Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism.

Feeding supplemental choline and Met during the periparturient period can have positive effects on cow performance; however, the mechanisms by which these nutrients affect performance and metabolism are unclear. The objective of this experiment was to determine if providing rumen-protected choline, rumen-protected Met, or both during the periparturient period modifies the choline metabolitic profile of plasma and milk, plasma AA, and hepatic mRNA expression of genes associated with choline, Met, and lipid metabolism. Cows (25 primiparous, 29 multiparous) were blocked by expected calving date and parity and randomly assigned to 1 of 4 treatments: control (no rumen-protected choline or rumen-protected Met); CHO (13 g/d choline ion); MET (9 g/d DL-methionine prepartum; 13.5 g/d DL-methionine, postpartum); or CHO + MET. Treatments were applied daily as a top dress from ∼21 d prepartum through 35 d in milk (DIM). On the day of treatment enrollment (d -19 ± 2 relative to calving), blood samples were collected for covariate measurements. At 7 and 14 DIM, samples of blood and milk were collected for analysis of choline metabolites, including 16 species of phosphatidylcholine (PC) and 4 species of lysophosphatidylcholine (LPC). Blood was also analyzed for AA concentrations. Liver samples collected from multiparous cows on the day of treatment enrollment and at 7 DIM were used for gene expression analysis. There was no consistent effect of CHO or MET on milk or plasma free choline, betaine, sphingomyelin, or glycerophosphocholine. However, CHO increased milk secretion of total LPC irrespective of MET for multiparous cows and in absence of MET for primiparous cows. Furthermore, CHO increased or tended to increase milk secretion of LPC 16:0, LPC 18:1, and LPC 18:0 for primi- and multiparous cows, although the response varied with MET supplementation. Feeding CHO also increased plasma concentrations of LPC 16:0 and LPC 18:1 in absence of MET for multiparous cows. Although milk secretion of total PC was unaffected, CHO and MET increased secretion of 6 and 5 individual PC species for multiparous cows, respectively. Plasma concentrations of total PC and individual PC species were unaffected by CHO or MET for multiparous cows, but MET reduced total PC and 11 PC species during wk 2 postpartum for primiparous cows. Feeding MET consistently increased plasma Met concentrations for both primi- and multiparous cows. Additionally, MET decreased plasma serine concentrations during wk 2 postpartum and increased plasma phenylalanine in absence of CHO for multiparous cows. In absence of MET, CHO tended to increase hepatic mRNA levels of betaine-homocysteine methyltransferase and phosphate cytidylyltransferase 1 choline, α, but tended to decrease expression of 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and peroxisome proliferator activated receptor α irrespective of MET. Although shifts in the milk and plasma PC profile were subtle and inconsistent between primi- and multiparous cows, gene expression results suggest that supplemental choline plays a probable role in promoting the cytidine diphosphate-choline and betaine-homocysteine S-methyltransferase pathways. However, interactive effects suggest that this response depends on Met availability, which may explain the inconsistent results observed among studies when supplemental choline is fed.

Comparison of prepartum low-energy or high-energy diets with a 2-diet far-off and close-up strategy for multiparous and primiparous cows.

