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 supply during the periparturient period enhances insulin signaling, amino acid transporters, and mechanistic target of rapamycin pathway proteins in adipose tissue of Holstein cows.

Enhanced postruminal supply of Met during the periparturient period increases dry matter intake and milk yield. In nonruminants, adipose tissue is responsive to AA supply, and can use AA as fuels or for protein synthesis regulated in part via insulin and mechanistic target of rapamycin (mTOR) signaling. Whether enhancing supply of Met has an effect on insulin and mTOR pathways in adipose tissue in peripartal cows is unknown. Multiparous Holstein cows were assigned from -28 to 60 d relative to parturition to a basal diet (control; 1.47 Mcal/kg of dry matter and 15.3% crude protein prepartum; 1.67 Mcal/kg and 17.7% crude protein postpartum) or the control plus ethyl-cellulose rumen-protected Met (RPM). The RPM was fed individually at a rate of 0.09% of dry matter intake prepartum and 0.10% postpartum. Subcutaneous adipose tissue harvested at -10, 10, and 30 d relative to parturition (days in milk) was used for quantitative PCR and Western blotting. A glucose tolerance test was performed at -12 and 12 d in milk to evaluate insulin sensitivity. Area under the curve for glucose in the pre- and postpartum tended to be smaller in cows fed Met. Enhanced Met supply led to greater overall mRNA abundance of Gln (SLC38A1), Glu (SLC1A1), l-type AA (Met, Leu, Val, Phe; SLC3A2), small zwitterionic α-AA (SLC36A1), and neutral AA (SLC1A5) transporters. Abundance of AKT1, RPS6KB1, and EIF4EBP1 was also upregulated in response to Met. A diet × day interaction was observed for protein abundance of insulin receptor due to Met cows having lower values at 30 d postpartum compared with controls. The diet × day interaction was significant for hormone-sensitive lipase due to Met cows having greater abundance at 10 d postpartum compared with controls. Enhanced Met supply upregulated protein abundance of insulin-responsive proteins phosphorylated (p)-AKT, peroxisome proliferator-activated receptor gamma, and fatty acid synthase. Overall abundance of solute carrier family 2 member 4 tended to be greater in cows fed Met. A diet × day interaction was observed for mTOR protein abundance due to greater values for RPM cows at 30 d postpartum compared with controls. Enhanced RPM supply upregulated overall protein abundance of solute carrier family 1 member 3, p-mTOR, and ribosomal protein S6. Overall, data indicate that mTOR and insulin signaling pathways in adipose tissue adapt to the change in physiologic state during the periparturient period. Further studies should be done to clarify whether the activation of p-AKT or increased availability of AA leads to the activation of mTOR.

Phosphorylation of AKT serine/threonine kinase and abundance of milk protein synthesis gene networks in mammary tissue in response to supply of methionine in periparturient Holstein cows.

