that signal through the gut-brain axis, increasing dietary fiber may prevent or reverse age-related dysregulation of microglia. Adult (3-4 months old) and aged (23-24 months old) male and female mice were given ad libitum access to a modified AIN-93M diet with 1% cellulose or the same diet with 2.5 or 5.0% inulin for 8 weeks. Several adult and aged male mice fed 0 or 5% inulin were randomly selected for whole brain single-cell RNA sequencing (scRNA-seq) and differential gene expression analysis to classify brain microglia according to gene expression profile; and identify additional genetic markers of aging as possible targets for dietary interventions. Microglia were isolated from remaining mice and expression of selected aging-, inflammatory-, and sensome-related genes was assessed by Fluidigm as was the ex vivo secretion of tumor necrosis factor-alpha (TNF-α). SCFAs were measured in samples collected from the cecum. Microglia from adult and aged mice segregated into distinct phenotypes according to their gene expression profile. In aged mice, a considerably greater proportion of the population of microglia was identified being "activated" and a considerably smaller proportion was identified being "quiescent." These findings using whole brain scRNA-seq were largely corroborated using highly purified microglia and Fluidigm analysis to assess a selected panel of genes. Aged mice compared to adults had lower levels of SCFA's in cecum. Dietary inulin increased SCFAs in cecum and mostly restored microglial cell gene expression and TNF-α secretion to that seen in adults. Sex differences were observed with females having lower levels of SCFAs in cecum and increased neuroinflammation. Overall, these data support the use of fiber supplementation as a strategy to counterbalance the age-related microglial dysregulation.

Treatment With the CSF1R Antagonist GW2580, Sensitizes Microglia to Reactive Oxygen Species.

Microglia activation and proliferation are hallmarks of many neurodegenerative disorders and may contribute to disease pathogenesis. Neurons actively regulate microglia survival and function, in part by secreting the microglia mitogen interleukin (IL)-34. Both IL-34 and colony stimulating factor (CSF)-1 bind colony stimulating factor receptor (CSFR)1 expressed on microglia. Systemic treatment with central nervous system (CNS) penetrant, CSFR1 antagonists, results in microglia death in a dose dependent matter, while others, such as GW2580, suppress activation during disease states without altering viability. However, it is not known how treatment with non-penetrant CSF1R antagonists, such as GW2580, affect the normal physiology of microglia. To determine how GW2580 affects microglia function, C57BL/6J mice were orally gavaged with vehicle or GW2580 (80mg/kg/d) for 8 days. Body weights and burrowing behavior were measured throughout the experiment. The effects of GW2580 on circulating leukocyte populations, brain microglia morphology, and the transcriptome of magnetically isolated adult brain microglia were determined. Body weights, burrowing behavior, and circulating leukocytes were not affected by treatment. Analysis of Iba-1 stained brain microglia indicated that GW2580 treatment altered morphology, but not cell number. Analysis of RNA-sequencing data indicated that genes related to reactive oxygen species (ROS) regulation and survival were suppressed by treatment. Treatment of primary microglia cultures with GW2580 resulted in a dose-dependent reduction in viability only when the cells were concurrently treated with LPS, an inducer of ROS. Pre-treatment with the ROS inhibitor, YCG063, blocked treatment induced reductions in viability. Finally, GW2580 sensitized microglia to hydrogen peroxide induced cell death. Together, these data suggest that partial CSF1R antagonism may render microglia more susceptible to reactive oxygen and nitrogen species.

Copper-Binding Peptides Attenuate Microglia Inflammation through Suppression of NF-kB Pathway.

SCOPE: Activation of microglia, the resident immune cells of the central nervous system, has been related to the etiology and progression of neurodegenerative diseases; thus, finding novel approaches to suppress the neuroinflammatory process is of utmost relevance. METHODS AND RESULTS: The anti-inflammatory activity of whey Cu-, Fe-, and Zn-binding peptides and their possible underlying mechanism of action were evaluated in microglia. Whey metal-binding peptides decreased nitric oxide production and tumor necrosis factor α (TNF-α) at mRNA and protein levels by stimulated BV-2 microglia in comparison to the control with no peptide treatment. The hydrophobicity, specific sequences, and possible synergistic effects seem to play a role. Cu-binding peptides (Cu-bp) presented anti-inflammatory activity both in BV-2 and primary microglia cultures. These peptides exert their action by suppressing nuclear factor kappa B (NF-kB) pathway since nuclear translocation of NF-kB p65 is decreased by roughly 30% upon Cu-bp treatment. Specific sequences identified in Cu-bp showed high affinity to bind NF-kB p65 by molecular docking (up to -8.8 kcal mol-1 ), corroborating the immunofluorescence studies. CONCLUSION: Cu-bp represent food-derived peptides that may be useful for neuroprotective purposes. Chelation of copper excess in the CNS and the bioavailability of such peptides, as well as their behavior in in vivo models, deserve further research for future applications.

