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.

Vitamin E analogs limit in vitro oxidant damage to bovine mammary endothelial cells.

Diseases that occur during the transition period are exacerbated when cows are unable to cope with an increased pro-oxidant load that results in oxidative stress. Dairy cattle are routinely supplemented with the vitamin E analog α-tocopherol to mitigate the severity of oxidative stress. Nonetheless, oxidative stress remains a disease predisposing condition for many dairy cattle. A better method of optimizing the antioxidant functions of vitamin E is needed. α-Tocopherol is only 1 of 8 analogs of vitamin E, all of which have varying antioxidant properties in other mammals, albeit a shorter physiological half-life compared with α-tocopherol. A primary bovine mammary endothelial cell oxidant challenge model was used to determine functions of certain vitamin E analogs. The aim of this study was to determine if other analogs, namely γ-tocopherol or γ-tocotrienol, have antioxidative functions in bovine cells and if these functions may protect cellular viability and endothelial function from oxidant damage. Physiological (10 µM) and supraphysiological (50 µM) concentrations of γ-tocopherol and γ-tocotrienol had a greater capacity to reduce accumulated reactive oxygen species derived from a nitric oxide donating pro-oxidant antagonist, when compared with α-tocopherol, after 30 min to 6 h of treatment. Further, γ-tocotrienol (10 µM) decreased cell cytotoxicity to a greater amount than other analogs at like concentrations, whereas γ-tocopherol (10 µM) reduced lipid peroxidation and apoptosis more effectively than other analogs. Last, α-tocopherol (5 and 10 µM) and γ-tocopherol (5 and 10 µM) significantly slowed pro-oxidant induced loss of endothelial cell barrier integrity over a 48-h period using an electrical cell-substrate impedance sensing system. Concerningly, γ-tocotrienol drastically reduced the endothelial barrier integrity at only 5 µM despite no apparent effect on cellular viability at like concentrations. γ-Tocotrienol, however, was also the only analog to show significant cytotoxicity and reductions in viability at supraphysiological doses (25 and 50 µM). Our results suggest that γ-tocopherol has antioxidant activities that reduces cellular damage and loss of function due to oxidant challenge as effectively as α-tocopherol. These data set the foundation for further investigation into the antioxidant properties of vitamin E analogs in other bovine cells types or whole animal models.

Invited review: Cytochrome P450 enzyme involvement in health and inflammatory-based diseases of dairy cattle.

Dairy cattle are at the greatest risk of developing diseases around the time of calving because of compromised immune responses and the occurrence of oxidative stress. Both the development of compromised immunity and oxidative stress are influenced directly or indirectly by the metabolism of polyunsaturated fatty acids (PUFA) and fat-soluble vitamins. The cytochrome P450 (CYP450) family of enzymes is central to the metabolism of both classes of these compounds, but to date, the importance of CYP450 in the health of dairy cattle is underappreciated. As certain CYP450 isoforms metabolize both PUFA and fat-soluble vitamins, potential interactions may occur between PUFA and fat-soluble vitamins that are largely unexplored. For example, one CYP450 that generates anti-inflammatory oxylipids from arachidonic acid additionally contributes to the activation of vitamin D. Other potential substrate interactions between PUFA and vitamins A and E may exist as well. The intersection of PUFA and fat-soluble vitamin metabolism by CYP450 suggest that this enzyme system could provide an understanding of how immune function and oxidant status interconnect, resulting in increased postpartum disease occurrence. This review will detail the known contributions of bovine CYP450 to the regulation of oxylipids with a focus on enzymes that may also be involved in the metabolism of fat-soluble vitamins A, D, and E that contribute to antioxidant defenses. Although the activity of specific CYP450 is generally conserved among mammals, important differences exist in cattle, such as the isoforms primarily responsible for activation of vitamin D that makes their specific study in cattle of great importance. Additionally, a CYP450-driven inflammatory positive feedback loop is proposed, which may contribute to the dysfunctional inflammatory responses commonly found during the transition period. Establishing the individual enzyme isoform contributions to oxylipid biosynthesis and the regulation of vitamins A, D, and E may reveal how the CYP450 family of enzymes can affect inflammatory responses during times of increased susceptibility to disease. Determining the potential effect of each CYP450 on disease susceptibility or pathogenesis may allow for the targeted manipulation of the CYP450 pathways to influence specific immune responses and antioxidant defenses during times of increased risk for health disorders.