Previous research demonstrated that nutrition during the far-off (early) dry period may be as important to transition success as nutrition during the close-up dry period. Our objectives were to determine if a low-energy, high-fiber diet fed throughout the dry period improved metabolic status and production of dairy cows compared with a higher-energy diet or a 2-diet system, and to compare responses of cows and heifers to those diets. Holstein cows (n = 25 with 10 primiparous per treatment) were assigned to each of 3 diets at 60 d before expected calving. Treatment LO [40.5% wheat straw; 5.6 MJ of net energy for lactation (NEL)/kg of DM] was designed to meet but not exceed National Research Council recommendations for ad libitum intake from dry-off until calving. Treatment HI was a high-energy diet (6.7 MJ of NEL/kg of DM) fed for ad libitum intake from dry-off until calving. For the LO+HI treatment, the LO diet was fed ad libitum from dry-off until 21 d before expected calving, followed by the HI diet until parturition. After parturition all cows were fed a lactation diet (7.0 MJ of NEL/kg of DM) through 63 d postpartum. Dry matter intake and body weight were greater for HI cows prepartum, but not postpartum. When LO+HI cows were switched to the HI diet, their dry matter intake increased to match that of HI cows. Cows fed HI had greater gain of body condition before calving but lost more postpartum. Energy balance postpartum was higher for LO cows than for HI cows. Milk production, protein content, and protein yield did not differ among diets. Milk fat content and yield were highest for HI cows, lowest for LO, and intermediate for LO+HI cows. The HI cows had lower serum nonesterified fatty acids prepartum than either LO or LO+HI, but greater concentrations postpartum. Serum ß-hydroxybutyrate did not differ prepartum, but was greater for HI than for LO or LO+HI postpartum. Serum glucose and insulin were lower for LO than HI and LO+HI prepartum; insulin was lower for LO and HI than for LO+HI postpartum. The LO cows had lower liver total lipid concentration postpartum than the HI cows and LO+HI cows. Primiparous cows generally responded to diets the same as multiparous cows. The LO+HI feeding strategy provided no benefit over the LO diet. Moreover, the high-energy diet, even when fed for only 19 d before calving in the LO+HI group, resulted in increased serum ß-hydroxybutyrate and liver total lipid concentrations compared with LO.

Production responses to rumen-protected choline and methionine supplemented during the periparturient period differ for primi- and multiparous cows.

The objective of this experiment was to examine production performance responses to feeding rumen-protected choline (RPC) or methionine (RPM), or both, during the periparturient period. Fifty-four Holstein cows (25 primiparous, 29 multiparous) were used in a randomized block design experiment with a 2 × 2 factorial treatment structure. Cows were blocked by expected calving date and parity and assigned to 1 of 4 treatments: CON (no RPC or RPM); RPC (13.0 g/d of choline ion); RPM (9 g/d of dl-methionine prepartum; 13.5 g/d of dl-methionine postpartum); or RPC + RPM. Treatments were applied once daily as a top-dress from 3 wk before through 5 wk after calving. Dry matter intake and milk production were recorded daily, and milk samples were obtained once weekly. Data were analyzed for primi- and multiparous cows separately, using a repeated-measures mixed model that included random effects of cow and block and fixed effects of RPC, RPM, week, and their interactions; week served as the repeated effect. Initial BW and previous lactation milk yield were included as covariates in the statistical model for multiparous cows. Feeding RPC without RPM increased milk yield for multiparous cows by 8.7 kg/d, but this increase was not observed when RPC was fed with RPM. In multiparous cows, feeding RPM increased milk fat concentration and tended to increase milk fat yield. Because of this, RPM increased fat-corrected milk (FCM) by 2.8 kg/d at wk 2 postpartum, and this increase was sustained through wk 5 postpartum. In contrast, RPM did not affect overall milk fat yield and concentration for primiparous cows. Feeding RPC increased milk yield for primiparous cows by 3.5 kg/d irrespective of RPM inclusion, which is contrary to observations in multiparous cows, where RPC increased milk yield only in the absence of RPM. These results indicate that responses to RPC during the periparturient period may be dependent upon supply of methionine. Our observations also demonstrate that primi- and multiparous cows respond differently to RPC and RPM supplemented individually or simultaneously during the periparturient period. This variation in response could have been mediated by putative differences in choline and methionine requirements of primiparous versus multiparous cows, or by differences in the levels of milk production between the 2 groups (36 vs. 25 kg of FCM/d). However, cows in this study did not experience severe negative energy balance (mean nadirs of -6.6 and -5.0 Mcal/d for multiparous and primiparous cows, respectively), which likely affected their responses to RPC and RPM.

Transcriptomic analysis of circulating neutrophils in metabolically stressed peripartal grazing dairy cows.

