Short communication: Supply of methionine during late pregnancy enhances whole-blood innate immune response of Holstein calves partly through changes in mRNA abundance in polymorphonuclear leukocytes.

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.

Glutathione metabolism and nuclear factor erythroid 2-like 2 (NFE2L2)-related proteins in adipose tissue are altered by supply of ethyl-cellulose rumen-protected methionine in peripartal Holstein cows.

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.

Methionine and choline supply alter transmethylation, transsulfuration, and cytidine 5'-diphosphocholine pathways to different extents in isolated primary liver cells from dairy cows.

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.

Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period.

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.

Ethyl-cellulose rumen-protected methionine alleviates inflammation and oxidative stress and improves neutrophil function during the periparturient period and early lactation in Holstein dairy cows.

The periparturient period is the most critical phase in the productive cycle of dairy cows and is characterized by impairment of the immune system. Our objective was to evaluate the effect of feeding ethyl-cellulose rumen-protected methionine (RPM) starting at d -28 from expected parturition through 60 d in milk on biomarkers of inflammation, oxidative stress, and liver function as well as leukocyte function. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or the control plus ethyl-cellulose RPM (Mepron, Evonik Nutrition & Care GmbH). Mepron was supplied from -28 to 60 d in milk at a rate of 0.09% and 0.10% dry matter during the prepartum and postpartum period. That rate ensured that the ratio of Lys to Met in the metabolizable protein was close to 2.8:1. Blood samples from 15 clinically healthy cows per treatment were collected at d -30, -14, 1, 7, 21, 30, and 60 and analyzed for biomarkers of liver function, inflammation, and oxidative stress. Neutrophil and monocyte function in whole blood was measured in vitro at -14, 1, 7, 21, and 30 d in milk. The statistical model included the random effect of block and fixed effect of treatment, time, and its interaction. Compared with control, ethyl-cellulose RPM increased plasma cholesterol and paraoxonase after parturition. Among the inflammation biomarkers measured, ethyl-cellulose RPM led to greater albumin (negative acute-phase protein) and lower haptoglobin than control cows. Although concentration of IL-1ß was not affected by treatments, greater IL-6 concentration was detected in response to ethyl-cellulose RPM. Cows supplemented with ethyl-cellulose RPM had greater plasma concentration of ferric-reducing antioxidant power, ß-carotene, tocopherol, and total and reduced glutathione, whereas reactive oxygen metabolites were lower compared with control cows. Compared with control, ethyl-cellulose RPM enhanced neutrophil phagocytosis and oxidative burst. Overall, the results indicate that ethyl-cellulose RPM supply to obtain a Lys-to-Met ratio of 2.8:1 in the metabolizable protein during the periparturient period and early lactation is an effective approach to help mitigate oxidative stress and inflammation as well as enhance liver and neutrophil function in dairy cows.

Ethyl-cellulose rumen-protected methionine enhances performance during the periparturient period and early lactation in Holstein dairy cows.

The onset of lactation in dairy cows is characterized by severe negative energy and protein balance. Increasing Met availability during this time may improve milk production, hepatic lipid metabolism, and immune function. The aim of this study was to evaluate the effect of feeding ethyl-cellulose rumen-protected methionine (RPM; Mepron, Evonik Nutrition and Care GmbH, Hanau-Wolfgang, Germany) on the performance of dairy cows during prepartum and early-lactation periods. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or an ethyl-cellulose RPM diet. Ethyl-cellulose RPM was supplied from -28 to 60 d relative to parturition at a rate of 0.09% and 0.10% of dry matter during the prepartum and postpartum periods, respectively. That rate ensured that the ratio of Lys to Met in metabolizable protein was close to 2.8:1. Cows fed ethyl-cellulose RPM had dry matter intakes (DMI) that were 1.2 kg/d greater during the prepartum period and consequently had overall greater cumulative DMI than cows in the control group. Compared with controls, during the fresh period (1-30 d in milk; DIM) feeding ethyl-cellulose RPM increased DMI by 1.7 kg/d, milk yield by 4.1 kg/d, fat yield by 0.17 kg/d, milk protein yield by 0.20 kg/d, 3.5% fat-corrected milk by 4.3 kg/d, and energy-corrected milk by 4.4 kg/d. Although ethyl-cellulose RPM supplementation increased milk protein content by 0.16 percentage units compared with the control during the fresh period, no differences were observed for milk fat, lactose, and milk urea nitrogen concentration. During the high-producing period (31-60 DIM), cows fed ethyl-cellulose RPM increased DMI and milk yield by 1.45 and 4.4 kg/d, respectively. Ethyl-cellulose RPM also increased fat yield by 0.19 kg/d, milk protein yield by 0.17 kg/d, 3.5% fat-corrected milk by 4.7 kg/d, and energy-corrected milk by 4.8 kg/d compared with controls. Ethyl-cellulose RPM supplementation reduced plasma fatty acids in the fresh period and decreased γ-glutamyl transferase, indicating better liver function. In conclusion, when lysine was adequate, feeding ethyl-cellulose RPM to achieve a ratio close to 2.8:1 in metabolizable protein improved dairy cow performance from parturition through 60 DIM. The greater milk production was, at least in part, driven by the greater voluntary DMI and better liver function.

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.

Corium molecular biomarkers reveal a beneficial effect on hoof transcriptomics in peripartal dairy cows supplemented with zinc, manganese, and copper from amino acid complexes and cobalt from cobalt glucoheptonate.

Supplying trace minerals in more bioavailable forms such as amino acid complexes (AAC) could help ameliorate the incidence of hoof disorders in peripartal dairy cows. The aim of this study was to evaluate the effects of supplementing metal AAC during the peripartal period on expression of 28 genes in corium tissue related to claw composition, oxidative stress, inflammation, chemotaxis, and transcriptional regulation. Forty-four multiparous Holstein cows received a common diet from -30 to 30 d relative to parturition and were assigned to receive an oral bolus containing either inorganic trace minerals (INO) or AAC (i.e., organic) Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1 ppm, respectively, in the total diet dry matter. Inorganic trace minerals were provided in sulfate form, and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 d in milk was evaluated before a hoof biopsy in a subset of cows (INO=10; AAC=9). Locomotion score did not differ between treatments in the prepartum or postpartum period. The incidence of heel horn erosion was lower in AAC cows, but the incidence of sole ulcers did not differ. Downregulation of KRT5, CTH, CALML5, and CYBB, and upregulation of BTD in AAC cows indicated a decrease in the need for activation of cellular pathways to regenerate corium tissue and increase biotin availability in the sole claw. These molecular changes in the sole could have been triggered by the lower incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, a transcription factor that regulates the antioxidant response, and the antioxidant enzyme SOD1. Among genes associated with inflammation, AAC cows had greater expression of TLR4, and lower expression of TLR2, IL1B, and TNF compared with INO cows. Supplementation with metal AAC during the peripartal period affected the expression of genes involved in composition, oxidative stress, and inflammation status in the corium. The hoof biopsy procedure used in the present study should be further perfected and implemented in future lameness research to expand our understanding of hoof biology in dairy cows.