Widespread basal cytochrome P450 expression in extrahepatic bovine tissues and isolated cells.

Periparturient cattle face increased risk of both metabolic and infectious diseases. Factors contributing to this predisposition include oxidized polyunsaturated fatty acids, also known as oxylipids, whose production is altered during the periparturient period and in diseased cattle. Alterations in the production of oxylipids derived from cytochrome P450 (CYP450) enzymes are over-represented during times of increased disease risk and clinical disease, such as mastitis. Many of these same CYP450 enzymes additionally regulate metabolism of fat-soluble vitamins, such as A, D, and E. These vitamins are essential to maintaining immune health, yet circulating concentrations are diminished near calving. Despite this, a relatively small amount of research has focused on the roles of CYP450 enzymes outside of the liver. The aim of this paper is to describe the relative gene expression of 11 CYP450 in bovine tissues and common in vitro bovine cell models. Eight tissue samples were collected from 3 healthy dairy cows after euthanasia. In vitro samples included primary bovine aortic and mammary endothelial cells and immortalized bovine kidney and mammary epithelial cells. Quantitative real-time-PCR was carried out to assess basal transcript expression of CYP450 enzymes. Surprisingly, CYP450 mRNA was widely expressed in all tissue samples, with predominance in the liver. In vitro CYP450 expression was less robust, with several cell types lacking expression of specific CYP450 enzymes altogether. Overall, cell culture models did not reflect expression of tissue CYP450. However, when CYP450 were organized by activity, certain cell types consistently expressed specific functional groups. These data reveal the widespread expression of CYP450 in individual organs of healthy dairy cows. Widespread expression helps to explain previous evidence of significant changes in CYP450-mediated oxylipid production and fat-soluble vitamin metabolism in organ microenvironments during periods of oxidative stress or disease. As such, these data provide a foundation for targeted functional experiments aimed at understanding the activities of specific CYP450 and associated therapeutic potential during times of increased disease risk.

Changes in bovine leukemia virus serological status and lymphocyte count between dry-off and early lactation in Michigan dairy cows.

This study addresses how the serological status of bovine leukemia virus (BLV) and lymphocyte count fluctuate from dry-off to early lactation in dairy cattle. Very few studies have investigated how BLV antibody status and lymphocyte count of cows changes longitudinally during the lactation cycle. Blood samples were collected from dairy cattle (n = 149) on 5 commercial dairy herds in Michigan at dry-off, close-up, and 7 to 10 d after calving. Plasma was analyzed for anti-BLV antibodies using a BLV-ELISA and whole blood was analyzed for lymphocyte counts. We found that BLV seroprevalence increased from dry-off (38.9%) to close-up (43.6%), then slightly decreased from close-up to 7 to 10 d after calving (43.0%). However, the change in seroprevalence was only significant from dry-off to close-up. Cows of third or higher parity were more likely to seroconvert than cows of lower parity and had the highest ELISA-negative prevalence of BLV. Lymphocyte counts were significantly higher in ELISA-positive animals, but only among second and third or greater parity animals. These results indicate that the use of lymphocyte counts as a disease severity monitoring tool for BLV should differ by parity group. Future studies should investigate if changes in seroprevalence are due to new infections or natural changes in antibody concentrations as the cow prepares for colostrum production. More accurate lymphocyte guidelines to be used for monitoring the progression of BLV should be created that consider parity and lactation stage.

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.

Diet starch concentration and starch fermentability affect markers of inflammatory response and oxidant status in dairy cows during the early postpartum period.