The high metabolic demand during the transition into lactation places cows at greater risk of metabolic and infectious disease than at any other time in their lactation cycle. Additionally, a change occurs in the innate immune response during this period, which contributes to increased risk of disease. In the current study, we compared the transcriptomes of neutrophils from dairy cows divergent in their metabolic health post-calving. Cows (n = 5 per risk group) were selected from a parent experiment (n = 45 cows). Those with high or low concentrations of plasma nonesterified fatty acids, plasma ß-hydroxybutyrate, and liver triacylglycerol in both wk 1 and 2 were deemed to be at "high risk" (HR) or "low risk" (LR) of metabolic dysfunction, respectively. Circulating neutrophils were isolated at 3 time points during the transition period (d 0 and wk 1 and 4 post-calving), and gene expression was analyzed using RNA sequencing. Differential gene expression between the risk groups was determined using edgeR (http://bioconductor.org), and pathway analysis was conducted using Ingenuity Pathway Analysis (Ingenuity Systems, Qiagen, Valencia, CA). Statistical analysis indicated no interaction between risk and week. Therefore, the overall effect of risk was analyzed across all time points. In total, 3,500 genes were differentially expressed between the HR and LR cows (false discovery rate < 0.05). Of these, 2,897 genes were identified by Ingenuity Pathway Analysis and used for pathway analysis. Of the relevant pathways identified, neutrophils isolated from HR cows showed downregulation of genes involved in the recruitment of granulocytes, interferon signaling, and apoptosis, and upregulation of genes involved in cell survival. The results indicate that metabolically stressed cows had reduced neutrophil function during the peripartum period, highlighting a potential relationship between subclinical metabolic disease and innate immune function that suggests that metabolic health negatively affects the innate immune system and may contribute to the state of immunosuppression during the peripartum period. In this way, the metabolic stress among the HR cows may reduce their ability to combat infection during the transition period.

Short communication: Recombinant bacteriophage endolysin PlyC is nontoxic and does not alter blood neutrophil oxidative response in lactating dairy cows.

Mastitis is the leading cause of antimicrobial use on dairy farms. The potential for antimicrobial resistance has led to the examination of alternative strategies for controlling mastitis. One such alternative is PlyC, a potent peptidoglycan hydrolase derived from the streptococcal C1 bacteriophage that causes targeted lysis of the cell wall of Streptococcus uberis. At a concentration of 1.0 µg/mL, recombinant PlyC can induce lytic activity, suggesting that a low dose may successfully eliminate infection. We evaluated the dose effect of PlyC (1-50 µg/mL) on cytotoxicity and oxidative response on bovine blood polymorphonuclear leukocytes (PMN) obtained from 12 healthy, mid-lactation primiparous dairy cows. Following incubation at 0.5 and 2 h, cytotoxicity was characterized by measuring lactate dehydrogenase release from isolated cells. Oxidative burst response was characterized as the intensity of chemiluminescence produced in the interaction of reactive oxygen species generated in response to 0 or 1.6 µg/mL of phorbol 12-myristate-13-acetate (PMA) with a luminescent substrate with and without addition to PlyC to the incubation matrix. Data were analyzed as a complete randomized block design using mixed model procedures. Cytotoxicity of PlyC was not affected by concentrations up to 50 µg/mL. As expected, PlyC cytotoxicity on PMN varied across incubation time with greater cell toxicity measured at 2 h of incubation as compared with 0.5 h and is primarily attributed to the short life of PMN ex vivo. Concentrations of PlyC up to 50 µg/mL did not affect oxidative response; however, oxidative response was affected by incubation time and PMA concentration. In summary, varying doses of PlyC are nontoxic as estimated by lactate dehydrogenase release from cells and do not appear to alter PMA-stimulated reactive oxygen species production in bovine PMN. These early observations support continued work on the potential for application of this novel agent in combating mastitis.

The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows.