Multifaceted role of one-carbon metabolism on immunometabolic control and growth during pregnancy, lactation and the neonatal period in dairy cattle.

Dairy cattle undergo dramatic metabolic, endocrine, physiologic and immune changes during the peripartal period largely due to combined increases in energy requirements for fetal growth and development, milk production, and decreased dry matter intake. The negative nutrient balance that develops results in body fat mobilization, subsequently leading to triacylglycerol (TAG) accumulation in the liver along with reductions in liver function, immune dysfunction and a state of inflammation and oxidative stress. Mobilization of muscle and gluconeogenesis are also enhanced, while intake of vitamins and minerals is decreased, contributing to metabolic and immune dysfunction and oxidative stress. Enhancing post-ruminal supply of methyl donors is one approach that may improve immunometabolism and production synergistically in peripartal cows. At the cellular level, methyl donors (e.g. methionine, choline, betaine and folic acid) interact through one-carbon metabolism to modulate metabolism, immune responses and epigenetic events. By modulating those pathways, methyl donors may help increase the export of very low-density lipoproteins to reduce liver TAG and contribute to antioxidant synthesis to alleviate oxidative stress. Thus, altering one-carbon metabolism through methyl donor supplementation is a viable option to modulate immunometabolism during the peripartal period. This review explores available data on the regulation of one-carbon metabolism pathways in dairy cows in the context of enzyme regulation, cellular sensors and signaling mechanisms that might respond to increased dietary supply of specific methyl donors. Effects of methyl donors beyond the one-carbon metabolism pathways, including production performance, immune cell function, mechanistic target or rapamycin signaling, and fatty acid oxidation will also be highlighted. Furthermore, the effects of body condition and feeding system (total mixed ration vs. pasture) on one-carbon metabolism pathways are explored. Potential effects of methyl donor supply during the pepartum period on dairy calf growth and development also are discussed. Lastly, practical nutritional recommendations related to methyl donor metabolism during the peripartal period are presented. Nutritional management during the peripartal period is a fertile area of research, hence, underscoring the importance for developing a systems understanding of the potential immunometabolic role that dietary methyl donors play during this period to promote health and performance.

The presence of an embryo affects day 14 uterine transcriptome depending on the nutritional status in sheep. b. Immune system and uterine remodeling.

Transcriptomics and bioinformatics were used to investigate the potential interactions of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on uterine immune system and remodeling. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition for 28 days: maintenance energy intake (control; 5 cyclic, 6 pregnant ewes) providing 7.8 MJ of metabolisable energy and 0.5 maintenance intake (undernourished; 6 cyclic, 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. Uterine gene expression was measured using Agilent 15 K Sheep Microarray chip on day 14 of estrus or pregnancy. Functional bioinformatics analyses were performed using PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. Pregnancy affected the expression of 18 genes in both control and undernourished ewes, underscoring the relevance for embryo-maternal interactions. Immune system evidenced by classical interferon stimulated genes were activated in control and -in a lesser extent-in undernourished pregnant vs cyclic ewes. Genes involved in uterine remodeling such as protein metabolism were also upregulated with the presence of an embryo in control and undernourished ewes. However, relevant genes for the adaptation of the uterus to the embryo were differentially expressed between pregnant vs cyclic ewes both in control and undernourished groups. Undernutrition alone led to an overall weak activation of immune system pathways both in cyclic and pregnant ewes. Data revealed that cellular and immune adaptations of the uterus to pregnancy are dependent on the nutritional status.

The embryo affects day 14 uterine transcriptome depending on nutritional status in sheep. a. Metabolic adaptation to pregnancy in nourished and undernourished ewes.