The main objective was to evaluate the effect of increasing the supply of Met around parturition on abundance and phosphorylation of insulin- and mechanistic target of rapamycin complex 1 (mTORC1)-related signaling proteins along with mRNA abundance of milk protein and fat synthesis-related genes in postpartal mammary tissue. A basal control diet (control) or the basal diet plus ethyl-cellulose rumen-protected Met (0.9 g/kg of dry matter intake; Mepron, Evonik Nutrition & Care GmbH, Hanau-Wolfgang, Germany) were fed (n = 30 cows/diet) from d -28 to 60 relative to parturition. Mammary tissue and blood plasma were harvested from the same cows (n = 5/diet) in the control and Met groups at d 21 postpartum for mRNA, protein, and AA analysis. Increasing the supply of Met led to greater milk protein percentage and milk yield along with greater ratio of phosphorylated (p-)AKT to total AKT. The ratio of p-mTORC1 to total mTORC1 did not differ, but ratio of p-RPS6 to total ribosomal protein S6 (RPS6) was lower in response to Met supply. These responses were associated with greater mRNA abundance of the signaling proteins Janus kinase 2 (JAK2) and insulin receptor substrate 1 (IRS1). Greater Met supply also upregulated mRNA abundance of high-affinity cationic (SLC7A1) and sodium-coupled AA transporters (SLC38A1, SLC38A2); leucyl-tRNA (LARS), valyl-tRNA (VARS), and isoleucyl-tRNA synthetases (IARS); glucose transport solute carrier family 2 member 3 (SLC2A1); glucose transport solute carrier family 2 member 3 (SLC2A3); and casein α-s1 (CSN1S1). The mRNA abundance of components of the unfolded protein response, such as x-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), were upregulated, and protein phosphatase 1, regulatory subunit 15A (PPP1R15A) was downregulated in response to greater Met supply. Overall, the data suggest that increased dry matter intake, greater phosphorylation status of AKT, upregulation of glucose and AA transporters, and transcripts of tRNases in response to enhanced Met supply might have compensated for a reduction in ribosome biogenesis due to a lower ratio of p-RPS6 to total RPS6. Together, these cellular responses constitute a mechanism whereby Met supply can regulate milk protein synthesis in early lactation.

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.

Hepatic 1-carbon metabolism enzyme activity, intermediate metabolites, and growth in neonatal Holstein dairy calves are altered by maternal supply of methionine during late pregnancy.

Maternal supply of methyl donors such as methionine (Met) during late pregnancy can affect offspring growth and development. The objective was to investigate the effect of postruminal Met supply during late pregnancy on 1-carbon, Met cycle, and transsulfuration pathways in the calf liver. During the last 28 d of pregnancy, cows were individually fed a control diet or the control diet plus rumen-protected dl-Met (MET; 0.09% dry matter intake). Liver samples obtained from calves (n = 14/group) at 4, 14, 28, and 50 d of age were used for metabolomics, real-time PCR, and enzyme activity analyses. Genes associated with 1-carbon metabolism, DNA methylation, and the cytidine 5'-diphosphocholine-choline pathway were analyzed via real-time PCR. Activity of betaine homocysteine methyltransferase, cystathionine ß-synthase, and 5-methyltetrahydrofolate homocysteine methyltransferase (MTR) was analyzed using 14C isotopes. Data were analyzed using a mixed model that included the fixed effects of maternal treatment, day, and their interaction, and the random effect was calf within maternal diet. Calves born to dams offered MET tended to have greater birth body weight and had overall greater body weight during the first 9 wk of life. However, no differences were detected for daily feed intake and average daily gain between groups. Concentrations of betaine and choline, reflecting Met cycle activity, at d 14 through 28 were greater in MET calves. Transsulfuration pathway intermediates also were altered in MET calves, with concentrations of cysteine sulfinic acid and hypotaurine (d 4 and 14) and taurine being greater (d 4, 14, 28, and 50). Despite the lack of differences in daily feed intake, the greater concentrations of the tricarboxylic acid cycle intermediates fumarate and glutamate along with NAD/NADH in MET calves indicated enhanced rates of energy metabolism. Although activity of betaine homocysteine methyltransferase was greater in MET calves at d 14, cystathionine ß-synthase was lower and increased at d 14 and 28, where it was greater compared with the control diet. Activity of MTR was lower at d 4 and 50 in MET calves. Among gene targets measured, MET calves had greater overall expression of MTR, phosphatidylethanolamine N-methyltransferase, and choline kinase α and ß. An interaction of maternal diet by time was detected for mRNA abundance of DNA methyltransferase 3α (involved in de novo methylation) due to greater values at d 4 and 14 in MET calves. Overall, the data indicate that enhanced postruminal supply of Met to cows during late pregnancy may program hepatic metabolism of the calf in the context of maintaining Met homeostasis, phosphatidylcholine and taurine synthesis, DNA methylation, and energy metabolism. These alterations potentially result in better efficiency of nutrient use, hence conferring the calf a physiologic advantage during a period of rapid growth and development. The precise biologic mechanisms remain to be established.