The 4 major tocopherol isoforms differ in their biochemical reactivity and cellular effects due to basic chemical structural differences. Alpha-tocopherol has been well studied regarding effects on bovine polymorphonuclear leukocyte (PMN) function and its involvement in respiratory burst. However, no studies to date have identified the effects of supplementing a mixed tocopherol oil (Tmix) particularly enriched in non-α tocopherol isoforms (i.e., γ- and δ-isoforms) on fundamental immunometabolic changes in dairy cows. Therefore, the objectives of this study were to determine whether short-term feeding of vegetable oil-derived Tmix alters specific biomarkers of metabolism, whole-blood leukocyte populations, respiratory burst, immunometabolic-related gene expression of PMN, or gene expression of isolated PMN when challenged with lipopolysaccharides (LPS). Clinically healthy multiparous lactating Holstein cows (n = 12; 179 ± 17 d in milk, 40.65 ± 3.68 kg of milk yield) were fed Tmix (620 g/d) for 7 consecutive days. Jugular blood (EDTA anticoagulant) was collected from all cows on d 0 before treatment initiation and again on d 7 after Tmix feeding. Total stimulated respiratory burst activity (RBA) and leukocyte populations were assessed in whole blood, and tocopherol isoform concentrations, metabolites, and hormones were measured in plasma. For gene expression analysis, isolated PMN from cows before and after Tmix feeding were incubated with LPS at a final concentration of either 0.0 or 1.5 µg/mL. Feeding of Tmix for 7 d increased the concentrations of α- and γ-tocopherol. The Tmix did not alter plasma insulin but decreased cholesterol. The Tmix did not alter whole-blood RBA or the leukocyte populations. The LPS challenge increased the expression of proinflammatory genes TNFA and IL6. However, Tmix treatment did not alter the patterns of LPS-affected expression of genes (e.g., TNFA, ITGB2, PPARA, and RXRA) associated with the immune or metabolic response. In conclusion, short-term feeding of Tmix may have no negative effect on animal health as Tmix increased α- and γ-tocopherol concentrations in blood and did not impair whole-blood RBA or alter leukocyte populations. The data provide further support that the α- and γ-tocopherol isoforms do not interfere with normal immune or metabolic function.

Regulatory effect of dietary intake of chromium propionate on the response of monocyte-derived macrophages from Holstein cows in mid lactation.

Chromium (Cr) has been reported to enhance immune function and improve insulin sensitivity and performance in beef and dairy cattle. However, its effect on bovine macrophage inflammatory and metabolic response is unknown. The objective of this study was to characterize the effect of dietary Cr on the inflammatory and metabolic response of polarized macrophages ex vivo. Twelve primiparous and 16 multiparous healthy Holstein cows in mid lactation (143 ± 37 d in milk) were enrolled in this study. Cows were fed a common total mixed ration once per day that was top-dressed with 200 g of ground corn containing 1 of 2 dietary treatments: control (CTL, no Cr supplementation) or Cr propionate (CrP, 8 mg of Cr/cow per day) for 35 d. At d 1, 17, and 35 of treatment, blood monocytes were isolated and cultured to obtain 3 monocyte-derived macrophage (MDM) phenotypes: M0 (non-polarized), M1 (pro-inflammatory; IFN-γ polarized) and M2 (anti-inflammatory; IL-4 polarized). The experiment was set in a randomized complete block design. Neither dry matter intake nor milk yield was affected by treatment. Plasma concentrations of metabolites and the metabolic and inflammatory response of MDM in spent media were not affected by treatment. Neither the whole blood cell population nor the specific proportion of leukocytes was affected by the main effect of treatment. However, we did observe a trend for fewer circulating neutrophils in cows fed CrP than in cows fed CTL for 35 d, which may be partly attributable to a greater influx of neutrophils into peripheral tissues, a reduced pro-inflammatory response during disease, or both; this warrants future study. Expression of IGFI was increased in MDM-M0, and expression of CXCL11 tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL. Expression of SLC2A3 also tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL at 17 d. Our results suggest that CrP has minimal effect on the inflammatory and metabolic response of MDM for Holstein dairy cows in mid lactation. Future studies are warranted to evaluate the differential regulation of Cr on the inflammatory and metabolic response of leukocytes from dairy cows at different stages of lactation and parity.

Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows.

