RESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos , Trastornos de Estrés por Calor , Animales , Antioxidantes/metabolismo , Carbono/metabolismo , Bovinos , Enfermedades de los Bovinos/metabolismo , Dieta/veterinaria , Suplementos Dietéticos , Femenino , Trastornos de Estrés por Calor/metabolismo , Trastornos de Estrés por Calor/veterinaria , Respuesta al Choque Térmico , Insulina/metabolismo , Lactancia/fisiología , Hígado/metabolismo , Metionina/metabolismo , Metionina Adenosiltransferasa , Leche/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Rumen/metabolismo , Superóxido Dismutasa-1 , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
Antioxidantes , Metionina , Tejido Adiposo , Animales , Bovinos , Dieta , Suplementos Dietéticos , Femenino , Peróxido de Hidrógeno , Insulina , LactanciaRESUMEN
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.
Asunto(s)
Metionina , Rumen , Animales , Animales Recién Nacidos , Bovinos , Cobalto , Dieta/veterinaria , Suplementos Dietéticos , Femenino , Ácido Fólico , Masculino , Neutrófilos , EmbarazoRESUMEN
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.
Asunto(s)
Carbono/metabolismo , Colina/metabolismo , Inmunidad Innata , Lactancia , Neutrófilos/inmunología , Neutrófilos/metabolismo , Animales , Antioxidantes/metabolismo , Bovinos , Enfermedades de los Bovinos/metabolismo , Dieta/veterinaria , Femenino , Redes Reguladoras de Genes , Respuesta al Choque Térmico , Inmunidad Innata/genética , Inflamación/veterinaria , Lactancia/fisiología , Recuento de Leucocitos/veterinaria , Metionina/metabolismo , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Bovinos/fisiología , Suplementos Dietéticos/análisis , Glicoproteínas/efectos de los fármacos , Ácidos Linoleicos Conjugados/farmacología , Leche/química , Animales , Peso Corporal , Dieta/veterinaria , Ingestión de Alimentos , Ácidos Grasos/metabolismo , Femenino , Glucolípidos/análisis , Glicoproteínas/análisis , Lactancia/efectos de los fármacos , Lactosa/metabolismo , Gotas Lipídicas , Leche/metabolismo , Proteínas de la Leche/metabolismo , Distribución AleatoriaRESUMEN
Enhancing the supply of arginine (Arg), a semi-essential amino acid, has positive effects on immune function in dairy cattle experiencing metabolic stress during early lactation. Our objective was to determine the effects of Arg supplementation on biomarkers of liver damage and inflammation in cows during early lactation. Six Chinese Holstein lactating cows with similar BW (508 ± 14 kg), body condition score (3.0), parity (4.0 ± 0), milk yield (30.6 ± 1.8 kg) and days in milk (20 ± days) were randomly assigned to three treatments in a replicated 3 × 3 Latin square design balanced for carryover effects. Each period was 21 days with 7 days for infusion and 14 days for washout. Treatments were (1) Control: saline; (2) Arg group: saline + 0.216 mol/day l-Arg; and (3) Alanine (Ala) group: saline + 0.868 mol/day l-Ala (iso-nitrogenous to the Arg group). Blood and milk samples from the experimental cows were collected on the last day of each infusion period and analyzed for indices of liver damage and inflammation, and the count and composition of somatic cells in milk. Compared with the Control, the infusion of Arg led to greater concentrations of total protein, immunoglobulin M and high density lipoprotein cholesterol coupled with lower concentrations of haptoglobin and tumor necrosis factor-α, and activity of aspartate aminotransferase in serum. Infusion of Ala had no effect on those biomarkers compared with the Control. Although milk somatic cell count was not affected, the concentration of granulocytes was lower in response to Arg infusion compared with the Control or Ala group. Overall, the biomarker analyses indicated that the supplementation of Arg via the jugular vein during early lactation alleviated inflammation and metabolic stress.
Asunto(s)
Arginina/administración & dosificación , Inflamación/veterinaria , Leche/metabolismo , Animales , Biomarcadores/análisis , Bovinos , Dieta/veterinaria , Femenino , Salud , Inflamación/tratamiento farmacológico , Infusiones Intravenosas , Lactancia , Hígado/metabolismo , Paridad , Embarazo , Distribución Aleatoria , Estrés FisiológicoRESUMEN
Rumen-protected betaine (RPB) can enhance betaine absorption in the small intestine of ruminants, while betaine can alter fat distribution and has the potential to affect the meat quality of livestock. Hence, we hypothesized that RPB might also affect the meat quality of lambs. Sixty male Hu sheep of similar weight (30.47 ± 2.04 kg) were selected and randomly subjected to five different treatments. The sheep were fed a control diet (control treatment, CTL); 1.1 g/day unprotected-betaine supplemented diet (UPB); or doses of 1.1 g/day (low RPB treatment; L-PB), 2.2 g/day (middle RPB treatment; M-PB) or 3.3 g/day (high RPB treatment; H-PB) RPB-supplemented diet for 70 days. Slaughter performance, meat quality, fatty acid and amino acid content in the longissimus dorsi (LD) muscle, shoulder muscle (SM) and gluteus muscle (GM) were measured. Compared with CTL, betaine (including UPB and RPB) supplementation increased the average daily weight gain (ADG) (P < 0.05) and average daily feed intake (P < 0.01) of lambs. Rumen-protected betaine increased ADG (P < 0.05) compared with UPB. With increasing RPB doses, the eye muscle area of the lambs linearly increased (P < 0.05). Compared with CTL, betaine supplementation decreased water loss (P < 0.05) in SM and increased pH24 in the SM (P < 0.05) and GM (P < 0.05). Compared with UPB, RPB decreased water loss in the GM (P < 0.01), decreased shear force (P < 0.05) in the LD and SM and increased the pH of the meat 24 h after slaughter (pH24). With increasing RPB doses, the shear force and b* value in the LD linearly decreased (P < 0.05), and the pH24 of the meat quadratically increased (P < 0.05). Compared with CTL, betaine supplementation increased the polyunsaturated fatty acid in the GM (P < 0.05). Compared with UPB, RPB supplementation decreased the saturated fatty acid (SFA) content in the LD (P < 0.05) and increased the unsaturated fatty acids (UFA), mono-unsaturated fatty acids and UFA/SFA ratio in the LD (P < 0.05). Compared with CTL, the content of histidine in the LD increased with betaine supplementation. Compared with UPB, RPB supplementation increased the content of total free amino acids and flavor amino acids in the LD of lambs (P < 0.05). With increasing RPB, the isoleucine and phenylalanine contents in the LD linearly increased (P < 0.05). Overall, the data collected indicated that the meat quality of lambs (especially in the LD) improved as a result of betaine supplementation, and RPB showed better effects than those of UPB.
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Aminoácidos/análisis , Betaína/administración & dosificación , Suplementos Dietéticos/análisis , Ácidos Grasos/análisis , Carne Roja/normas , Ovinos/fisiología , Alimentación Animal/análisis , Animales , Peso Corporal , Dieta/veterinaria , Ácidos Grasos Insaturados/análisis , Masculino , Músculo Esquelético/metabolismo , Distribución Aleatoria , Rumen/metabolismo , Aumento de PesoRESUMEN
Synthetic zeolites are used to control the availability of dietary minerals (e.g., Ca, Mg, and P) in dairy cows. Due to calcium demand increasing with lactation onset, most cows become hypocalcemic immediately postpartum, which likely contributes to poorer immune function because calcium is important for immune cell signaling. To overcome postpartum hypocalcemia, we fed transition cows synthetic zeolite A (sodium aluminosilicate) precalving and hypothesized that it would alter calcium and thus neutrophil function during the transition period. Multiparous Holstein-Friesian cows in late gestation were randomly allocated to an untreated control group (n = 10) or a treatment group in which each cow received 500 g of zeolite A daily (n = 10) for 14 d prior to the expected calving date (actual duration = 17 ± 3 d prepartum). The cows grazed pasture, and each was supplemented with 2 kg/d of maize silage (dry matter basis), with or without zeolite, until calving. Blood samples for neutrophil isolation and analysis of plasma indicators of mineral status, energy status, liver function, and inflammation were collected pretreatment (covariate; d -19); on d -14 and -7 precalving; on the day of calving (d 0); and on d 1, 4, 7, and 28 postcalving. Neutrophils were isolated and gene expression was analyzed using microfluidic gene expression arrays. Neutrophil respiratory burst was assessed using stimulation with phorbol 12-myristate 13-acetate and flow cytometry. Plasma calcium and phosphorus revealed a treatment by time interaction; cows offered zeolite had greater plasma calcium concentrations at d 0, 1, and 4 postcalving and plasma phosphorus concentrations were lower in zeolite-treated cows during the precalving period until d 1 postcalving compared with control animals. Zeolite treatment downregulated neutrophil gene expression of CXCR4 and S100A8 and tended to lower gene expression for other immune mediators (CXCR1, IFNG, S100A12, and S100A9) compared with the control. Zeolite treatment did not affect neutrophil respiratory burst or expression of the other genes investigated. Plasma concentrations of cytokine IL-6 were reduced with zeolite treatment, which was most evident immediately postcalving (d 0, 1, and 7). Overall, feeding zeolite precalving had few effects on neutrophil gene expression and function; however, the lower gene expression of neutrophil inflammatory mediators may be due to altered availability of dietary minerals prepartum and indicates that zeolite A may control inflammation during the transition period.
