Postruminal choline supply during negative nutrient balance alters components of hepatic mTOR signaling and plasma amino acids in lactating Holstein cows.

Choline requirements for dairy cattle are unknown. However, enhanced postruminal supply of choline may increase flux through the methionine cycle to spare Met for other functions such as protein synthesis and phosphatidylcholine (PC) synthesis during periods of negative nutrient balance (NNB). The objective was to investigate the effects of postruminal choline supply during a feed restriction-induced NNB on hepatic abundance and phosphorylation of mTOR (mechanistic target of rapamycin)-related signaling proteins, hepatic lipidome and plasma AA. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 DIM) were used in a replicated 5 × 5 Latin square design with 4 d of treatment and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements to induce NNB) with abomasal infusion of water (R0) or restriction plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected via biopsy on d 5 after infusions ended and used for Western blot analysis to measure proteins involved in mTOR signaling and untargeted lipidomics. Blood was collected on d 1 to 5 for plasma AA analysis. Statistical contrasts for protein and AA data were A0 versus R0 (CONT1), R0 versus the average of choline dose (CONT2) and tests of linear and quadratic effects of choline dose. Analysis of lipidomic data were performed with the web-based metabolomic processing tool MetaboAnalyst 5.0. Ratios of p-RPS6KB1:tRPS6KB1, p-EEF2:tEEF2, and p-EIF2:tEIF2 were greater with R (CONT1). Among those, supply of choline led to decreases in p-EEF2:tEEF2 (CONT2), p-EIF2:tEIF2 and tended to decrease p-EIF4BP1:tEIF4BP1. However, the effect was quadratic only for p-EEF2:tEEF2 and p-EIF2A:tEIF2A, reaching a nadir at 6.25 to 12.5 g/d choline ion. The ratio of p-RPS6KB1:tRPS6KB1 was not affected by supply of choline and was close to 2-fold greater at 25 g/d choline versus A0. Plasma Met concentration decreased with R (CONT1), but increased linearly with choline. Restriction also increased plasma 3-methyl-histidine (CONT1). The partial least squares discriminant analysis model of liver lipids distinguished treatments, with 13.4% of lipids being modified by treatment. One-way ANOVA identified 109 lipids with a false discovery rate ≤0.05. The largest group identified was PC species; all 35 detected decreased with R versus A0, but there were few differences among choline treatments. Overall, data suggested that dephosphorylation of EEF2 and EIF2A due to enhanced choline supply potentially helped maintain or increase protein synthesis during NNB. While activation of mTOR was not altered by choline, this idea of increased protein synthesis is partly supported by the increased circulating Met. However, enhanced postruminal choline had limited effects on the species of lipid produced during a period of NNB.

Feeding a Saccharomyces cerevisiae fermentation product improves udder health and immune response to a Streptococcus uberis mastitis challenge in mid-lactation dairy cows.

BACKGROUND: We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product (NTK) in response to a mastitis challenge. Eighteen mid-lactation multiparous Holstein cows (n = 9/group) were fed the control diet (CON) or CON supplemented with 19 g/d NTK for 45 d (phase 1, P1) and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis (phase 2, P2). After 36-h, mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2 (9 d post challenge). Cows were then followed until day 75 (phase 3, P3). Milk yield (MY) and dry matter intake (DMI) were recorded daily. Milk samples for somatic cell score were collected, and rectal and udder temperature, heart and respiration rate were recorded during the challenge period (P2) together with blood samples for metabolite and immune function analyses. Data were analyzed by phase using the PROC MIXED procedure in SAS. Biopsies were used for transcriptomic analysis via RNA-sequencing, followed by pathway analysis. RESULTS: DMI and MY were not affected by diet in P1, but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON. NTK reduced rectal temperature, somatic cell score, and temperature of the infected quarter during the challenge. Transcriptome data supported these findings, as NTK supplementation upregulated mammary genes related to immune cell antibacterial function (e.g., CATHL4, NOS2), epithelial tissue protection (e.g. IL17C), and anti-inflammatory activity (e.g., ATF3, BAG3, IER3, G-CSF, GRO1, ZFAND2A). Pathway analysis indicated upregulation of tumor necrosis factor α, heat shock protein response, and p21 related pathways in the response to mastitis in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows. CONCLUSIONS: Overall, results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.