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.

Hepatic phosphorylation status of serine/threonine kinase 1, mammalian target of rapamycin signaling proteins, and growth rate in Holstein heifer calves in response to maternal supply of methionine.

The study investigated whether methionine supply during late pregnancy is associated with liver mammalian target of rapamycin (MTOR) pathway phosphorylation, plasma biomarkers, and growth in heifer calves born to cows fed a control diet (CON) or the control diet plus ethylcellulose rumen-protected methionine (MET; 0.09% of dry matter intake) for the last 28 d prepartum. Calves were fed and managed similarly during the first 56 d of age. Plasma was harvested at birth and 2, 7, 21, 42, and 50 d of age and was used for biomarker profiling. Liver biopsies were harvested at 4, 14, 28, and 50 d of age and used for protein expression. Body weight, hip height, hip width, wither height, body length, rectal temperature, fecal score, and respiratory score were measured weekly. Starter intake was measured daily, and average daily gain was calculated during the first 8 wk of age. During the first 7 wk of age, compared with calves in the CON group, calves in the MET group had greater body weight, hip height, wither height, and average daily gain despite similar daily starter intake. Concentration of methionine in plasma was lower at birth but increased markedly at 2 and 7 d of age in MET calves. Plasma insulin, glucose, free fatty acids, and hydroxybutyrate did not differ. A greater ratio of phosphorylated α-serine/threonine kinase (AKT):total AKT protein expression was detected in MET calves, namely due to differences at 4 d of age. The phosphorylated MTOR:total MTOR ratio also was greater in MET calves due to differences at 28 and 50 d (8 d postweaning). The decrease in phosphorylated MTOR:total MTOR between 14 and 28 d in CON calves agreed with the increase in phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1):total EIF4EBP1 ratio during the same time frame. The overall expression of phosphorylated ribosomal protein S6 kinase B1 (RPS6KB1):total RPS6KB1 and phosphorylated eukaryotic translation elongation factor 2 (EEF2):total EEF2 was lower in MET calves. Regardless of methionine supply prepartum, there was an 11-fold temporal decrease from 4 to 50 d in phosphorylated AKT:total AKT. Similarly, regardless of methionine supply, there were overall decreases in phosphorylation ratios of AKT, MTOR, RPS6KB1, and eukaryotic translation initiation factor 2A (EIF2A) over time. Data provide evidence of a positive effect of methionine supply during the last month of pregnancy on rates of growth during the first 7 wk of age. Phosphorylation status of some components of the MTOR pathway in neonatal calf liver also was associated with greater maternal supply of methionine. Thus, the data suggest that molecular mechanisms in the liver might be programmed by supply of methionine during late pregnancy. The exact mechanisms coordinating the observed responses remain to be determined.

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.

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.

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.

Association of aqueous hydrogen concentration with methane production in continuous cultures modulated to vary pH and solids passage rate.