Our objective was to evaluate the effects of diet starch concentration and starch fermentability on inflammatory response markers and oxidant status during the early postpartum (PP) period and its carryover effects. Fifty-two multiparous Holstein cows were used in a completely randomized block design experiment with a 2 × 2 factorial arrangement of treatments. Treatments were starch concentration and starch fermentability of diets; diets were formulated to 22% (low starch, LS) or 28% (high starch, HS) starch with dry-ground corn (DGC) or high-moisture corn (HMC) as the primary starch source. Treatments were fed from 1 to 23 d PP and then switched to a common diet until 72 d PP to measure carryover (CO) effects. Treatment period (TP) diets were formulated to 22% forage neutral detergent fiber and 17% crude protein. The diet for the CO period was formulated to 20% forage neutral detergent fiber, 17% crude protein, and 29% starch. Coccygeal blood was collected once a week during the TP and every second week during the CO period. Liver and adipose tissue biopsies were performed within 2 d PP and at 20 ± 3 d PP. Blood plasma was analyzed for concentrations of albumin, haptoglobin, reactive oxygen and nitrogen species (RONS), and antioxidant potential (AOP), with lipopolysaccharide-binding protein (LBP) and TNFα evaluated during the TP only. Oxidative stress index (OSi) was calculated as RONS/AOP. Abundance of mRNA from genes involved in inflammation and glucose metabolism in liver and genes involved in lipogenesis in adipose tissue were determined. Data were analyzed separately for the TP and CO periods. During the TP, treatments interacted to affect concentrations of TNFα, haptoglobin, and LBP, with HMC increasing their concentrations for HS (9.38 vs. 7.45 pg/mL, 0.45 vs. 0.37 mg/mL, and 5.94 vs. 4.48 µg/mL, respectively) and decreasing their concentrations for LS (4.76 vs. 12.9 pg/mL, 0.27 vs. 0.41 mg/mL, and 4.30 vs. 5.87 µg/mL, respectively) compared with DGC. Effects of treatments diminished over time for LBP and haptoglobin with no differences by the end of the TP and no main CO effects of treatment for haptoglobin. The opposite treatment interaction was observed for albumin, with HMC tending to decrease its concentration for HS (3.24 vs. 3.34 g/dL) and increase its concentration for LS (3.35 vs. 3.29 g/dL) compared with DGC, with no carryover effect. Feeding DGC increased the OSi during the first week of the TP compared with HMC, with this effect diminishing over time; during the CO period HMC increased OSi for HS and decreased it for LS compared with DGC, with this effect diminishing toward the end of CO. Feeding HMC increased the abundance of genes associated with inflammation and gluconeogenesis in liver for HS and decreased it for LS compared with DGC. Feeding HS increased the mRNA abundance of genes associated with adipose tissue lipogenesis compared with LS. Results during the TP suggest that feeding LS-DGC and HS-HMC elicited a more pronounced inflammatory response and induced an upregulation of genes associated with inflammation and gluconeogenesis in liver, without effects on OSi, but effects on plasma markers of inflammation diminished during the CO period.

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.

Reduced serum vitamin D concentrations in healthy early-lactation dairy cattle.