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Suplementos Dietéticos , Regulación de la Expresión Génica/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Zeolitas/administración & dosificación , Alimentación Animal/análisis , Animales , Bovinos , Dieta/veterinaria , Femenino , Lactancia/fisiología , Leche/metabolismo , Neutrófilos/metabolismo , Periodo Posparto , Embarazo , Ensilaje , Zeolitas/síntesis química , Zeolitas/farmacologíaRESUMEN
Mastitis, inflammation of the udder, is one of the most common diseases hampering milk yield of dairy cows. Methionine (Met) and arginine (Arg) are key nutrients with potential to regulate inflammation and oxidative stress. The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide (LPS) stimulation in primary bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 replicates per treatment) were pre-incubated for 12 h in media with the following amino acid combinations: ideal profile of amino acids (control; Con), increased Met supply (incMet), increased Arg supply (incArg), and increased supply of Met and Arg (incMetArg). Subsequently, cells were challenged with or without LPS (1 µg/mL) and incubated for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS was attenuated in the incArg cultures. Although challenge with LPS led to lower abundance of proteins related to the antioxidant response (NFE2L2, NQO1, GPX1), lower levels of ATG7, and lower mRNA abundance of GPX3, we found little effect in cultures with incMet or incArg. Cultures with incMet, incArg, or incMetArg led to attenuation of the upregulation of SOD2 and NOS2 induced by LPS. Abundance of phosphorylated p65 (RELA) was greater after LPS stimulation, but the response was attenuated in cultures with incMet. The greater ratio of pRELA to total RELA in responses to LPS was also attenuated in cultures with incMetArg. The greater mRNA abundance of the proinflammatory cytokine IL1B induced by LPS was attenuated in cultures with incMet, and the same trend induced by LPS on CXCL2 was also alleviated in cultures with incArg. Overall, the data suggest that greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS through controlling the abundance of proinflammatory cytokines and chemokines and activity of NF-κB. Little benefit on oxidative stress induced by LPS challenge in BMEC was detected with greater supply of Met and Arg.
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Arginina/uso terapéutico , Células Epiteliales/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Mastitis Bovina/prevención & control , Metionina/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Bovinos , Suplementos Dietéticos , Femenino , Inmunidad Innata/efectos de los fármacos , Inflamación/metabolismo , Inflamación/veterinaria , Lipopolisacáridos , Glándulas Mamarias Animales/citología , Metionina/metabolismo , Leche , Fosforilación , ARN Mensajero/metabolismoRESUMEN
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.
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Bovinos/sangre , Colina/administración & dosificación , Metionina/administración & dosificación , Neutrófilos/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa , Animales , Antioxidantes/metabolismo , Bovinos/inmunología , Bovinos/fisiología , Colina/genética , Colina/metabolismo , Dieta/veterinaria , Regulación hacia Abajo , Femenino , Expresión Génica , Inmunidad Innata/genética , Inflamación/genética , Inflamación/veterinaria , Lactancia/efectos de los fármacos , Metionina/genética , Metionina/metabolismo , Neutrófilos/inmunología , Estrés Oxidativo/genética , ARN Mensajero/metabolismoRESUMEN
The supply of methionine (Met) in late pregnancy can alter mRNA abundance of genes associated with metabolism and immune response in liver and polymorphonuclear leukocytes (PMN) of the neonatal calf. Whether prenatal supply of Met elicits postnatal effects on systemic inflammation and innate immune response of the calf is not well known. We investigated whether enhancing the maternal supply of Met via feeding ethyl-cellulose rumen-protected Met (RPM) was associated with differences in calf innate immune response mRNA abundance in PMN and systemic indicators of inflammation during the first 50 d of life. Calves (n = 14 per maternal diet) born to cows fed RPM at 0.09% of diet dry matter per day (MET) for the last 28 ± 2 d before calving or fed a control diet with no added Met (CON) were used. Blood for biomarker analysis and isolation of PMN for innate immune function assays and mRNA abundance was harvested at birth (before colostrum feeding) and at 7, 21 and 50 d of age. Whole blood was challenged with enteropathogenic bacteria (Escherichia coli 0118:H8) and phagocytosis and oxidative burst of neutrophils and monocytes were quantified via flow cytometry. Although concentration of haptoglobin and activity of myeloperoxidase among calves from both maternal groups increased markedly between 0 and 7 d of age followed by a decrease to baseline at d 21 the responses were lower in MET compared with CON calves. Nitric oxide concentration decreased markedly between 0 and 7 d regardless of maternal group but MET calves tended to have lower overall concentrations during the study. In vitro phagocytosis in stimulated neutrophils increased markedly over time in both CON and MET calves but responses were overall greater in MET calves. Oxidative burst in both neutrophils and monocytes increased over time regardless of maternal treatment. The mRNA abundance of lactate dehydrogenase (LDHA) signal transducer and activator of transcription 3 (STAT3) and S100 calcium binding protein A8 (S100A8) in PMN was overall greater in MET calves. Overall data suggest that increasing the maternal supply of Met during late pregnancy could affect the neonatal calf inflammatory status and innate immune response. Although changes in mRNA abundance could play a role in coordinating the immune response the exact mechanisms merit further study.
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Bovinos , Dieta/veterinaria , Inmunidad Innata/efectos de los fármacos , Metionina/farmacología , Neutrófilos/inmunología , ARN Mensajero/metabolismo , Animales , Bovinos/inmunología , Suplementos Dietéticos , Femenino , Inflamación/prevención & control , Inflamación/veterinaria , Recuento de Leucocitos , Hígado/metabolismo , Metionina/metabolismo , Fagocitosis , Embarazo , Complicaciones del Embarazo/prevención & control , Complicaciones del Embarazo/veterinaria , Rumen/metabolismoRESUMEN
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.
Asunto(s)
Bovinos/inmunología , Redes Reguladoras de Genes , Inmunidad/genética , Metionina/farmacología , Neutrófilos/inmunología , Animales , Bovinos/genética , Células Cultivadas , Dieta/veterinaria , Suplementos Dietéticos , Femenino , Lactancia/fisiología , Lipopolisacáridos/inmunología , Metionina/administración & dosificación , Leche/química , Rumen/metabolismoRESUMEN
Enhancing the supply of rumen-protected Met (RPM) during the peripartum period alleviates inflammation and oxidative stress status in dairy cows. We tested the hypothesis that RPM could increase abundance of genes and proteins related to glutathione (GSH) metabolism and the antioxidant transcription factor nuclear factor erythroid 2-like 2 (NFE2L2) in subcutaneous adipose tissue. Multiparous Holstein cows were fed a basal diet [control prepartum diet = 1.47 Mcal/kg of dry matter (DM) and 15.3% crude protein; control postpartum diet = 1.67 Mcal/kg of DM and 17.7% crude protein] or the control plus ethyl-cellulose RPM at a rate of 0.09 and 0.10% of DM intake before expected calving and after calving, respectively. Sixty cows were assigned to treatments based on parity, previous 305-d milk yield, and body condition score at 28 d from parturition. Diets were fed from -28 to 30 d. Biopsies of subcutaneous adipose tissue collected on d -10, 10, and 30 relative to parturition from 7 cows in each group were used for measuring concentrations of GSH, reactive oxygen species, superoxide dismutase, malondialdehyde, and mRNA and protein abundance (Western blotting). A repeated-measures ANOVA was used for statistics. The statistical model included the random effect of block and fixed effects of treatment, time, and its interaction. There was a diet × time effect for reactive oxygen species due to lower concentrations in Met versus control cows specifically at d -10. Cows fed Met also had lower concentrations of malondialdehyde in subcutaneous adipose tissue. Compared with controls, overall mRNA abundance of the GSH metabolism-related genes cystathionine-ß-synthase (CBS), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), and glutathione peroxidase 1 (GPX1) was greater in cows fed Met. Furthermore, supply of Met resulted in an overall upregulation of protein abundance of glutathione peroxidase (GPX) 1, GPX3, glutathione S-transferase mu 1 (GSTM1), and glutathione S-transferase α 4 (GSTA4), all related to GSH metabolism. There was a diet × time effect for protein abundance of NFE2L2 and its repressor Kelch-like ECH associated protein 1 (KEAP1) due to lower values at 30 d in cows fed Met versus controls. The abundance of phosphorylated NFE2L2 was lower at 30 d in response to Met. Overall, the data suggest that exogenous Met may play a role in activating GSH metabolism and the antioxidant NFE2L2 pathways in subcutaneous adipose tissue.
Asunto(s)
Bovinos/fisiología , Suplementos Dietéticos , Glutatión/metabolismo , Inflamación/veterinaria , Metionina/administración & dosificación , Factor 2 Relacionado con NF-E2/metabolismo , Tejido Adiposo/metabolismo , Animales , Antioxidantes/metabolismo , Celulosa/análogos & derivados , Celulosa/química , Dieta/veterinaria , Femenino , Inflamación/prevención & control , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Metionina/química , Leche/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Periodo Periparto , Fosforilación , Periodo Posparto/efectos de los fármacos , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Rumen/metabolismoRESUMEN
Heat stress (HS) causes reductions in milk production, but it is unclear whether this effect is due to reduced number or functional capacity (or both) of mammary cells. Methionine supplementation improves milk protein, whereas Arg is taken up in excess by mammary cells to produce energy and nonessential AA that can be incorporated into milk protein. To evaluate molecular mechanisms by which mammary functional capacity is affected by HS and Met or Arg, mammary alveolar (MAC-T) cells were incubated at thermal-neutral (37°C) or HS (42°C) temperatures. Treatments were optimal AA profiles (control; Lys:Met = 2.9:1.0; Lys:Arg = 2.1:1.0), control plus Met (Lys:Met = 2.5:1.0), or control plus Arg (Lys:Arg = 1.0:1.0). After incubation for 6 h, cells were harvested and RNA and protein were extracted for quantitative real-time PCR and Western blotting. Protein abundance of mechanistic target of rapamycin (MTOR), eukaryotic initiation factor 2a, serine-threonine protein kinase (AKT), 4E binding protein 1 (EIF4EBP1), and phosphorylated EIF4EBP1 was lower during HS. The lower phosphorylated EIF4EBP1 with HS would diminish translation initiation and reduce protein synthesis. Both Met and Arg had no effect on MTOR proteins, but the phosphorylated EIF4EBP1 decreased by AA, especially Arg. Additionally, Met but not Arg decreased the abundance of phosphorylated eukaryotic elongation factor 2, which could be positive for protein synthesis. Although HS upregulated the heat shock protein HSPA1A, the apoptotic gene BAX, and the translation inhibitor EIF4EBP1, the mRNA abundance of PPARG, FASN, ACACA (lipogenesis), and BCL2L1 (antiapoptotic) decreased. Greater supply of Met or Arg reversed most of the effects of HS occurring at the mRNA level and upregulated the abundance of HSPA1A. In addition, compared with the control, supply of Met or Arg upregulated genes related to transcription and translation (MAPK1, MTOR, SREBF1, RPS6KB1, JAK2), insulin signaling (AKT2, IRS1), AA transport (SLC1A5, SLC7A1), and cell proliferation (MKI67). Upregulation of microRNA related to cell growth arrest and apoptosis (miR-34a, miR-92a, miR-99, and miR-184) and oxidative stress (miR-141 and miR-200a) coupled with downregulation of fat synthesis-related microRNA (miR-27ab and miR-221) were detected with HS. Results suggest that HS has a direct negative effect on synthesis of protein and fat, mediated in part by coordinated changes in mRNA, microRNA, and protein abundance of key networks. The positive responses with Met and Arg raise the possibility that supplementation with these AA during HS might have a positive effect on mammary metabolism.
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Arginina/farmacología , Bovinos , Glándulas Mamarias Animales/citología , Metionina/farmacología , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Animales , Arginina/administración & dosificación , Caseínas/metabolismo , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Respuesta al Choque Térmico , Calor , Metionina/administración & dosificación , Proteínas de la Leche/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia ArribaRESUMEN
Periparturient dairy cows are likely subject to altered intracellular reduction-oxidation (redox) balance due to the high metabolic rates and physiological adaptations occurring around parturition. Such conditions could induce oxidative damage. In nonruminants, it is well established that nuclear factor erythroid 2 like 2 (NFE2L2) is a critical transcription factor for maintaining cellular redox balance by inducing adaptive responses against oxidative stress (OS) that can otherwise lead to uncontrolled inï¬ammation. Tea polyphenols (TP), the major polyphenolic constituents of green tea, are potent antioxidants that could exert protective effects on bovine mammary epithelial cells (BMEC) by scavenging free radicals. We used NFE2L2 short interfering RNA (siRNA) to downregulate NFE2L2 expression in cultured BMEC to investigate whether TP could inhibit H2O2-induced OS by activating the NFE2L2/heme oxygenase-1 (HMOX1) pathway. Isolated BMEC were exposed to H2O2 (600 µM) for 6 h to induce OS. Optimal doses of TP (0, 60, 80, and 100 µg/mL) were evaluated by pretreatment of BMEC for 0, 2, 4, 6, 8, 12, and 24 h, followed by a H2O2 (600 µM) challenge for 6 h. The BMEC were transfected with NFE2L2-siRNA for 48 h, pretreated with 100 µg/mL of TP for 12 h, then challenged by 600 µM H2O2 for 6 h. Results revealed that after H2O2 exposure a concentration of TP of 100 µg/mL during a 12-h incubation led to greater cell viability, protein, and mRNA abundance of NFE2L2, and lower intracellular reactive oxygen species (ROS) accumulation. In addition, transfection with NFE2L2-siRNA decreased abundance of NFE2L2 and HMOX1 in spite of exogenous TP supplementation, whereas ROS production was increased in response to exogenous H2O2 (600 µM). Overall, TP had beneficial effects on redox balance in BMEC, slowing down cellular OS-related injury through decreasing the production of ROS and enhancing mechanisms controlled at least in part by the NFE2L2/HMOX1 pathway.
Asunto(s)
Bovinos/metabolismo , Células Epiteliales/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Glándulas Mamarias Animales/citología , Factor 2 Relacionado con NF-E2/metabolismo , Polifenoles/farmacología , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Peróxido de Hidrógeno/farmacología , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Polifenoles/química , Té/químicaRESUMEN
Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5'-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 µM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 µM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase ß (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine ß-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study.
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Colina/metabolismo , Citidina Difosfato Colina/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Metionina/metabolismo , Animales , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Bovinos , Células Cultivadas , Dieta/veterinaria , Suplementos Dietéticos/análisis , Femenino , Lactancia , Proteínas de la Leche/metabolismo , Parto , EmbarazoRESUMEN
Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (-EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase ß-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase ß-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower ß-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.