One-carbon, carnitine, and glutathione metabolism-related biomarkers in peripartal Holstein cows are altered by prepartal body condition.

We investigated how prepartal body condition score (BCS) alters key hepatic enzymes associated with 1-carbon, carnitine, and glutathione metabolism and the related biomarkers in liver tissue and plasma of periparturient dairy cows. Twenty-six multiparous Holstein dairy cows were retrospectively selected according to BCS at 4 wk prepartum and divided into high (HighBCS, BCS ≥ 3.50) and normal (NormBCS, BCS ≤ 3.25) BCS groups (n = 13 each). Blood plasma samples were obtained at -30, -10, 7, 15, and 30 d relative to calving. Liver tissue biopsies were performed at -15, 7, and 30 d relative to calving, and samples were used to assess protein abundance via Western blot assay. Cows in the HighBCS group lost ∼1 unit of BCS between -4 and 4 wk around calving, while NormBCS cows lost ∼0.5 unit in the same period. Prepartal dry matter intake (DMI, kg/d) did not differ between groups. Compared with NormBCS cows, HighBCS cows had higher postpartal DMI and milk yield (+5.34 kg/d). In addition, greater overall plasma concentrations of fatty acids and activity of the neutrophil-enriched enzyme myeloperoxidase were observed in HighBCS compared with NormBCS cows. Despite similar reactive oxygen metabolite concentrations in both groups at 30 d, HighBCS cows had lower overall concentrations of ß-carotene and tocopherol, explaining the lower (BCS × Time) antioxidant capacity (ferric reducing ability of plasma). The HighBCS cows also had greater liver malondialdehyde concentrations and superoxide dismutase activity at 30 d. Overall, compared with NormBCS cows, HighBCS cows had lower hepatic protein abundance of the 1-carbon metabolism enzymes cystathionine-ß-synthase, betaine-homocysteine methyltransferase, and methionine adenosyltransferase 1 A (MAT1A), as well as the glutathione metabolism-related enzymes glutathione S-transferase α 4 and glutathione peroxidase 3 (GPX3). A lower protein abundance of glutathione S-transferase mu 1 (GSTM1) at -15 and 7 d was also observed. Regardless of BCS, cows had increased abundance of GSTM1 and GPX3 between -15 and 7 d around calving. A marked decrease of gamma-butyrobetaine dioxygenase 1 from -10 to 7 d in HighBCS compared with NormBCS cows suggested a decrease in de novo carnitine synthesis that was partly explained by the lower abundance of MAT1A. Overall, data suggest biologic links between BCS before calving, milk yield, immune response, and hepatic reactions encompassing 1-carbon metabolism, carnitine, and antioxidant synthesis.

Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows.

Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥ 3.5) or normal BCS (NBCS; n = 11, BCS ≤ 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at -15, 7, and 30 d relative to parturition were used for mRNA (real time-PCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC), with P ≤ 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS × Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at -15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in HBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC36A1), and sodium-coupled amino acid transporters (SLC38A2) were greater in HBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS × Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.

Body condition alters glutathione and nuclear factor erythroid 2-like 2 (NFE2L2)-related antioxidant network abundance in subcutaneous adipose tissue of periparturient Holstein cows.

