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.

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.

Inflammation and oxidative stress transcription profiles due to in vitro supply of methionine with or without choline in unstimulated blood polymorphonuclear leukocytes from lactating Holstein cows.

Neutrophils are the most important polymorphonuclear leukocytes (PMNL), representing the front-line defense involved in pathogen clearance upon invasion. As such, they play a pivotal role in immune and inflammatory responses. Isolated PMNL from 5 mid-lactating Holstein dairy cows were used to evaluate the in vitro effect of methionine (Met) and choline (Chol) supplementation on mRNA expression of genes related to the Met cycle and innate immunity. The target genes are associated with the Met cycle, cell signaling, inflammation, antimicrobial and killing mechanisms, and pathogen recognition. Treatments were allocated in a 3 × 3 factorial arrangement, including 3 Lys-to-Met ratios (L:M, 3.6:1, 2.9:1, or 2.4:1) and 3 levels of supplemental Chol (0, 400, or 800 µg/mL). Three replicates per treatment group were incubated for 2 h at 37°C and 5% atmospheric CO2. Both betaine-homocysteine S-methyltransferase and choline dehydrogenase were undetectable, indicating that PMNL (at least in vitro) cannot generate Met from Chol through the betaine pathway. The PMNL incubated without Chol experienced a specific state of inflammatory mediation [greater interleukin-1ß (IL1B), myeloperoxidase (MPO), IL10, and IL6] and oxidative stress [greater cysteine sulfinic acid decarboxylase (CSAD), cystathionine gamma-lyase (CTH), glutathione reductase (GSR), and glutathione synthase (GSS)]. However, data from the interaction L:M × Chol indicated that this negative state could be overcome by supplementing additional Met. This was reflected in the upregulation of methionine synthase (MTR) and toll-like receptor 2 (TLR2); that is, pathogen detection ability. At the lowest level of supplemental Chol, Met downregulated GSS, GSR, IL1B, and IL6, suggesting it could reduce cellular inflammation and enhance antioxidant status. At 400 µg/mL Chol, supplemental Met upregulated PMNL recognition capacity [higher TLR4 and L-selectin (SELL)]. Overall, enhancing the supply of methyl donors to isolated unstimulated PMNL from mid-lactating dairy cows leads to a low level of PMNL activation and upregulates a cytoprotective mechanism against oxidative stress. Enhancing the supply of Met coupled with adequate Chol levels enhances the gene expression of PMNL pathogen-recognition mechanism. These data suggest that Chol supply to PMNL exposed to low levels of Met effectively downregulated the entire repertoire of innate inflammatory-responsive genes. Thus, Met availability in PMNL during an inflammatory challenge may be sufficient for mounting an appropriate biologic response.

In vitro methionine supplementation during lipopolysaccharide stimulation modulates immunometabolic gene network expression in isolated polymorphonuclear cells from lactating Holstein cows.

Methionine (Met) is one of the 2 most limiting amino acids for milk production in dairy cow diets. The accepted "ideal" ratio of lysine (Lys) to Met (L:M) when formulating diets is 3:1. However, blood from cows fed corn silage-based diets without supplemental rumen-protected Met averages approximately 3.6:1 L:M. Recent in vivo research on cattle immunonutrition has revealed that the immune system could benefit from greater Met supply. To study more closely the effects of different L:M ratios, blood polymorphonuclear cells (PMN) were isolated from 5 Holstein cows in mid-lactation (238 ± 20 d postpartum, 33.8 ± 3.8 kg of milk/d; mean ± SD). The PMN were incubated at 3 different levels of L:M (3.6:1, 2.9:1, or 2.4:1) and stimulated with lipopolysaccharide (LPS) at either 0 or 50 µg/mL for 2 h at 37°C. Target genes were associated with cytokines, pathogen recognition, nuclear receptors, killing mechanisms, and Met and glutathione metabolism. Data were subjected to ANOVA using PROC MIXED in SAS, with L:M, LPS, and their interaction as fixed effects. Stimulation with LPS upregulated genes related to cytokines (IL1B, TNF, IL10 and IL6) and nuclear receptors, including nuclear factor kappa B (NFKB1) and glucocorticoid receptor (NR3C1), and downregulated the mRNA abundance of chemokine receptor 1 (CXCR1), lysozyme (LYZ) and glutathione reductase (GSR). A linear decrease was observed in the mRNA abundance of TNF when L:M was decreased. A similar response was observed for interleukin-1 receptor-associated kinase 1 (IRAK1) and NFKB1 abundance in cells stimulated with LPS (linear effect). A linear increase of LYZ mRNA expression as L:M decreased was detected in unstimulated cells. Furthermore, a decrease in L:M led to a linear decrease of superoxide dismutase 1 (SOD1) mRNA abundance in cells challenged with LPS. Overall, LPS challenge triggered the activation of isolated PMN from mid-lactation cows. However, data suggest the use of a shorter incubation time to capture the peak response and not the resolution of the inflammatory response as in the present study. Our results indicate a possible involvement of Met in modulating PMN inflammatory and oxidative stress status and in helping the resolution of inflammation after initial stimulation.