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Calcio/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/citología , Metionina/metabolismo , Receptores de Aminoácidos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Valina/metabolismo , Animales , Caseínas/genética , Caseínas/metabolismo , Bovinos , Dimerización , Femenino , Glándulas Mamarias Animales/metabolismo , Fosforilación , Biosíntesis de Proteínas , Receptores de Aminoácidos/química , Receptores de Aminoácidos/genética , Transducción de SeñalRESUMEN
Polymorphonuclear leukocytes (PMNL) are the first responders upon pathogen invasion and hence play an important role in inflammatory and immune responses. Rumen-protected methionine (MET) and choline (CHOL) during the peripartal period affect the immune response and inflammatory status in dairy cows to different extents. We aimed to examine the effect of MET and CHOL supply on expression of genes regulating key PMNL functions and associations with whole-blood immune challenge. Thirty multiparous Holstein cows from a larger cohort randomly assigned from -21 to 30 d relative to parturition to a basal control (CON) diet, CON plus MET at a rate of 0.08% of dry matter, or CON plus CHOL at 60 g/d were used. Blood was sampled at -10, 7, and 30 d relative to parturition for inflammatory biomarker analyses and PMNL isolation. Neutrophil and monocyte phagocytosis and oxidative burst in vitro were assessed in whole blood at 1, 7, and 28 d. Although neutrophil and monocyte phagocytosis did not differ, oxidative burst in neutrophils and monocytes was greater in MET-supplemented cows relative to CON cows. Compared with CON, PMNL adhesion and migration-related genes (ITGAM, ITGB2, ITGA4) were downregulated in response to MET and CHOL. Expression of CADM1 and SELL was also lower in MET-supplemented cows compared with CON cows but not in CHOL cows. In contrast, compared with CON cows, the expression of ICAM1 was lower in CHOL but not MET cows. Similar to adhesion and migration-related genes, cows receiving MET- or CHOL-supplemented diets had lower expression of inflammation-related genes (IL1ß, IL10RA, NFKB1, STAT3, TLR2). However, expression of IRAK1 and TLR4 was lower in MET- but not CHOL-supplemented cows. Plasma taurine concentration was greater in MET cows compared with CHOL and CON cows, suggesting a better redox status in plasma. In agreement with plasma taurine, oxidative stress-related genes (CBS, CTH, GPX1, GSS, SOD2) in PMNL were lower in response to MET and to CHOL supply. Overall, immunometabolic gene expression profile and blood biomarker analyses suggest an overall better redox status in PMNL during the transition period in response to MET and CHOL supply. These adaptations in PMNL might be beneficial for mounting a better bactericidal response upon challenge.
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Bovinos/fisiología , Colina/farmacología , Suplementos Dietéticos , Metionina/farmacología , Animales , Biomarcadores/sangre , Bovinos/inmunología , Dieta/veterinaria , Femenino , Regulación de la Expresión Génica , Inflamación/veterinaria , Neutrófilos/inmunología , Estrés Oxidativo , Parto , Embarazo , Distribución Aleatoria , Rumen/metabolismoRESUMEN
The periparturient period is the most critical period during the lactation cycle of dairy cows and is characterized by increased oxidative stress status. The objective of this experiment was to evaluate the effect of supplementing rumen-protected methionine on nuclear factor erythroid 2-like 2 (NFE2L2, formerly NRF2) protein and target gene expression in the mammary gland during the early postpartal period. Multiparous Holstein cows were used in a block design experiment with 30 cows per treatment. Treatments consisting of a basal control diet (control) or the basal diet plus rumen-protected methionine (methionine) were fed from d -28 to 60 relative to parturition. Mammary tissue biopsies were harvested on d 21 postpartum from 5 cows per treatment. Compared with control, methionine increased dry matter intake, milk yield, and milk protein content. Among plasma parameters measured, methionine led to greater methionine and lower reactive oxygen metabolites. Compared with control, methionine supply resulted in greater mRNA abundance of the NFE2L2 target genes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), malic enzyme 1 (ME1), ferrochelatase (FECH), ferritin heavy chain 1 (FTH1), and NAD(P) H quinone dehydrogenase 1 (NQO1) in the mammary tissue. In addition, methionine upregulated the mRNA abundance of NFE2L2, NFKB1, MAPK14 and downregulated KEAP1. The ratio of phosphorylated NFE2L2 to total NFE2L2 protein, and total heme oxygenase 1 (HMOX1) protein were markedly greater in response to methionine supply. In contrast, total protein abundance of Kelch-like ECH-associated protein 1 (KEAP1), which sequesters NFE2L2 in the cytosol and reduces its activity, was lower with methionine. Besides the consistent positive effect of methionine supply on systemic inflammation and oxidative stress status, the present data indicate a positive effect also on antioxidant mechanisms within the mammary gland, which are regulated, at least in part, via phosphorylation of NFE2L2 and its target genes. The exact mechanisms for these responses merit further study.
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Bovinos , Redes Reguladoras de Genes , Metionina/administración & dosificación , Factor 2 Relacionado con NF-E2/química , Animales , Dieta , Suplementos Dietéticos , Femenino , Lactancia , Hígado , Factor 2 Relacionado con NF-E2/metabolismo , Fosforilación , RumenRESUMEN
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.