This study investigated the effects of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on the expression of uterine indicators of metabolism in ewes. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition for 28 days: maintenance energy intake (control; 5 cyclic and 6 pregnant ewes) providing 7.8 MJ of metabolisable energy, and 0.5 maintenance intake (undernourished; 6 cyclic and 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. RNA from intercaruncular uterine tissue was harvested at slaughter on Day 14 of estrous cycle or pregnancy, and hybridized to the Agilent 15K Sheep Microarray chip. Functional bioinformatics analyses were performed using PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. The presence of the embryo upregulated expression of genes encoding peptide and monocarboxylate transporters regardless of nutritional treatment, although the degree of gene expression was lower in undernourished ewes. Genes encoding enzymes involved in glycolysis were downregulated both in pregnant control and undernourished ewes, probably as a compensatory mechanism for the increased glucose transport to the uterus. Compared with control cyclic ewes, control pregnant ewes had greater expression of genes involved in oxidation of fatty acids, suggesting increased uterine energy demands. This was not observed in undernourished pregnant animals when compared to undernourished cyclic ewes; nevertheless, those animals had lower uterine expression of enzymes involved in fatty acid biosynthesis. The presence of the embryo upregulated genes involved in electron transport probably as a result of increased energy demands for pregnancy. Overall, the data indicate that depending on the nutritional status of ewe, pregnancy alters gene expression of metabolic pathways related to energy generation in the uterus. An impairment in nutrient transport and metabolism in the uterus of pregnant undernourished ewes may explain the greater embryo mortality associated with undernutrition.

Rumen-protected methionine supplementation during the peripartal period alters the expression of galectin genes associated with inflammation in peripheral neutrophils and secretion in plasma of Holstein cows.

The work described in this research communication aimed to investigate whether rumen-protected methionine (Met) supplementation during the periparturient period would affect the expression of galectins in blood-derived neutrophils, and secretion of galectins, IL (interleukin)-1ß, IL-6, myeloperoxidase (MPO), and glucose in plasma. Because supplementation of rumen-protected Met would alleviate inflammation and oxidative stress during the peripartal period, we hypothesized that enhancing Met supply would benefit the innate immune response at least in part by altering the expression of galectin genes associated with neutrophil activity and inflammation. Galectins (Gal) have an immuno-modulating effect acting like cell-surface receptors whose activation often results in signaling cascades stimulating cells such as neutrophils. This study revealed an association between Met supplementation and galectin expression and secretion. This implies that galectin expression and secretion can be modulated by Met supplementation. Further studies are needed to evaluate the regulation of galectin gene expression for therapeutic and dietary intervention in the peripartal cow.

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.

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.

Dietary Egg Protein Prevents Hyperhomocysteinemia via Upregulation of Hepatic Betaine-Homocysteine S-Methyltransferase Activity in Folate-Restricted Rats.

BACKGROUND: Hyperhomocysteinemia is associated with increased cardiovascular disease risk. Whole eggs contain several nutrients known to affect homocysteine regulation, including sulfur amino acids, choline, and B vitamins. OBJECTIVE: The aim of this study was to determine the effect of whole eggs and egg components (i.e., egg protein and choline) with respect to 1) homocysteine balance and 2) the hepatic expression and activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine ß-synthase (CBS) in a folate-restricted (FR) rat model of hyperhomocysteinemia. METHODS: Male Sprague Dawley rats (n = 48; 6 wk of age) were randomly assigned to a casein-based diet (C; n = 12), a casein-based diet supplemented with choline (C + Cho; 1.3%, wt:wt; n = 12), an egg protein-based diet (EP; n = 12), or a whole egg-based diet (WE; n = 12). At week 2, half of the rats in each of the 4 dietary groups were provided an FR (0 g folic acid/kg) diet and half continued on the folate-sufficient (FS; 0.2 g folic acid/kg) diet for an additional 6 wk. All diets contained 20% (wt:wt) total protein. Serum homocysteine was measured by HPLC and BHMT and CBS expression and activity were evaluated using real-time quantitative polymerase chain reaction, Western blot, and enzyme activity. A 2-factor ANOVA was used for statistical comparisons. RESULTS: Rats fed FR-C exhibited a 53% increase in circulating homocysteine concentrations compared with rats fed FS-C (P < 0.001). In contrast, serum homocysteine did not differ between rats fed FS-C and FR-EP (P = 0.078). Hepatic BHMT activity was increased by 45% and 40% by the EP (P < 0.001) and WE (P = 0.002) diets compared with the C diets, respectively. CONCLUSIONS: Dietary intervention with egg protein prevented elevated circulating homocysteine concentrations in a rat model of hyperhomocysteinemia, due in part to upregulation of hepatic BHMT. These data may support the inclusion of egg protein for dietary recommendations targeting hyperhomocysteinemia prevention.

Cellular Mechanisms and Epigenetic Changes: Role of Nutrition in Livestock.