Investigations of the temporal changes in mammary gene expression that occur during sudden diet change have been limited by the use of mammary tissue as the source of RNA because of the invasive nature of mammary biopsy procedures. However, the cytosolic crescent, present in 1% of the largest milk fat globules, contains mammary epithelial cell RNA that has become trapped between the inner and outer milk fat globule membranes during final formation and secretion of milk fat into the lumen of the mammary alveoli. We hypothesized that cytosolic crescent RNA extracted from milk fat could be used as an alternative source of mammary epithelial cell RNA to measure the immediate temporal changes in gene expression as a result of changes in diet. In this experiment, feed restriction was used to mimic the state of negative energy balance observed in early lactation and induce a rapid change in milk fat yield and lipogenic gene expression. Ten multiparous Holstein dairy were fed a basal diet ad libitum during a 14-d preliminary period followed by a 4-d experimental period where 5 cows remained on ad libitum feeding and 5 cows were fed at 60% of their d 8-14 intakes (restricted) on d 15 to 18 and then returned to ad libitum feeding on d 19 to 21. Milk samples were collected from each milking on d 13 to 20 and the milk fat was immediately isolated, mixed with Trizol LS, and stored at -80°C for subsequent extraction of RNA that was used for measurement of gene expression. Feed restriction tended to increase milk fat percentage. However, total milk and milk fat production were reduced by 21 and 18%, respectively. Consistent with increased use of body fat for milk synthesis, serum nonesterified fatty acids increased 6-fold (0.78 mEq/L in the feed restriction vs. 0.13 mEq/L ad libitum group), whereas the milk fatty acids

Effects of precalving body condition and prepartum feeding level on gene expression in circulating neutrophils.

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (-1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalving compared with those offered 75% of maintenance requirements. Results indicate that precalving body condition and changes in prepartum energy lead to altered gene expression of circulating neutrophils, highlighting the importance of transition cow nutrition for peripartum health.

The Impact of Intramammary Escherichia coli Challenge on Liver and Mammary Transcriptome and Cross-Talk in Dairy Cows during Early Lactation Using RNAseq.

Our objective was to identify the biological response and the cross-talk between liver and mammary tissue after intramammary infection (IMI) with Escherichia coli (E. coli) using RNAseq technology. Sixteen cows were inoculated with live E. coli into one mammary quarter at ~4-6 weeks in lactation. For all cows, biopsies were performed at -144, 12 and 24 h relative to IMI in liver and at 24 h post-IMI in infected and non-infected (control) mammary quarters. For a subset of cows (n = 6), RNA was extracted from both liver and mammary tissue and sequenced using a 100 bp paired-end approach. Ingenuity Pathway Analysis and the Dynamic Impact Approach analysis of differentially expressed genes (overall effect False Discovery Rate≤0.05) indicated that IMI induced an overall activation of inflammation at 12 h post-IMI and a strong inhibition of metabolism, especially related to lipid, glucose, and xenobiotics at 24 h post-IMI in liver. The data indicated in mammary tissue an overall induction of inflammatory response with little effect on metabolism at 24 h post-IMI. We identified a large number of up-stream regulators potentially involved in the response to IMI in both tissues but a relatively small core network of transcription factors controlling the response to IMI for liver whereas a large network in mammary tissue. Transcriptomic results in liver and mammary tissue were supported by changes in inflammatory and metabolic mediators in blood and milk. The analysis of potential cross-talk between the two tissues during IMI uncovered a large communication from the mammary tissue to the liver to coordinate the inflammatory response but a relatively small communication from the liver to the mammary tissue. Our results indicate a strong induction of the inflammatory response in mammary tissue and impairment of liver metabolism 24h post-IMI partly driven by the signaling from infected mammary tissue.

Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows.

Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n=5; high risk) and low (n=5; low risk) concentrations of nonesterified fatty acids, ß-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category; however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows' greater plasma ß-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state.

Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils.

Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (-1wk; n=46), day of calving (d 0; n=46), and postcalving at wk 1 (n=46), wk 2 (n=45), and wk 4 (n=43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of functional changes in neutrophils of grazing dairy cows over the transition period and align with studies in cows of conventional total mixed ration systems. This altered function may predispose cows to disease over the transition period and is likely to be a natural change in function due to parturition.

Short communication: Amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro.

Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50µg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1, coding for the enzyme pyruvate dehydrogenase α 1, tended to increase with AA supplementation. Due to the lower concentration of tumor necrosis factor-α in media coupled with a downregulation of several proinflammatory genes, we concluded that AA, rather than Gln, alter the inflammatory response of bovine blood PMN. Independent from Gln, blood PMN from cows in early lactation may use certain AA as their primary carbon source for energy than cows in later lactation. Evaluating cows during the early postpartum period will provide additional information on the effect of stage of lactation and nutrient supplementation on PMN function.

TRIENNIAL LACTATION SYMPOSIUM: Nutrient partitioning during intramammary inflammation: A key to severity of mastitis and risk of subsequent diseases?

In early lactation, susceptibility to disease is greatest, impacting cow health and productivity and leading to economic losses. Mastitis is the most economically costly disease to the dairy industry and is most frequent at this time. The objective of this paper is to discuss the energetic fuels used by leukocytes in the metabolic response during mastitis that may reveal potential mechanisms linking mastitis with the development of subsequent metabolic diseases for dairy cows during lactation. Glucose and glutamine are the primary fuels used by leukocytes and are essential substrates for optimal leukocyte function. Yet because these substrates are in high demand to support milk synthesis during early lactation, their supply to leukocytes may be compromised and may partly contribute to immunosuppression observed at this time. Production-related metabolic diseases during early lactation, such as ketosis and hepatic lipidosis, can also adversely affect health and productivity. Risk of subsequent disease for cows during mastitis has not been fully elucidated. Regardless of stage of lactation and physiological state, increases in circulating NEFA and glucose and decreases in ketones during an intramammary inflammation in dairy cows have been reported. In addition, previous work indicates that hepatic metabolism may be impaired during inflammation. These results indicate a potential link between mastitis and the risk of subsequent metabolic disease for dairy cows during lactation. This paper will discuss the complex relationships between metabolism and immune function and how these immunometabolic interactions relate mastitis with increased risk of subsequent disease during early lactation.

Hepatic metabolic response of Holstein cows in early and mid lactation is altered by nutrient supply and lipopolysaccharide in vitro.

The metabolic response of the liver during periods of inflammation is poorly understood. The objective of this study was to characterize the effects of nutrient supply and lipopolysaccharide (LPS) challenge on hepatic intermediate metabolism of early- and mid-lactation cows by employing gas chromatography-mass spectrometry with stable isotope tracer. Twelve multiparous Holstein-Friesian cows in early (n = 6; 12 ± 4.2 d in milk) and mid (n = 6; 115 ± 13.5 d in milk) lactation were used for this study. Liver biopsies were performed on all cows. Liver slices (40-60 mg) were incubated in a 37°C water bath for 2 h with either control (phosphate buffered saline), pyruvate (PYR; 1mM unlabeled pyruvate and 1mM [(13)C3]pyruvate), pyruvate + propionate (PYR+PRO; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM sodium propionate), or pyruvate + AA (PYR+AA; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM AA solution), and LPS (0.0 or 0.2 µg/mL) was added to flasks per treatment. Enrichment of isotopomers in metabolic equilibrium with Krebs cycle intermediates was assessed. Pyruvate fluxes and the enzymatic activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) and phosphoenol pyruvate carboxykinase (PEPCK) were calculated. Media were analyzed for concentrations of tumor necrosis factor-α (TNF-α), glucose, and haptoglobin. Data were analyzed as randomized block (stage of lactation) design in a factorial arrangement of nutrient treatments by LPS dose. Challenge with LPS increased the mRNA abundance of TNF-α, haptoglobin, and serum amyloid A 2, and the concentration of TNF-α in media. Challenge with LPS increased mRNA abundance of PC but reduced the enrichment of (13)C1[M1] and (13)C2[M2]alanine and tended to reduce the enzymatic activity of PEPCK. Incubation with PYR+PRO and PYR+AA increased the flux of pyruvate to acetyl CoA. However, only PYR+PRO increased the enzymatic activity of PEPCK and PDH versus PC and decreased the mRNA abundance of PC. Cows in early lactation tended to receive a greater contribution of pyruvate to the oxaloacetate flux via the lower PDH versus PC activity and a higher mRNA abundance of PC than cows in mid lactation. Our results suggest that regardless of stage of lactation and nutrient supplement, hepatic gluconeogenesis was impaired during inflammation. Further research examining how various nutrients support liver function and improve the immunometabolic response of liver during inflammation is warranted.

Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro.

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, ß-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50µg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

Are free glucose and glucose-6-phosphate in milk indicators of specific physiological states in the cow?