Cattle obtain vitamin D by ingestion or cutaneous exposure to UV light. Dairy cattle diets are frequently supplemented with vitamin D to compensate for limited sun exposure or during times of increased metabolic demands, such as the periparturient period, to maintain calcium homeostasis. Whether housing and supplemental vitamin D practices supply adequate amounts of vitamin D to optimally support the transition from gestation to lactation in dairy cattle is unknown. Our objective was to determine how serum vitamin D concentrations of dairy cows change with season, age, parity, and stage of lactation. Clinically healthy cows (n = 183) from 5 commercial dairies were enrolled in the study. Serum samples were collected at dry off, within 7 d of entering the close-up group, and within 7 d after calving (calving+7). Vitamin D status was determined by measuring serum 25-hydroxyvitamin D [25(OH)D] by radioimmunoassay. We performed repeated-measures mixed-effects linear regression to determine the effects of season, age, parity, and lactation stage (dry off, close-up, and calving+7) on 25(OH)D concentrations in serum. Bivariable analysis indicated that parity, age, and season were not associated with serum 25(OH)D concentrations. Sample period affected 25(OH)D concentrations, with the highest 25(OH)D levels at dry off (99.7 ± 1.9 ng/mL) followed by close up (93.8 ± 2.1 ng/mL), with the lowest levels at calving+7 (82.6 ± 1.7 ng/mL). These data showed a large depletion of 25(OH)D in dairy cattle postpartum compared with late prepartum, although the biological significance of this change in these healthy cattle is unclear. Consumption of serum 25(OH)D by immune system functions and calcium homeostasis in early lactation likely caused the reduction in serum 25(OH)D concentrations after calving. These results suggest that determining whether serum 25(OH)D concentrations are associated with the incidence of transition period disease is an appropriate next step. Assessing the effects of enhanced vitamin D supplementation of cows in early lactation on postpartum diseases may be warranted.

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.

Supplementation of linoleic acid (C18:2n-6) or α-linolenic acid (C18:3n-3) changes microbial agonist-induced oxylipid biosynthesis.

Oxylipids are derived from polyunsaturated fatty acids (PUFA) in cellular membranes and the relative abundance or balance may contribute to disease pathogenesis. Previous studies documented unique oxylipid profiles from cows with either coliform or Streptococcus uberis mastitis, suggesting that lipid mediator biosynthesis may be dependent on the type of microbial-derived agonist. Changing the fatty acid content of peripheral blood leukocytes also may be critical to the relative expression of oxylipid profiles and the outcome of bacterial infection. No information is available in dairy cows describing how changing cellular PUFA content will modify oxylipids in the context of a microbial agonist challenge. Therefore, the hypothesis for the current study was that PUFA supplementation would change bovine leukocyte fatty acid content and respective oxylipid profiles from ex vivo microbial agonist-challenged leukocytes. Fatty acid content of leukocytes and plasma was quantified in (1) samples from cows not supplemented with PUFA, (2) cows supplemented with linoleic acid (LnA), and (3) cows supplemented with α-linolenic acid (ALA). Plasma oxylipids were assessed after S. uberis or lipopolysaccharide exposure and was compared with unstimulated oxylipid profiles. Fatty acid supplementation with ALA significantly increased ALA content of blood leukocytes and plasma relative to LnA. Fatty acid supplementation affected several S. uberis-induced oxylipids, but only S. uberis-induced 15-oxoETE was greater with ALA supplementation compared with LnA. Notably, only LPS-induced 5,6 LXA4 was altered with fatty acid supplementation, but no significant effect of LnA vs. ALA treatment was identified. Future studies are needed to understand how leukocyte activation and membrane PUFA availability collectively contribute to differential oxylipid profiles.

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows.

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d -6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d -6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na+ and K+) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na+ and K+ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.

Nutritional strategies to optimize dairy cattle immunity.

Dairy cattle are susceptible to increased incidence and severity of both metabolic and infectious diseases during the periparturient period. A major contributing factor to increased health disorders is alterations in bovine immune mechanisms. Indeed, uncontrolled inflammation is a major contributing factor and a common link among several economically important infectious and metabolic diseases including mastitis, retained placenta, metritis, displaced abomasum, and ketosis. The nutritional status of dairy cows and the metabolism of specific nutrients are critical regulators of immune cell function. There is now a greater appreciation that certain mediators of the immune system can have a reciprocal effect on the metabolism of nutrients. Thus, any disturbances in nutritional or immunological homeostasis can provide deleterious feedback loops that can further enhance health disorders, increase production losses, and decrease the availability of safe and nutritious dairy foods for a growing global population. This review will discuss the complex interactions between nutrient metabolism and immune functions in periparturient dairy cattle. Details of how either deficiencies or overexposure to macro- and micronutrients can contribute to immune dysfunction and the subsequent development of health disorders will be presented. Specifically, the ways in which altered nutrient metabolism and oxidative stress can interact to compromise the immune system in transition cows will be discussed. A better understanding of the linkages between nutrition and immunity may facilitate the design of nutritional regimens that will reduce disease susceptibility in early lactation cows.

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.

Cabergoline inhibits prolactin secretion and accelerates involution in dairy cows after dry-off.

Dairy cattle require a dry period between successive lactations to ensure optimal milk production. Because prolactin (PRL) is necessary for the initiation and maintenance of milk production, strategies that can inhibit PRL secretion might hasten the involution process. The objective of this study was to determine the effect of the PRL release inhibitor cabergoline on markers of mammary gland involution during the early dry period. To assess the effect of cabergoline treatment on mammary gland involution, 14 Holstein dairy cows in late lactation were treated with either a single i.m. administration of 5.6mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n=7) or placebo (n=7) at the time of dry-off. Blood samples and mammary secretion samples were collected 6d before dry-off and again 1, 2, 3, 4, 8, and 14d following the abrupt cessation of lactation. Blood samples were used to determine plasma PRL concentrations. Mammary secretion samples were used to determine somatic cell count, milk fat, lactose, true protein content, and concentrations of α-lactalbumin, lactoferrin, and citrate. Following the cessation of lactation, changes in mammary secretion composition indicated diminished milk synthesis, including reduced concentrations of α-lactalbumin, citrate, and lactose. In contrast, milk somatic cell count, percent total protein, percent fat content, and lactoferrin concentrations significantly increased as involution progressed. Cabergoline treatment decreased the plasma PRL concentrations during the first week of dry-off, compared with the control treatment. No significant differences in citrate, α-lactalbumin, or protein content were observed between treatment groups. The most dramatic changes in secretion composition as a consequence of cabergoline treatment occurred during the first week of the dry period, when lactose concentrations and the citrate:lactoferrin molar ratio were lower and lactoferrin concentrations higher than in the control cows. Cabergoline treatment also tended to increase fat content and somatic cell count more rapidly following dry-off compared with the control group. These changes in mammary secretion composition following the abrupt cessation of lactation indicate that cabergoline treatment facilitated dry-off and effectively accelerated mammary gland involution.

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.

Short communication: Characterizing metabolic and oxidant status of pastured dairy cows postpartum in an automatic milking system.

The periparturient period represents a stressful time for dairy cows as they transition from late gestation to early lactation. Undesirable fluctuations in metabolites and impaired immune defense mechanisms near parturition can severely affect cow health and have residual effects on performance and longevity. Metabolic and oxidative stress profiles of multiparous and primiparous dairy cows in traditional parlor and feeding systems are well characterized, but status of these profiles in alternative management systems, such as grazing cows managed with an automatic milking system (AMS), are poorly characterized. Therefore, the objective of this case study was to characterize the metabolic and oxidant status of pastured cows milked with an AMS. It was hypothesized that primiparous and multiparous cows milked with an AMS would experience changes in oxidative and metabolic status after parturition; however, these changes would not impair cow health or production. Blood was collected from 14 multiparous and 8 primiparous Friesian-cross dairy cows at 1, 7, 14, and 21 d relative to calving for concentrations of insulin, glucose, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, reduced glutathione, oxidized glutathione, and antioxidant potential. Milk production and milking frequency data were collected postpartum. Milk production differed on d 7 and 14 between primiparous and multiparous cows and frequency was not affected by parity. Primiparous cows had higher levels of glucose than multiparous cows. No differences in insulin, NEFA, or ß-hydroxybutyrate concentrations were noted between multiparous and primiparous cows postpartum, though days relative to calving significantly affected insulin and NEFA. Primiparous cows also had higher antioxidant potential than multiparous cows during the postpartum period. Results from this study show that, although responses were within expected ranges, periparturient multiparous cows responded differently than periparturient primiparous cows with respect to metabolic and oxidative measures during the postpartum period at this pastured-AMS dairy, suggesting different management strategies may need to be considered with primiparous and multiparous cows.