In the context of physiologic responses that determine the growth, development, and health status of livestock, the role of epigenetics and the underlying cellular mechanisms it affects remain to be fully elucidated. Although recent work has provided evidence that maternal dietary energy level, carbohydrate type, or intestinal supply of methyl donors can elicit molecular changes in tissues of the embryo, fetus, or neonate, there are few data linking epigenetics with biochemical and physiologic outcomes. Therefore, efforts linking the epigenome with physiologic and developmental outcomes offer exciting opportunities for discoveries that can impact efficiency of nutrient use and well-being of livestock.

Hepatic Cystathionine ß-Synthase Activity Is Increased by Greater Postruminal Supply of Met during the Periparturient Period in Dairy Cows.

BACKGROUND: Postruminal supply of Met during the periparturient period enhances production efficiency (feed conversion to milk) in dairy cows partly through alleviation of oxidant and inflammatory status. Whether alterations in hepatic 1-carbon metabolism (major contributor of antioxidants) and/or energy metabolism contribute to these beneficial effects is unknown. OBJECTIVES: To investigate alterations in hepatic 1-carbon and energy metabolism and associations with plasma amino acids (AAs) and production efficiency in response to enhanced postruminal supply of Met. METHODS: Holstein cows (n = 30 per group) were fed during the last 28 d of pregnancy a control diet (CON) or the control plus ethylcellulose rumen-protected Met (MET; 0.9 g/kg of dry matter intake). Plasma (n = 15 per group) and liver tissue (n = 10 per group) were collected throughout the periparturient period to evaluate AA profiles, activity of the tricarboxylic acid cycle, and 1-carbon metabolism via mRNA abundance, enzyme activity, and targeted metabolomics. RESULTS: Cows in the MET group had greater overall (27%, P = 0.027) plasma Met concentrations, but had similar total plasma AA concentrations. Although mRNA abundance of 1-carbon metabolism enzymes did not differ, hepatic activity of cystathionine ß-synthase (CBS) (51.2 compared with 44.4 mmol/h/mg protein; P = 0.032) and concentration (19%, P = 0.048) of the cellular antioxidant glutathione were greater overall in the MET group. mRNA abundance of aconitase 2 and fumarate hydratase was greater overall (P = 0.049), and phosphoenolpyruvate carboxykinase 1 tended (P = 0.093) to be greater overall in cows fed MET. There was a tendency (P ≤ 0.093) for greater overall hepatic concentrations of malic acid, α-ketoglutaric acid, and isocitric acid in cows fed MET. CONCLUSIONS: Greater activity of CBS in response to enhanced postruminal supply of Met likely contributes to alleviating oxidant status by increasing concentrations of glutathione. Hence, transsulfuration plays an important role in the observed improvements in production efficiency of dairy cows during the periparturient period.

Effects of chronic heat stress on lactational performance and the transcriptomic profile of blood cells in lactating dairy goats.

High temperature is a major stress that negatively affects welfare, health, and productivity of dairy animals. Heat-stressed animals are more prone to disease, suggesting that their immunity is hindered. Although productive and physiologic responses of dairy animals to heat stress are well known, there is still limited information on the response at the transcriptome level. Our objective was to evaluate the changes in performance and blood transcriptomics of dairy goats under heat stress. Eight multiparous Murciano-Granadina dairy goats in mid-lactation were assigned to 1 of 2 climatic treatments for 35 d. Treatments and temperature-humidity index (THI) were: (1) thermal neutral (TN: n = 4; 15-20 °C, 40-45%, THI = 59-65), and (2) heat stress (HS: n = 4; 12 h at 37 °C-40%, THI = 86; 12 h at 30 °C-40%, THI = 77). Rectal temperature, respiratory rate, feed intake and milk yield were recorded daily. Additionally, milk composition was evaluated weekly. Blood samples were collected at d 35 and RNA was extracted for microarray analyses (Affymetrix GeneChip Bovine Genome Array). Differences in rectal temperature and respiratory rate between HS and TN goats were maximal during the first 3 d of the experiment, reduced thereafter, but remained significant throughout the 35-d experimental period. Heat stress reduced feed intake, milk yield, milk protein and milk fat contents by 29, 8, 12, and 13%, respectively. Microarray analysis of blood revealed that 55 genes were up-regulated, whereas 88 were down-regulated by HS. Bioinformatics analysis using the Dynamic Impact Approach revealed that 31 biological pathways were impacted by HS. Pathways associated with leukocyte transendothelial migration, cell adhesion, hematopoietic cell lineage, calcium signaling, and PPAR signaling were negatively impacted by HS, whereas nucleotide metabolism was activated. In conclusion, heat stress not only negatively affected milk production in dairy goats, but also resulted in alterations in the functionality of immune cells, which would make the immune system of heat-stressed goats less capable of fending-off diseases.

Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows.

BACKGROUND: G-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues. RESULTS: Quantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40, MCFA receptor GPR84, and niacin receptor HCAR2/3. Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable. CONCLUSIONS: The data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

Supplemental Smartamine M in higher-energy diets during the prepartal period improves hepatic biomarkers of health and oxidative status in Holstein cows.

BACKGROUND: Feeding higher-energy prepartum is a common practice in the dairy industry. However, recent data underscore how it could reduce performance, deepen negative energy balance, and augment inflammation and oxidative stress in fresh cows. We tested the effectiveness of rumen-protected methionine in preventing the negative effect of feeding a higher-energy prepartum. Multiparous Holstein cows were fed a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), or were switched to a higher-energy (OVE, 1.54 Mcal/kg DM), or OVE plus Smartamine M (OVE + SM; Adisseo NA) during the last 21 d before calving. Afterwards cows received the same lactation diet (1.75 Mcal/kg DM). Smartamine M was top-dressed on the OVE diet (0.07% of DM) from -21 through 30 d in milk (DIM). Liver samples were obtained via percutaneous biopsy at -10, 7 and 21 DIM. Expression of genes associated with energy and lipid metabolism, hepatokines, methionine cycle, antioxidant capacity and inflammation was measured. RESULTS: Postpartal dry matter intake, milk yield, and energy-corrected milk were higher in CON and OVE + SM compared with OVE. Furthermore, milk protein and fat percentages were greater in OVE + SM compared with CON and OVE. Expression of the gluconeogenic gene PCK1 and the lipid-metabolism transcription regulator PPARA was again greater with CON and OVE + SM compared with OVE. Expression of the lipoprotein synthesis enzyme MTTP was lower in OVE + SM than CON or OVE. Similarly, the hepatokine FGF21, which correlates with severity of negative energy balance, was increased postpartum only in OVE compared to the other two groups. These results indicate greater liver metabolism and functions to support a greater production in OVE + SM. At 7 DIM, the enzyme GSR involved in the synthesis of glutathione tended to be upregulated in OVE than CON-fed cows, suggesting a greater antioxidant demand in overfed cows. Feeding OVE + SM resulted in lower similar expression of GSR compared with CON. Expression of the methionine cycle enzymes SAHH and MTR, both of which help synthesize methionine endogenously, was greater prepartum in OVE + SM compared with both CON and OVE, and at 7 DIM for CON and OVE + SM compared with OVE, suggesting greater Met availability. It is noteworthy that DNMT3A, which utilizes S-adenosylmethionine generated in the methionine cycle, was greater in OVE and OVE + SM indicating higher-energy diets might enhance DNA methylation, thus, Met utilization. CONCLUSIONS: Data indicate that supplemental Smartamine M was able to compensate for the negative effect of prepartal energy-overfeeding by alleviating the demand for intracellular antioxidants, thus, contributing to the increase in production. Moreover Smartamine M improved hepatic lipid and glucose metabolism, leading to greater liver function and better overall health.

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.

The objective of this study was to profile plasma amino acids (AA) and derivatives of their metabolism during the periparturient period in response to supplemental rumen-protected methionine (MET) or rumen-protected choline (CHOL). Forty cows were fed from -21 through 30 days around parturition in a 2 × 2 factorial design a diet containing MET or CHOL. MET supply led to greater circulating methionine and proportion of methionine in the essential AA pool, total AA, and total sulfur-containing compounds. Lysine in total AA also was greater in these cows, indicating a better overall AA profile. Sulfur-containing compounds (cystathionine, cystine, homocystine, and taurine) were greater in MET-fed cows, indicating an enriched sulfur-containing compound pool due to enhanced transsulfuration activity. Circulating essential AA and total AA concentrations were greater in cows supplied MET due to greater lysine, arginine, tryptophan, threonine, proline, asparagine, alanine, and citrulline. In contrast, CHOL supply had no effect on essential AA or total AA, and only tryptophan and cystine were greater. Plasma 3-methylhistidine concentration was lower in response to CHOL supply, suggesting less tissue protein mobilization in these cows. Overall, the data revealed that enhanced periparturient supply of MET has positive effects on plasma AA profiles and overall antioxidant status.