The objective of this study was to evaluate the effects of altering pH and solids passage rate (kp) on concentration of aqueous H2 [H2(aq)], CH4 production, volatile fatty acids (VFA) production, and fiber digestibility in a continuous culture fermentation system. The present study was conducted as a 2 × 2 factorial treatment arrangement in a Latin square design using continuous culture fermentors (n = 4). Our continuous culture system was converted to a closed system to measure CH4 and H2 emission while measuring H2(aq) concentration and VFA production for complete stoichiometric assessment of fermentation pattern. Treatments were control pH (CpH; ranging from 6.3 to 6.9) or low pH (LpH; 5.8 to 6.4) factorialized with solids kp that was adjusted to be either low (Lkp; 2.5%/h) or high (Hkp; 5.0%/h); liquid dilution was maintained at 7.0%/h. Fermentors were fed once daily (40 g of dry matter; 50:50 concentrate:forage diet). Four periods lasted 10 d each, with 3 d of sample collection. The main effect of LpH increased nonammonia nitrogen flow, and both LpH and Hkp increased nonammonia nonbacterial N flow. We observed a tendency for Hkp to increase bacterial N flow per unit of nonstructural carbohydrates and neutral detergent fiber degraded. The main effect of LpH decreased H2(aq) by 4.33 µM compared with CpH. The main effect of LpH decreased CH4 production rate from 5 to 9 h postfeeding, and Hkp decreased CH4 production rate from 3 to 9 h postfeeding. We found no effect of LpH on daily CH4 production or CH4 produced per gram of neutral detergent fiber degraded, but Hkp decreased daily CH4 production by 33.2%. Both the main effects of LpH and Hkp decreased acetate molar percentage compared with CpH and Lkp, respectively. The main effect of both LpH and Hkp increased propionate molar percentage, decreasing acetate-to-propionate ratio from 2.62 to 2.34. We noted no treatment effects on butyrate molar percentage or total VFA production. The results indicate increasing kp and decreasing pH decreased acetate-to-propionate ratio, but only increasing kp decreased CH4 production; lack of differences for LpH might be a result of compensatory methanogenesis during the second half of the day postfeeding.

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.

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows.

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off 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.

Relationship between the magnitude of the inbreeding coefficient and milk traits in Holstein and Jersey dairy bull semen used in Brazil.

Artificial insemination has been used to improve production in Brazilian dairy cattle; however, this can lead to problems due to increased inbreeding. To evaluate the effect of the magnitude of inbreeding coefficients on predicted transmitting abilities (PTAs) for milk traits of Holstein and Jersey breeds, data on 392 Holstein and 92 Jersey sires used in Brazil were tabulated. The second-degree polynomial equations and points of maximum or minimal response were estimated to establish the regression equation of the variables as a function of the inbreeding coefficients. The mean inbreeding coefficient of the Holstein bulls was 5.10%; this did not significantly affect the PTA for percent milk fat, protein percentage and protein (P = 0.479, 0.058 and 0.087, respectively). However, the PTAs for milk yield and fat decreased significantly after reaching inbreeding coefficients of 6.43 (P = 0.034) and 5.75 (P = 0.007), respectively. The mean inbreeding coefficient of Jersey bulls was 6.45%; the PTAs for milk yield, fat and protein, in pounds, decreased significantly after reaching inbreeding coefficients of 15.04, 9.83 and 12.82% (P < 0.001, P = 0.002, and P = 0.001, respectively). The linear regression was only significant for fat and protein percentages in the Jersey breed (P = 0.002 and P = 0.005, respectively). The PTAs of Holstein sires were more affected by smaller magnitudes of inbreeding coefficients than those of Jersey sires. It is necessary to monitor the inbreeding coefficients of sires used for artificial insemination in breeding schemes in Brazil, since the low genetic variability of the available sires may lead to reduced production.

Flint corn grain processing and citrus pulp level in finishing diets for feedlot cattle.

