Oxylipids are associated with higher disease risk in postpartum cows.

Postpartum diseases are a major animal welfare and economic concern for dairy producers. Dysregulated inflammation, which may begin as soon as the cessation of lactation, contributes to the development of postpartum diseases. The ability to regulate inflammation and mitigate postpartum health diseases relies, in part, on the production of inflammatory mediators known as oxylipids. The objective of this study was to examine associations between oxylipids and postpartum diseases. Plasma samples were collected from 16 cattle via coccygeal venipuncture at the following time points: 6 d before dry-off; dry-off (d 0); 1, 2, 6, and 12 d after dry-off; 14 ± 3 d before the expected calving date; and 7 ± 2 d after calving. After calving, cows were grouped according to if clinical disease was undetected throughout the sampling period (n = 7) or if they developed a disease postpartum (n = 9). Liquid chromatography-tandem mass spectrometry was used to analyze plasma concentrations of 63 oxylipid species. Of the 32 oxylipids detected, concentrations of 7 differed between cows with no detected disease and diseased cows throughout the sampling period. Thus, a variable oxylipid profile was demonstrated through 2 major physiological transitions of a lactation cycle. Further, the information gained from this pilot study using a small number of animals with diverse diseases from a single herd suggests that it may be possible to use oxylipids at early mammary involution to alert dairy producers of cows at risk for disease after calving. Future studies should be performed in larger populations of animals, including cows from diverse geographies and dairying styles, and focus on specific diseases to evaluate the utility of oxylipids as biomarkers. Furthermore, it is important to determine the clinical implications of variable oxylipid concentrations throughout the lactation cycle and if the oxylipid profile can be modulated to improve inflammatory outcomes.

Peripheral markers of oxidative stress in Labrador retrievers with copper-associated hepatitis.

OBJECTIVES: To evaluate biomarkers of oxidative stress in dogs with copper-associated hepatitis (CAH) as compared with healthy controls, and to evaluate if these markers correlate with hepatic copper concentrations and hepatic histopathologic features. MATERIALS AND METHODS: Prospective study. Plasma reactive metabolite concentrations, plasma antioxidant potential, and plasma and urine isoprostane concentrations were determined in Labrador retrievers with copper-associated hepatitis (n=9) as well as in breed- and sex-matched (n=9) and age- and sex-matched (n=9) healthy control populations. Possible correlations between markers of oxidative stress and hepatic histopathological features also were investigated. RESULTS: Reactive metabolites (median, range) were over twofold greater in dogs with copper-associated hepatitis (87.2 RFU/µL, 60.9 to 185.6 RFU/µL) as compared to breed- and sex-matched (38.2 RFU/µL, 22.4 to 116.8 RFU/µL) and age- and sex-matched controls (32.0 RFU/µL, 18.5 to 127.4 RFU/µL). Antioxidant potential was decreased in copper-associated hepatitis dogs (6.5 TE/µL, 5.1 to 7.7 TE/µL) as compared to breed- and sex-matched controls (8.2 TE/µL, 5.3 to 11.8 TE/µL). Both reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio were positively correlated with hepatic copper concentrations. Plasma and urine isoprostanes were variable and not significantly different between populations. CLINICAL SIGNIFICANCE: Labrador retrievers with copper-associated hepatitis have altered oxidant status. Plasma reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio could be useful biomarkers. However, neither plasma nor urine isoprostanes were useful biomarkers for copper-associated hepatitis.

Evaluation of natural plant extracts as antioxidants in a bovine in vitro model of oxidative stress.

Oxidative stress contributes to many inflammatory-based diseases of dairy cattle especially during periods of increased metabolic activity such as around calving. Endothelial cells play a key role in maintaining normal inflammatory responses, but they are especially susceptible to macromolecule damage during times of oxidative stress. Therefore, bovine aortic endothelial cells (BAEC) were used to study the effect of natural tannin-based extracts on oxidative stress that may improve health and well-being of cattle. Tannins are secondary metabolites in plants with potent antioxidant activity that have been used as natural feed additives for food-producing animals. However, there is little information on how tannin-rich plant extracts may affect oxidative stress in dairy cattle. The objective of this study was to evaluate the antioxidant effect of pomegranate (Punica granatum; PMG), tara (Caesalpinia spinosa; TA), chestnut (Castanea sativa; CH), and gambier (Uncaria gambir; GM) natural extracts using an in vitro BAEC model of oxidative stress. Natural extracts were tested at a concentration of 80 µg/mL. Viability, apoptosis, intracellular reactive oxygen species, and isoprostanes were determined on cultured BAEC treated with different plant natural extracts. No changes in cell viability was detected following PMG and GM treatments. In contrast, there was a 30% reduction of BAEC viability following treatment with CH or TA extracts. Intracellular reactive oxygen species production was significantly less abundant in cells treated with natural extracts than with the lipopolysaccharide control. Moreover, antioxidant activity varied according to the tested extract, showing a reduction of 63, 45, 51, and 27% in PMG, GM, CH, and TA, respectively. The formation of isoprostanes as a consequence of lipid peroxidation after induction of oxidative stress also were significantly decreased in PMG-treated cells when compared with the untreated cells. Theses findings suggest that PMG extract has the potential to mitigate oxidative stress without detrimental effects on cell viability. Further in vitro and in vivo research is warranted to explore the antioxidant potential of PMG extract as a dietary supplement to control oxidative stress in dairy cattle.

Serum vitamin D concentrations at dry-off and close-up predict increased postpartum urine ketone concentrations in dairy cattle.

Vitamin D is commonly supplemented to dairy cows as vitamin D3 to support calcium homeostasis and in times of low sunlight exposure. Vitamin D has beneficial immunomodulatory and anti-inflammatory properties. Serum 25-hydroxyvitamin D [25(OH)D] concentrations fluctuated during lactation, with the lowest concentrations measured in healthy cows within 7 d of calving. However, it is unknown if serum 25(OH)D concentrations measured during the previous lactation are associated with transition diseases or health risk factors in dairy cattle. We collected serum samples from 279 dairy cattle from 5 commercial dairy herds in Michigan at dry-off, close-up, and 2-10 d in milk (DIM). Vitamin D concentrations were determined by measuring serum 25(OH)D by radioimmunoassay. Total serum calcium was measured by colorimetric methods. Body condition scores (BCS) were assigned at the time of blood collection. Clinical disease incidence was monitored until 30 d postparturition. Separate bivariable logistic regression analyses were used to determine if serum 25(OH)D at dry-off, close-up, and 2-10 DIM was associated with various clinical diseases including mastitis, lameness, and uterine disorders (classified as metritis, retained placenta, or both) and increased urine ketone concentrations at P < 0.05. Among all significant bivariable analyses, multivariable logistic regression analyses were built to adjust for potential confounding variables including parity, BCS, season, and calcium. Receiver operator characteristic (ROC) curve analyses were used to determine optimal concentrations of serum 25(OH)D. We found that higher serum 25(OH)D concentrations at dry-off and close-up predicted increased urine ketone concentrations in early lactation, even after adjustment for confounders. Alternatively, we found that lower serum 25(OH)D at 2-10 DIM was associated with uterine diseases. Optimal concentrations for serum 25(OH)D at dry-off and close-up for lower risk of increased urine ketone concentrations were below 103.4 and 91.1 ng/mL, respectively. The optimal concentration for serum 25(OH)D at 2-10 DIM for uterine diseases was above 71.4 ng/mL. These results indicate that serum 25(OH)D at dry-off and close-up may be a novel predictive biomarker for increased urine ketone concentrations during early lactation. Increased urine ketone concentrations are not necessarily harmful or diagnostic for ketosis but do indicate development of negative energy balance, metabolic stress, and increased risk of early lactation diseases. Predicting that dairy cattle are at increased risk of disease facilitates implementation of intervention strategies that may lower disease incidence. Future studies should confirm these findings and determine the utility of serum 25(OH)D concentrations as a predictive biomarker for clinical and subclinical ketosis.

Cohort-level disease prediction by extrapolation of individual-level predictions in transition dairy cattle.

Dairy cattle experience metabolic stress during the transition from late gestation to early lactation resulting in higher risk for several economically important diseases (e.g. mastitis, metritis, and ketosis). Metabolic stress is described as a physiological state composed of 3 processes: nutrient metabolism, oxidative stress, and inflammation. Current strategies for monitoring transition cow nutrient metabolism include assessment of plasma non-esterified fatty acids and beta-hydroxybutyrate concentrations around the time of calving. Although this method is effective at identifying cows with higher disease risk, there is often not enough time to implement intervention strategies to prevent health disorders from occurring around the time of calving. Previously, we published predictive models for early lactation diseases at the individual cow level at dry-off. However, it is unknown if predictive probabilities from individual-level models can be aggregated to the cohort level to predict cohort-level incidence. Therefore, our objective was to test different data aggregation methods using previously published models that represented the 3 components of metabolic stress (nutrient metabolism, oxidative stress, and inflammation). We included 277 cows from five Michigan dairy herds for this prospective cohort study. On each farm, two to four calving cohorts were formed, totaling 18 cohorts. We measured biomarker data at dry-off and followed the cows until 30 days post-parturition for cohort disease incidence, which was defined as the number of cows: 1) having one or more clinical transition disease outcome, and/or 2) having an adverse health event (abortion or death of calf or cow) within each cohort. We tested three different aggregation methods that we refer to as the p-central, p-dispersion, and p-count methods. For the p-central method, we calculated the averaged predicted probability within each cohort. For the p-dispersion method, we calculated the standard deviation of the predicted probabilities within a cohort. For the p-count method, we counted the number of cows above a specified threshold of predicted probability within each cohort. We built four sets of models: one for each aggregation method and one that included all three aggregation methods (p-combined method). We found that the p-dispersion method was the only method that produced viable predictive models. However, these models tended to overestimate incidence in cohorts with low observed counts and underestimate risk in cohorts with high observed counts.

Cohort-level disease prediction using aggregate biomarker data measured at dry-off in transition dairy cattle: A proof-of-concept study.

During the transition from late gestation to early lactation, dairy cattle are at increased risk for disease. Herd-level monitoring for disease risk involves evaluating multiple factors, including food intake, cow density, and biomarkers of nutrient metabolism. Biomarkers that are measured include non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB), which are usually measured in a subset of the herd (i.e. cohort). If a certain proportion of cows in the cohort are above a specific threshold for a biomarker, the cohort is considered at high risk of disease. Few previous studies have investigated other methods to aggregate individual cow-level data to the cohort level. We designed a proof-of-concept study to determine if biomarker aggregation methods may be useful to predict cohort incidence of adverse health events including 1) clinical diseases: mastitis, metritis, retained placenta, ketosis, lameness, pneumonia, milk fever, displaced abomasum, 2) and abortion or death of the calf or the cow. The study design was a prospective cohort study that used cows (N = 277) from five Michigan commercial dairy herds. Multiple cohorts of cows (two to four cohorts per farm, 18 total) were enrolled that shared the same dry-off date. We tested three different methods (central, dispersion, and count) to aggregate individual cow data (i.e. biomarkers and covariates) measured at dry-off. The central method consisted of calculating the average value of each variable within a cohort, and the dispersion method involved taking the standard deviation or mean absolute deviation about the median of each variable within a cohort. The count method consisting of counting the number of cows above a specific threshold for each variable within a cohort. We used best subsets selection to select a bouquet of candidate models for each aggregation method and averaged the predictions over the model set. We built 4 sets of Poisson regression models: one for each aggregation method and a combined model that included all three methods. We evaluated the models based on goodness-of-fit, model calibration using scoring rules, and comparison of observed versus predicted counts. The central and the combined method produced models that had good fit and model calibration. These results indicate that it may be possible to use aggregate measures to predict cohort disease incidence as early as dry-off. The next step is to test biomarker aggregation methods in studies with larger sample sizes.

Oxylipid profiles of dairy cattle vary throughout the transition into early mammary gland involution.

Successful lactation in multiparous dairy cattle relies on a well-managed dry period that allows the mammary gland to remodel and regenerate between lactations. Oxylipids are potent inflammatory mediators that are capable of regulating all aspects of inflammation. Although an oxylipid profile has been documented for periparturient and lactating cattle, little work has been done to define the profile of cows in the early dry period. Therefore, our group aimed to characterize the oxylipid profile in healthy cows during the transition into early mammary gland involution. Plasma samples were collected from 10 healthy Holstein dairy cows via coccygeal venipuncture 6 d before dry-off (d -6), at dry-off (d 0), and 1 (d +1), 2 (d +2), 6 (d +6), and 12 (d +12) d after the dry-off date. Liquid chromatography-mass spectrometry was used to quantify select monounsaturated fatty acids, polyunsaturated fatty acids, and saturated fatty acids, whereas oxylipids were quantified using liquid chromatography-tandem mass spectrometry. The results of this study revealed a unique profile of pro- and anti-inflammatory oxylipids throughout the transition from late lactation into the dry period. Many compounds reached the highest concentrations of the study at d +1, d +2, or d +12, whereas others reached the lowest concentrations at d +12. The characterization of this profile allows for further understanding of the physiology of early mammary involution. Future studies should investigate how the oxylipid profile of early mammary involution may affect the health and productivity of dairy cows.

Predictive models for early lactation diseases in transition dairy cattle at dry-off.

During the transition period, dairy cattle undergo tremendous metabolic and physiological changes to prepare for milk synthesis and secretion. Failure to sufficiently regulate these changes may lead to metabolic stress, which increases risk of transition diseases. Metabolic stress is defined as a physiological state consisting of 3 components: aberrant nutrient metabolism, oxidative stress, and inflammation. Current monitoring methods to detect cows experiencing metabolic stress involve measuring biomarkers for nutrient metabolism. However, these biomarkers, including non-esterified fatty acids, beta-hydroxybutyrate, and calcium are typically measured a few weeks before to a few days after calving. This is a retroactive approach, because there is little time to integrate interventions that remediate metabolic stress in the current cohort. Our objective was to determine if biomarkers of metabolic stress measured at dry-off are predictive of transition disease risk. We designed a prospective cohort study carried out on 5 Michigan dairy farms (N = 277 cows). We followed cows from dry-off to 30 days post-calving. Diseases and adverse outcomes were grouped in an aggregate outcome that included mastitis, metritis, retained placenta, ketosis, lameness, pneumonia, milk fever, displaced abomasum, abortion, and death of the calf or the cow. We used best subsets selection to select candidate models for four different sets of models: one set for each component of metabolic stress (nutrient metabolism, oxidative stress, and inflammation), and a combined model that included all 3 components. We used model averaging to obtain averaged predicted probabilities across each model set. We hypothesized that the averaged predictions from the combined model set with all 3 components of metabolic stress would be more effective at predicting disease than each individual component model set. The area under the curve estimated using receiver operator characteristic curves for the combined model set (0.93; 95% confidence interval [CI] = 0.90-0.96) was significantly higher compared with averaged predictions from the inflammation (0.87; 95% CI = 0.83-0.91), oxidative stress (0.78; 95% CI = 0.72-0.84), and nutrient metabolism (0.73; 95% CI = 0.67-0.79) model sets (p < 0.05). Our results indicate that it may be possible to detect cattle at risk for some transition diseases as early as dry-off. This has important implications for disease prevention, as earlier identification of cows at risk of health disorders will allow for earlier implementation of intervention strategies. A limitation of the current study is that we did not perform external validation. Future validation studies are needed to confirm our findings.

Changes in biomarkers of nutrient metabolism, inflammation, and oxidative stress in dairy cows during the transition into the early dry period.

Metabolic stress occurs in dairy cows when physiologic homeostasis is disrupted as a consequence of aberrant nutrient metabolism, chronic inflammation, and oxidative stress. Early-lactation cows that suffer from metabolic stress are susceptible to health disorders that cause significant production losses. However, there is little information regarding the occurrence and effect of metabolic stress during involution. Therefore, the purpose of this study was to investigate well-known biomarkers associated with metabolic stress in early-lactation cows at various time points during the early dry period when dairy cows also are subjected to dramatic changes in physiologic homeostasis. Our group conducted a descriptive study by collecting serum and whole-blood samples from the coccygeal vein of 29 healthy dairy cows at a commercial dairy herd. Sampling points included d -6, 0, +1, +2, +6, and +12 relative to dry-off date. Samples were used to quantify biomarkers related to nutrient metabolism, oxidative stress, and inflammation that included calcium, nonesterified fatty acids, ß-hydroxybutyrate, albumin, haptoglobin, cortisol, reactive oxygen and nitrogen species, antioxidant potential, oxidant status index, and isoprostanes. Additionally, whole-blood leukocyte differentials for total leukocyte, neutrophils, lymphocytes, eosinophils, and monocytes were analyzed. Within altered nutrient metabolism biomarkers, calcium and nonesterified fatty acid concentrations changed most from d 0 to d +2 during the sampling period. Indicators of oxidant status, such as reactive oxygen and nitrogen species, antioxidant potential, and oxidant status index, generally increased throughout the sampling period except at d +2, suggesting altered redox status throughout early involution. In contrast, isoprostane concentrations fluctuated throughout the study, demonstrating that indicators of oxidative damage occurred more sporadically during the sampling period. Therefore, many of the biomarkers associated with early-lactation metabolic stress also changed during the transition from late lactation to the early dry period, but not to the same magnitude and duration previously reported in periparturient cows. Future studies should be directed toward assessing whether the magnitude and duration of biomarker expression can affect the health and well-being of cows during the early dry period.

Short communication: Markers of oxidant status and inflammation relative to the development of claw lesions associated with lameness in early lactation cows.

Lameness is a major health disorder of dairy cattle and evidence suggests that it may be associated with oxidative stress (OS) during the transition period. Some debate exists, however, as to whether OS precedes the development of lameness or if OS occurs as a consequence of lameness. The purpose of this study was to test whether cows showing claw lesions during early lactation had a greater pro-oxidant and inflammatory status throughout the dry period or at the start of the lactation. Blood samples were taken from 30 cows from the same herd at dry off, movement to the close-up pen, and between 3 and 7 d in milk. Sera were analyzed for concentrations of haptoglobin, serum amyloid A, reactive oxygen and nitrogen species, and antioxidant potential. Blood samples also were subjected to total and differential white blood cell counts. Animals were monitored through 120 d in milk and grouped ex post into the following health categories: (1) exclusively hoof lesions; (2) other production diseases; or (3) nondiseased. Changes in oxidant status and inflammatory markers were significantly different with respect to metabolic and physiologic adaptations to calving and lactation. No differences in oxidant status, acute phase protein concentrations, or leukocyte populations were observed between the hoof lesions and the nondiseased categories. Thus, any associations between OS and lameness likely occurs closer to the onset of clinical signs or as a consequence of inflammatory responses due to localized tissue injury.

15-F2t-Isoprostane Concentrations and Oxidant Status in Lactating Dairy Cattle with Acute Coliform Mastitis.

BACKGROUND: Severe mammary tissue damage during acute coliform mastitis in cattle is partially caused by oxidative stress. Although considered a gold standard biomarker in some human conditions, the utility of 15-F2t-Isoprostanes (15-F2t-Isop) in detecting oxidative stress in dairy cattle has not been validated. HYPOTHESIS: Concentrations of 15-F2t-Isop in plasma, urine, and milk correlate with changes in oxidant status during severe coliform mastitis in cattle. ANIMALS: Eleven lactating Holstein-Friesian dairy cows in their 3rd-6th lactation. METHODS: A case-control study using cows with acute coliform mastitis and matched healthy controls were enrolled into this study. Measures of inflammation, oxidant status, and redox status in plasma and milk samples were quantified using commercial assays. Plasma, urine, and milk 15-F2t-Isop were quantified by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and ELISA assays. Data were analyzed by Wilcoxon rank sum tests (α = 0.05). RESULTS: Plasma 15-F2t-Isop quantified by LC-MS/MS was positively correlated with systemic oxidant status (r = 0.83; P = .01). Urine 15-F2t-Isop quantified by LC-MS/MS did not correlate with systemic oxidant status, but was negatively correlated with redox status variables (r = -0.83; P = .01). Milk 15-F2t-Isop quantified by LC-MS/MS was negatively correlated (r = -0.86; P = .007) with local oxidant status. Total 15-F2t-Isop in milk quantified by a commercial ELISA (cbELISA) was positively correlated with oxidant status in milk (r = 0.98; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Free plasma 15-F2t-Isop quantified by LC-MS/MS and total milk 15-F2t-Isop quantified by cbELISA are accurate biomarkers of systemic and mammary gland oxidant status, respectively. Establishing reference intervals for free and total 15-F2t-Isops for evaluating oxidative stress in dairy cows should currently be based on the LC-MS/MS method.

Quantification of bovine oxylipids during intramammary Streptococcus uberis infection.

Streptococcus uberis mastitis results in severe mammary tissue damage in dairy cows due to uncontrolled inflammation. Oxylipids are potent lipid mediators that orchestrate pathogen-induced inflammatory responses, however, changes in oxylipid biosynthesis during S. uberis mastitis are unknown. Thus, the current objective was to determine how oxylipid concentrations change in milk and mammary tissues during different stages of S. uberis mastitis. Increased arachidonic acid and linoleic acid-derived oxylipids were significantly increased in S. uberis-infected bovine mammary tissue. Linoleic acid metabolites, hydroxyoctadecadienoic acid (HODE) and oxooctadecadienoic acid, predominated in tissue and milk. Furthermore, in vitro exposure of bovine mammary endothelial cells to 13-hydroperoxyoctadecadienoic acid, upstream metabolite of HODE, significantly increased cyclooxygenase-2 expression, but 13-HODE exposure had no effect. The findings in the current study indicate lipidomic profiling may explain some of the dynamics of inflammation during bacterial challenge, however continued research is necessary to determine sample compartments which best reflect disease pathogenesis.

Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide infusion in horses.

REASONS FOR PERFORMING THE STUDY: Endotoxaemia contributes to morbidity and mortality in horses with colic due to inflammatory cascade activation. Effective therapeutic interventions are limited for these horses. Ethyl pyruvate (EP), an anti-inflammatory agent that alters the expression of proinflammatory cytokines, improved survival and organ function in sepsis and gastrointestinal injury in rodents and swine. Therapeutic efficacy of EP is unknown in endotoxaemic horses. OBJECTIVES: Determine the effects of EP on signs of endotoxaemia and expression of proinflammatory cytokines following administration of lipopolysaccharide (LPS) in horses. METHODS: Horses received 30 ng/kg bwt LPS in saline to induce signs of endotoxaemia. Next, horses received lactated Ringer's solution (LRS), (n = 6), 150 mg/kg bwt EP in LRS, (n = 6), or 1.1 mg/kg bwt flunixin meglumine (FM), (n = 6). Controls received saline followed by LRS (n = 6). Physical examinations, behaviour pain scores and blood for clinical pathological testing and gene expression were obtained at predetermined intervals for 24 h. RESULTS: Lipopolysaccharide infusion produced clinical and clinicopathological signs of endotoxaemia and increased expression of tumour necrosis factor alpha (TNFα), interleukin 6 (IL-6) and IL-8 (P<0.001) compared with controls. Leucopenia and neutropenia occurred in all horses that received LPS. Horses treated with EP and FM had significantly (P<0.0001) reduced pain scores compared with horses receiving LPS followed by LRS. Flunixin meglumine was significantly more effective at ameliorating fever compared with EP. Both EP and FM significantly diminished TNFα expression. Ethyl pyruvate significantly decreased, but FM significantly increased, IL-6 expression. Neither EP nor FM altered IL-8 expression. CONCLUSIONS AND POTENTIAL RELEVANCE: Ethyl pyruvate administered following LPS diminished the clinical effects of endotoxaemia and decreased proinflammatory gene expression in horses. Ethyl pyruvate suppressed expression of proinflammatory cytokines better than FM. However, FM was a superior anti-pyretic compared with EP. Ethyl pyruvate may have therapeutic applications in endotoxaemic horses.

Enhanced n-3 phospholipid content reduces inflammatory responses in bovine endothelial cells.

Uncontrolled inflammation contributes to the increased incidence and severity of infectious diseases in periparturient dairy cattle. The objective of this study was to determine if increasing n-3 fatty acid (FA) content and altering the profile of vasoactive eicosanoids could attenuate endothelial cell inflammatory responses. Bovine aortic endothelial cells (BAEC) were cultured with free FA mixtures that mimic the plasma NEFA composition during the first week of lactation of dairy cows or with a free FA mixture supplemented with a higher proportion of n-3 FA, including eicosapentaenoic and docosahexaenoic acids. The effects of increasing the docosahexaenoic and eicosapentaenoic acid content of BAEC on the expression of proinflammatory mediators and eicosanoid biosynthesis was assessed. Culturing BAEC with enriched concentrations of n-3 FA decreased the expression of proinflammatory cytokines, adhesion molecules, and reactive oxygen species with a concomitant increase in the biosynthesis of proresolving eicosanoids, including resolvins, protectins, and lipoxins. This study showed for the first time that increasing the n-3 FA content of endothelial cell phospholipids could alter the expression of eicosanoids and control the magnitude of inflammatory responses. Future studies are necessary to elucidate the mechanisms by which resolvins, protectins, and lipoxins may modify endothelial inflammatory pathways necessary to reduce the severity and duration of disease in periparturient cows.

Glucose transporter and hypoxia-associated gene expression in the mammary gland of transition dairy cattle.

Glucose is an important energy substrate, especially needed by dairy cows postpartum to support the onset of lactation. The prioritization and regulation of glucose uptake is accomplished, in part, by changes in expression of cellular glucose transport molecules (GLUT) within the mammary gland. The objectives of this study were to (1) evaluate the expression and cell-type specific localization of GLUT and hypoxia-associated genes that may regulate GLUT expression over the transition period and through lactation in bovine mammary tissue and (2) determine functionality of GLUT on primary bovine mammary endothelial cells (BMEC). Mammary tissue biopsies were taken from cows at 15 d before calving and again at 1, 15, 30, 60, 120, and 240 d post-parturition for quantitative real-time PCR analysis of GLUT and hypoxia-associated genes. Additional mammary tissue samples were used to localize GLUT within the cells of the lobulo-alveolar system via fluorescence microscopy. Cultures of primary bovine mammary endothelial cells were used to confirm the functionality of GLUT with a fluorescent glucose analog uptake assay. Significant increases in GLUT1 gene expression were observed during early lactation, whereas both GLUT3 and GLUT4 gene expression increased during late lactation. The gene expression for 2 receptors of vascular endothelial growth factor increased significantly during early lactation and remained increased throughout lactation when compared with gene expression during the transition period. All GLUT were detected on cultured BMEC and were capable of internalizing glucose through GLUT-mediated mechanisms. These data suggest mammary vascular tissues express GLUT during lactation and BMEC express functional glucose transporters. A better understanding of glucose uptake at the endothelial level may prove to be critical to improve glucose absorption from the blood for utilization by mammary epithelial cells.

Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses.

REASONS FOR PERFORMING THE STUDY: Endotoxaemia causes substantial morbidity and mortality in horses with colic and sepsis. Ethyl pyruvate is a novel anti-inflammatory medication that improved survival in preclinical models of severe sepsis endotoxaemia and intestinal ischaemia and reperfusion in rodents, swine, sheep and dogs and may be a useful medication in horses. HYPOTHESIS: Ethyl pyruvate has no adverse effects in normal horses and is biologically active based on suppression of proinflammatory gene expression in endotoxin stimulated whole blood, in vitro. METHODS: Physical and neurological examinations, behaviour scores, electrocardiograms and clinicopathological tests were performed on 5 normal healthy horses receiving 4 different doses of ethyl pyruvate. Doses included 0, 50, 100 and 150 mg/kg bwt administered in a randomised crossover design with a 2 week washout period between doses. Biological efficacy was assessed by stimulating whole blood with endotoxin from the horses that received ethyl pyruvate prior to and 1 and 6 h after drug infusion. Gene expression for TNFα, IL-1ß and IL-6 was assessed. RESULTS: There were no effects of drug or dose (0, 50, 100 or 150 mg/kg bwt) on any of the physical or neurological examination, behaviour factors, electrocardiogram or clinical pathological results collected from any of the horses. All parameters measured remained within the normal reference range. There was a significant reduction in TNFα, IL-1ß and IL-6 gene expression in endotoxin stimulated whole blood from horses 6 h after receiving 150 mg/kg bwt ethyl pyruvate. There were no detectable effects on gene expression of any of the other doses of ethyl pyruvate tested. CONCLUSION: We were unable to detect any detrimental effects of ethyl pyruvate administration in normal horses. Ethyl pyruvate significantly decreased proinflammatory gene expression in endotoxin stimulated blood 6 h after drug administration. CLINICAL RELEVANCE: Ethyl pyruvate may be a safe, effective medication in endotoxaemic horses.

Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period.

The incidence and severity of mastitis can be high during the period of transition from pregnancy to lactation when dairy cattle are susceptible to oxidative stress. Oxidative stress may contribute to the pathogenesis of mastitis by modifying the expression of proinflammatory genes. The overall goal of this study was to determine the relationship between critical antioxidant defense mechanisms and proinflammatory markers in normal bovine mammary tissue during the periparturient period. Mammary tissue samples were obtained from 12 cows at 35, 20, and 7 d before expected calving and during early lactation (EL, 15 to 28 d in milk). Enzyme activities for cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase were relatively low during the dry period, but increased during EL, whereas activity of thioredoxin reductase 1 did not change significantly as a function of time. In contrast, gene expression for these antioxidant selenoproteins and for heme oxygenase-1 gradually decreased as parturition approached and then increased during EL. The expression of intercellular vascular adhesion molecule-1 and vascular cell adhesion molecule-1 followed a similar trend where mRNA abundance gradually declined as parturition approached with a slight rebound in EL. Gene expression of the pro-oxidant, 15-lipoxygenase 1, which is known to increase during times of oxidative stress, also increased dramatically in mammary tissue from EL cows. Expression of the proinflammatory cytokines, IL-1beta, IL-6, and IL-8 did not change significantly during the periparturient period. Strong positive correlations were found between several antioxidant enzymes (cytosolic glutathione peroxidase, thioredoxin reductase 1, and heme oxygenase-1) and vascular adhesion molecules (intercellular vascular adhesion molecule-1, vascular cell adhesion molecule-1) suggesting a protective response of these antioxidants to an enhanced proinflammatory state. Ability to control oxidative stress through manipulation of key antioxidant enzymes in the future may modify the proinflammatory state of periparturient cows and reduce incidence and severity of some diseases such as mastitis.

Platelet activating factor production and proinflammatory gene expression in endotoxin-challenged bovine mammary endothelial cells.

The bovine mammary gland responds to gram-negative pathogens by stimulating the production of cytokines and other proinflammatory mediators that orchestrate the migration of leukocytes into tissues. Platelet activating factor (PAF), interleukin 1 beta (IL-1 beta), IL-8, and intercellular adhesion molecule 1 (ICAM1) are among the several inflammatory factors involved in the early activation and migration of leukocytes into the mammary gland during the initial stages of coliform mastitis. Several different cell types within the mammary gland are capable of expressing these potent pro-inflammatory mediators. The objective of this study was to characterize the expression profile of vascular-derived inflammatory molecules that may play a role in the pathogenesis of bovine coliform mastitis. Isolated bovine mammary gland endothelial cells were stimulated in culture for up to 12 h with endotoxin obtained from Escherichia coli, and the temporal expression of proinflammatory cytokines and adhesion molecules relative to endogenous PAF biosynthesis was evaluated. Results from the in vitro time course experiment showed that vascular-derived PAF biosynthesis began as early as 30 min and peaked at 1 h following endotoxin challenge. The biosynthesis of PAF preceded the endotoxin-induced IL-1 beta, IL-8, and ICAM1 mRNA expression that increased after 1 h and reached peak expression between 4 and 12 h following stimulation. Inhibiting the effects of endogenous PAF with a receptor antagonist suggests that vascular-derived PAF is an early proinflammatory mediator that plays at least a partial role in the subsequent expression of IL-1 beta, IL-8, and ICAM1 during endotoxin challenge. Furthermore, endotoxin-induced PAF biosynthesis by bovine mammary gland endothelial cells is regulated to some extent by phospholipase D activity and phosphatidic acid production. The results from this study support the contention that mammary gland endothelial cells can contribute to the production of important proinflammatory mediators that are typically associated with coliform mastitis.

Shifts in thioredoxin reductase activity and oxidant status in mononuclear cells obtained from transition dairy cattle.

Measures of oxidative status were examined in 14 dairy cows during the transition period. Blood samples were obtained approximately 21 d before expected calving, at calving, and again at 21 d in milk (DIM). Plasma samples were used to determine lipid hydroperoxide concentrations. Total white blood cells were used to determine the oxidative status of glutathione. Peripheral blood mononuclear cell (PBMC) lysates were used to determine the total antioxidant potential and enzymatic activities of glutathione peroxidase (GPX) and thioredoxin reductase (TrxR1). Both plasma lipid hydroperoxide concentrations and GPX activity in PBMC increased at calving and during the first 21 DIM when compared with prepartum samples. Conversely, the total antioxidant potential and TrxR activity declined in PBMC during the first 21 DIM, even though both GPX activity and the glutathione-to-GSSG ratio remained elevated during this time period. Results from this study support previous findings that report increased GPX activity when reactive oxygen metabolites, including lipid hydroperoxides, increase in transition dairy cows. The significant decrease in TrxR activity with a concomitant decrease in total antioxidant potential in PBMC during this same stage of lactation, however, would suggest that this selenoprotein is not able to rebound during periods of oxidative stress to the same extent as GPX1. This study shows for the first time that TrxR may be an important antioxidant defense mechanism in PBMC that is compromised during the periparturient period.