Association between polyunsaturated fatty acid-derived oxylipid biosynthesis and leukocyte inflammatory marker expression in periparturient dairy cows.

Peripheral blood mononuclear leukocytes from periparturient cows can have exacerbated inflammatory responses that contribute to disease incidence and severity. Oxylipids derived from the oxygenation of polyunsaturated fatty acids (PUFA) can regulate the magnitude and duration of inflammation. Although PUFA substrate for oxylipid biosynthesis in leukocytes is known to change across the periparturient period, the plasma oxylipid profile and how this profile relates to leukocyte inflammatory phenotype is not clear. The objective of this study was to determine if a relationship exists between the profile of pro- and antiinflammatory plasma oxylipids and the inflammatory phenotype of peripheral blood leukocytes during the periparturient period. Seven multiparous Holsteins were sampled from the prepartum period through peak lactation. Plasma oxylipids were measured by liquid chromatography-mass spectrometry, peripheral leukocyte mRNA expression was measured by quantitative PCR, and PUFA content of peripheral blood mononuclear cells was measured by gas chromatography-mass spectrometry. Concentrations of several hydroxyl products of linoleic and arachidonic acid changed over time. Linoleic acid and arachidonic acid concentrations in leukocytes increased during early lactation, suggesting that substrate availability for hydroxyoctadecadienoic and hydroxyeicosatetraenoic acid biosynthesis may influence the oxylipid profile. Leukocyte mRNA expressions of IL-12B, IL-1B, inducible nitric oxide synthase 2, and cyclooxygenase 2 were correlated with several plasma oxylipids. These are the first observations linking leukocyte inflammatory gene responses to shifts in oxylipid biosynthesis in periparturient dairy cows.

Comparison of supplementation of n-3 fatty acids from fish and flax oil on cytokine gene expression and growth of milk-fed Holstein calves.

The ability to reduce incidence of disease in calves and improve early vaccination strategies is of particular interest for dairy producers. The n-3 fatty acids have been reported to reduce inflammatory diseases in humans but limited research has been done in calves. The objective of this study was to compare supplementation of n-3 fatty acids from fish and flax oil on gene expression of whole blood cells and growth of milk-fed Holstein calves. Forty-eight Holstein bull calves from a commercial dairy were randomly assigned to 1 of 3 diets beginning at 4d old: (1) control milk replacer (MR) with all pork fat, (2) MR with 2% flax oil, and (3) MR with 2% fish oil. All MR were 17% fat, 27% crude protein on a dry matter (DM) basis, with all protein from whey sources. Calves were each fed 654g DM of MR daily for the first 25d and then 327g/d for d26, 27, and 28. On d28, calves were challenged with a Pasteurella vaccine and the temperature response to the vaccine was recorded. Milk and feed intake and fecal scores were recorded daily, and body weight and hip width were recorded weekly. Blood was collected from all calves on d25. One tube of collected blood was incubated with endotoxin (lipopolysaccharide; LPS) for 2h and frozen with a second tube of control blood. Quantitative real-time PCR was used to assess the effects of LPS stimulation on cytokine gene expression. During the 28 d, calves supplemented with flax oil had a greater growth rate and feed efficiency than calves fed fish oil (0.52±0.02 vs. 0.48±0.02g of gain:g of feed). Fish oil tended to decrease LPS stimulation of tumor necrosis factor-α expression. Flax oil, but not fish oil, decreased the expression of IL-4 and tended to decrease expression of osteopontin and IL-8. Flax oil tended to reduce the increase in rectal temperature in response to a Pasteurella vaccine. In conclusion, our data support the idea that supplementation with n-3 fatty acids affects cytokine gene expression.