Four trials were conducted to evaluate the effects of flint corn processing and the replacement of corn with citrus pulp (CiP) in diets for Nellore feedlot cattle. In a 103-d finishing trial, 216 Nellore bulls (350 ± 24 kg initial BW) were used in a randomized complete block design with a 2 × 4 factorial arrangement of treatments. Factors included 2 processing methods, either ground corn (GC) or steam-flaked corn (FC), with CiP replacing each corn type at 4 levels (0, 25, 50, and 75% of DM). All diets contained 12% sugarcane bagasse and 88% concentrate (DM basis). Treatments were also evaluated in metabolism trials, in which 10 ruminally cannulated Nellore steers (389 ± 37 kg) were assigned to 2 independent but simultaneous 5 × 5 Latin squares, each using 1 method of corn processing (GC and FC). Interactions ( < 0.05) between corn processing and CiP inclusion level were observed for final BW, DMI, ADG, G:F, and HCW. With FC-based diets, added CiP linearly decreased final BW ( = 0.04), whereas with GC-based diets, added CiP quadratically increased final BW ( = 0.002). With FC-based diets, the inclusion of CiP linearly increased DMI ( = 0.03) and linearly decreased ADG ( = 0.03) and G:F ( = 0.001). Increasing CiP in GC-based diets quadratically increased DMI ( = 0.001), ADG ( = 0.005), and HCW ( = 0.003). In FC-based diets, CiP inclusion had no effect on HCW ( = 0.21). Dressing percent, LM area, and 12th-rib fat were not affected by diet ( ≥ 0.05). For steers fed GC diets, CiP inclusion in the diet quadratically decreased the molar proportion of isovalerate ( = 0.001) but linearly increased ruminal butyrate ( = 0.006). No differences ( ≥ 0.16) were observed for total VFA concentrations, acetate:propionate ratio, and ruminal NH-N as CiP replaced GC. For steers fed FC diets, the molar proportion of acetate linearly increased ( = 0.002) whereas the proportion of propionate was linearly decreased ( < 0.001), resulting in a linear increase ( = 0.001) in the acetate:propionate ratio. Replacing corn with CiP linearly reduced NEm ( = 0.001) and NEg ( < 0.001) of FC-based diets but did not affect ( = 0.15) NE values of CG-based diets. Steam flaking flint corn improved cattle performance in this trial more than has been reported for dent corn in the published literature.

Green leaf allowance and dairy ewe performance grazing on tropical pasture.

The objective of this study was to explain the influence of green leaf allowance levels on the performance of dairy ewes grazing a tropical grass. Seventy-two lactating ewes grazed Aruana guineagrass (Panicum maximum Jacq. cv. Aruana) for 80 d. The treatments were 4 daily levels of green leaf allowance (GLA) on a DM basis corresponding to 4, 7, 10, and 13 kg DM/100 kg BW, which were named low, medium-low, medium-high, and high level, respectively. The experimental design was completely randomized with 3 replications. During the experimental period, 4 grazing cycles were evaluated in a rotational stocking grazing method (4 d of grazing and 16 d of rest). There was a linear effect of GLA on forage mass, and increasing GLA resulted in increased total leaf mass, reaching an asymptotic plateau around the medium-high GLA level. The stem mass increased with increased GLA, and a pronounced increase was observed between medium-high and high GLAs. Increasing GLA increased both forage disappearance rate and postgrazing forage mass. Leaf proportion increased with GLA, peaking at the medium-high level, and the opposite occurred for stem proportions, which reduced until medium-high GLA level, followed by an increase on high GLA. Forage CP decreased linearly with GLA, and increasing GLA from low to high reduced CP content by 31%. On the other hand, NDF increased 14% and ADF increased 26%, both linearly in response to greater GLA levels. Total digestible nutrients decreased linearly by 8% when GLA increased from low to high level. Milk yield increased, peaking at medium-high GLA (1.75 kg ewe(-1) d(-1)) and decreased at high GLA level (1.40 kg ewe(-1) d(-1)). Milk composition was not affected by the GLA levels. There was a reduction in stocking rate from 72 to 43 ewes/ha when GLA increased from low to high level. Productivity (milk yield kg ha(-1) d(-1)) increased as GLA increased, peaking at medium-low level (115 kg ha(-1) d(-1)). Although this tropical grass showed the same pattern in responses to GLA levels as reported in the literature with temperate pastures, the magnitude of the process changed and the maximum response in milk yield from lactating dairy ewes grazing a tropical pasture would be achieved with higher forage allowances than in temperate pastures. Overall, Aruana guineagrass grazed by lactating dairy ewes should be managed to provide 7 to 10 GLA in kg DM/100 kg BW according to the production goals.