Dairy cows with high body condition score (BCS) in late prepartum are more susceptible to oxidative stress (OS). Nuclear factor erythroid 2-like 2 (NFE2L2) is a major antioxidant transcription factor. We investigated the effect of precalving BCS on blood biomarkers associated with OS, inflammation, and liver function, along with mRNA and protein abundance of targets related to NFE2L2 and glutathione (GSH) metabolism in s.c. adipose tissue (SAT) of periparturient dairy cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥3.5) or normal BCS (NBCS; n = 11, BCS ≤3.17) on d 28 before parturition. Cows were fed a corn silage- and wheat straw-based total mixed ration during late prepartum, and a corn silage- and alfalfa hay-based total mixed ration postpartum. Blood samples obtained at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with inflammation, including albumin, ceruloplasmin, haptoglobin, and myeloperoxidase, as well as OS, including ferric reducing ability of plasma (FRAP), reactive oxygen species (ROS), and ß-carotene. Adipose biopsies harvested at -15, 7, and 30 d relative to parturition were analyzed for mRNA (real-time quantitative PCR) and protein abundance (Western blotting) of targets associated with the antioxidant transcription regulator nuclear factor, NFE2L2, and GSH metabolism pathway. In addition, concentrations of GSH, ROS and malondialdehyde were measured. High BCS cows had lower prepartum dry matter intake expressed as a percentage of body weight along with greater BCS loss between -4 and 4 wk relative to parturition. Plasma concentrations of ROS and FRAP increased after parturition regardless of treatment. Compared with NBCS, HBCS cows had greater concentrations of FRAP at d 7 postpartum, which coincided with peak values in those cows. In addition, NBCS cows experienced a marked decrease in plasma ROS after d 7 postpartum, while HBCS cows maintained a constant concentration by d 30 postpartum. Overall, ROS concentrations in SAT were greater in HBCS cows. However, overall mRNA abundance of NFE2L2 was lower and cullin 3 (CUL3), a negative regulator of NFE2L2, was greater in HBCS cows. Although HBCS cows had greater overall total protein abundance of NFE2L2 in SAT, ratio of phosphorylated NFE2L2 to total NFE2L2 was lower, suggesting a decrease in the activity of this antioxidant system. Overall, mRNA abundance of the GSH metabolism-related genes glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), and transaldolase 1 (TALDO1), along with protein abundance of glutathione S-transferase mu 1 (GSTM1), were greater in HBCS cows. Data suggest that HBCS cows might experience greater systemic OS after parturition, while increased abundance of mRNA and protein components of the GSH metabolism pathway in SAT might help alleviate tissue oxidant status. Data underscored the importance of antioxidant mechanisms at the tissue level. Thus, targeting these pathways in SAT during the periparturient period via nutrition might help control tissue remodeling while allowing optimal performance.

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.

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

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

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.

Higher plane of nutrition pre-weaning enhances Holstein calf mammary gland development through alterations in the parenchyma and fat pad transcriptome.

BACKGROUND: To reduce costs of rearing replacement heifers, researchers have focused on decreasing age at breeding and first calving. To increase returns upon initiation of lactation the focus has been on increasing mammary development prior to onset of first lactation. Enhanced plane of nutrition pre-weaning may benefit the entire replacement heifer operation by promoting mammary gland development and greater future production. METHODS: Twelve Holstein heifer calves (< 1 week old) were reared on 1 of 2 dietary treatments (n = 6/group) for 8 weeks: a control group fed a restricted milk replacer at 0.45 kg/d (R, 20% crude protein, 20% fat), or an accelerated group fed an enhanced milk replacer at 1.13 kg/d (EH, 28% crude protein, 25% fat). At weaning (8 weeks), calves were euthanized and sub-samples of mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested upon removal from the body. Total RNA from both tissues was extracted and sequenced using the Illumina HiSeq2500 platform. The Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis (IPA) were used for pathway analysis and functions, gene networks, and cross-talk analyses of the two tissues. RESULTS: When comparing EH vs R 1561 genes (895 upregulated, 666 downregulated) and 970 genes (506 upregulated, 464 downregulated) were differentially expressed in PAR and MFP, respectively. DIA and IPA results highlight a greater proliferation and differentiation activity in both PAR and MFP, supported by an increased metabolic activity. When calves were fed EH, the PAR displayed transcriptional signs of greater overall organ development, with higher ductal growth and branching, together with a supportive blood vessel and nerve network. These activities were mediated by intracellular cascades, such as AKT, SHH, MAPK, and Wnt, probably activated by hormones, growth factors, and endogenous molecules. The analysis also revealed strong communication between MFP and PAR. CONCLUSION: The transcriptomics and bioinformatics approach highlighted key mechanisms that mediate the mammary gland response to a higher plane of nutrition in the pre-weaning period.