A total of 3200 milk samples from Holstein and Jersey cows were analysed for free glucose and glucose-6-phosphate (G6P) by an enzymatic-fluorometric method that requires no pre-treatment. The cows were primiparous as well as multiparous, and samples were taken throughout the entire lactation period. In addition, lactose, protein, fat, citrate and ß-hydroxybutyrate were determined and comparisons between these variables were made. Data were analysed using GLM model for the effect of parity, breed, time from last milking and stage of lactation on variations in parameters in milk. Pearson's correlations were generated between milk variables. P<0.05 was considered significant. Concentration of free glucose and G6P were on average 331 and 81 µM, respectively. Time from last milking (stay in the gland cistern) did not increase the concentration of these monosaccharides, indicating that they are not hydrolysis product from lactose post secretion, but rather reflecting the energy status of the mammary epithelial cells pre-secretion. Wide variation in range of these metabolites, that is, from 90 to 630 µM and 5 to 324 µM, for glucose and G6P, respectively, was observed. During the first 21 weeks in milk, free glucose increased whereas G6P decreased. Concentration of free glucose in milk is greater for primiparous than multiparous cows and greater for Holstein than Jersey cows. Concentration of G6P was not affected by parity or breed. The use of free glucose and G6P as indicators of physiological conditions and risk of disease is warranted for use as potential biomarkers for in-line surveillance systems on-farm.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro.

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×10(6) PMNL) and chemotaxis (6×10(6) PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×10(6) PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at -80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=-2.14], NFKBIA (FC=1.82), IL1B (FC=-2.16), TNFA (FC=-9.43), and SOD2 (FC=-1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=-3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

Postpartal immunometabolic gene network expression and function in blood neutrophils are altered in response to prepartal energy intake and postpartal intramammary inflammatory challenge.

The effect of over-feeding energy prepartum on blood polymorphonuclear neutrophil (PMN) response remains unclear. Cows fed controlled (CON; 1.34Mcal/kg of dry matter) or excess energy (OVE; 1.62Mcal/kg dry matter) during the dry period (~45d before expected calving date) received an intramammary (IM) challenge with Escherichia coli lipopolysaccharide (LPS) during the postpartal period to determine the effects of IM LPS and prepartal diet on the expression of key genes associated with immunometabolic response in blood PMN. Feed intake and daily milk yield were recorded throughout the study period. At 7d in milk (DIM), all cows received LPS (200µg) into 1 rear mammary quarter. Blood PMN were isolated at 7, 14, and 30 DIM, as well as before (0h) and after (12h) IM LPS challenge for gene expression analysis using quantitative real time PCR. Phagocytosis capabilities in vitro were assessed at 7, 14, and 30 DIM. Data were analyzed using the MIXED procedure of SAS with repeated measures. No differences in feed intake and milk yield were observed between OVE- and CON-fed cows. As expected, IM LPS challenge altered the expression of genes associated with the immune response (e.g., 1.9- and 1.8-fold for SELL and TLR2, respectively), metabolism (e.g., 1.8- and -1.8-fold for LDHA and SLC2A1, respectively), and transcription (e.g., 1.1- and 1.7-fold for NCOR1 and PPARD, respectively). At 12h postchallenge, an upregulation of TLR2 (1.8-fold), HIF1A (1.9-fold), and NFKB1 (1.5-fold) was observed for OVE rather than CON. At 7 DIM, S100A9 tended (2.2-fold) to be upregulated for OVE rather than CON. At 14 DIM, OVE resulted in lower PMN phagocytosis and an upregulation of NCOR2 (1.6-fold) and RXRA (1.9-fold) compared with CON-fed cows. At 30 DIM, an upregulation of MPO (3.5-fold) and PLA2G4A (1.5-fold) and a tendency for RXRA (1.7-fold) was observed for OVE- rather than CON-fed cows. Our results suggest that IM LPS challenge altered gene expression associated with metabolism in PMN and that OVE impaired PMN phagocytosis and increased the expression of immunometabolic genes after IM LPS challenge and during the postpartal period. The current study provides new linkages among prepartal feed energy intake, metabolism, and immune response of blood PMN and risk of disease during early lactation.