Transcriptional profiling of swine mammary gland during the transition from colostrogenesis to lactogenesis using RNA sequencing.

BACKGROUND: Colostrum and milk are essential sources of antibodies and nutrients for the neonate, playing a key role in their survival and growth. Slight abnormalities in the timing of colostrogenesis/lactogenesis potentially threaten piglet survival. To further delineate the genes and transcription regulators implicated in the control of the transition from colostrogenesis to lactogenesis, we applied RNA-seq analysis of swine mammary gland tissue from late-gestation to farrowing. Three 2nd parity sows were used for mammary tissue biopsies on days 14, 10, 6 and 2 before (-) parturition and on day 1 after (+) parturition. A total of 15 mRNA libraries were sequenced on a HiSeq2500 (Illumina Inc.). The Dynamic Impact Approach and the Ingenuity Pathway Analysis were used for pathway analysis and gene network analysis, respectively. RESULTS: A large number of differentially expressed genes were detected very close to parturition (-2d) and at farrowing (+ 1d). The results reflect the extraordinary metabolic changes in the swine mammary gland once it enters into the crucial phases of lactogenesis and underscore a strong transcriptional component in the control of colostrogenesis. There was marked upregulation of genes involved in synthesis of colostrum and main milk components (i.e. proteins, fat, lactose and antimicrobial factors) with a pivotal role of CSN1S2, LALBA, WAP, SAA2, and BTN1A1. The sustained activation of transcription regulators such as SREBP1 and XBP1 suggested they help coordinate these adaptations. CONCLUSIONS: Overall, the precise timing for the transition from colostrogenesis to lactogenesis in swine mammary gland remains uncharacterized. However, our transcriptomic data support the hypothesis that the transition occurs before parturition. This is likely attributable to upregulation of a wide array of genes including those involved in 'Protein and Carbohydrate Metabolism', 'Immune System', 'Lipid Metabolism', 'PPAR signaling pathway' and 'Prolactin signaling pathway' along with the activation of transcription regulators controlling lipid synthesis and endoplasmic reticulum biogenesis and stress response.

Supplementation with rumen-protected methionine or choline during the transition period influences whole-blood immune response in periparturient dairy cows.

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

Supplemental methionine, choline, or taurine alter in vitro gene network expression of polymorphonuclear leukocytes from neonatal Holstein calves.

Isolated PMNL from neonatal calves were used to evaluate the effect of Met, choline, and taurine supplementation on mRNA expression of genes related to the Met cycle and innate immunity. Five neonatal Holstein calves (3 wk old) were used for PMNL isolation and in vitro culture. The selected genes were related to the 1-carbon and Met cycles, cell signaling and cytokine mediators, inflammation, antimicrobial and killing mechanism associated genes, immune mediators, adhesion, and pathogen recognition. The results indicated that supplementation of Met, choline, and taurine increased homocysteine synthesis through upregulation of SAHH. Furthermore, the lower expression of CXCR1, IL10, IL6, IRAK1, NFKB1, NR3C1, SELL, TLR4, and TNFA indicated that all treatments mitigated the inflammatory activation of blood PMNL. As indicated by the modulation of GCLC and GPX1, choline and taurine supplementation also affected the antioxidant system. However, data indicate that oversupplementation could alter the inflammatory and oxidative status, suggesting the existence of cytotoxicity thresholds. Overall, multiple biological processes in calf PMNL related to inflammatory response and cytoprotection against oxidative stress were affected by Met, choline, and taurine supplementation. These data underscore an important role of these compounds in pre-weaning calf nutritional management.

Better postpartal performance in dairy cows supplemented with rumen-protected methionine compared with choline during the peripartal period.

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

Rumen-protected methionine compared with rumen-protected choline improves immunometabolic status in dairy cows during the peripartal period.

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

The effect of calving in the summer on the hepatic transcriptome of Holstein cows during the peripartal period.

The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum.