Genetic variation in bovine mononuclear leukocyte responses to dexamethasone

The objective of this study was to determine whether bovine mononuclear leukocytes exhibit genetic variability prior to and after a glucocorticoid hormone challenge in vivo. Test animals included 60 pedigreed Holstein bulls treated on 3 consecutive days with dexamethasone and 5 untreated control bulls. Eight indicator traits of leukocyte responsiveness to dexamethasone included the percentages of circulating B cells, T cells (CD4, CD8, and workshop cluster 1 molecule expressed by bovine gammadelta T cell), major histocompatibility complex (MHC) I and II expressing cells, and mean expressions of surface MHC I and MHC II on circulating cells. Blood for this work was collected from each test bull 10 times before, during, and after dexamethasone administration, with corresponding samples taken for control bulls. Random regression models with treatment-specific serial correlation were applied to the leukocyte data sets to estimate genetic and nongenetic sources of variation in baseline and recovery aspects of the traits. All traits responded predictably to glucocorticoid challenge. Genetic variation was observed in baseline measurements of all traits, with heritability estimates ranging from 0.21 +/- 0.03 to 0.60 +/- 0.06. Genetic variation in linear recovery from nadir values following dexamethasone administration was significant only for percentage CD4, percentage CD8, and for surface expression of MHC II. The genetic covariance between basal and linear recovery was positive and significant for percentage CD4, percentage CD8, and MHC II expression. The bovine lymphocyte antigen DRB3.2 locus accounted for significant proportions of total variation in percentage MHC II cells and MHC I expression. These results suggest that genetic variability exists for important basal and glucocorticoid-modified phenotypes of bovine mononuclear leukocytes, implying that immunocompetence traits impacted by this stress hormone may be enhanced by genetic selection.     

L-selectin and beta2-integrin expression on circulating bovine polymorphonuclear leukocytes during endotoxin mastitis

The aim of this in vivo study was to examine the effect of intramammarily administered endotoxin (lipopolysaccharide, LPS) on the expression of L-selectin (CD62L) and the beta2-integrin subunits CD11b and CD18 on circulating bovine PMN. Six early lactating cows were infused with Escherichia coli LPS. The adhesion molecules under study were stained at the cell surface and analyzed flow cytometrically. In addition, some of the clinical parameters associated with adhesion molecule mobilization such as fever, blood cortisol levels, somatic cell count (SCC), and total and differential blood leukocyte count were measured. In analogy with observations during clinical coliform mastitis, a progressive decrease of CD62L expression levels was observed early after LPS infusion, concomitantly with a continuous rise of CD11b and CD18 density. However, no correlation was found between the kinetics of CD11b and CD18 density. The initial changes in adhesion molecule expression paralleled the decrease in blood PMN numbers, together with the increase in rectal temperature, cortisol levels, SCC, and number of circulating immature PMN. In conclusion, intramammarily administered LPS seems to play an important role in modulating adhesion receptor expression on circulating bovine PMN. Interestingly, in contrast to coliform mastitis, the net CD18 variation is not principally influenced by CD11b upregulation during endotoxin administration. The knowledge of adhesion molecule kinetics in relation to the different parameters evaluated in the present study contributes to an improved understanding of the inflammatory reaction.     

The class II major histocompatibility complex molecule BoLA-DR is expressed by endothelial cells of the bovine corpus luteum

Cells expressing class II major histocompatibility complex (MHC) molecules are found within the corpus luteum (CL) of several species. Expression and localization of class II MHC molecules in the bovine CL were examined in the present study. Immunohistochemical evaluation revealed class II MHC molecules on single cells in early CL (days 4 and 5 post-estrus). Two class II MHC-expressing cell types were observed in midcycle CL (days 10-12 post-estrus), single cells similar to those observed in the early CL, and endothelial cells. Not all endothelial cells expressed class II MHC, and further investigation revealed expression of only one type of class II MHC molecule, DR, on endothelial cells. Class II MHC was also localized to endothelial cells in late CL (day 18 post-estrus). Steroidogenic luteal cells were negative for class II MHC throughout the estrous cycle. Quantitative RT-PCR revealed higher (P < 0.05) concentrations of mRNA encoding the alpha-subunit of DR (DRA) in late CL when compared with those in the early CL. DRA mRNA abundance was also measured in cultures of mixed luteal and luteal endothelial (CLENDO) cells, in the presence or absence of tumor necrosis factor-alpha (TNF). No differences were found in the DRA mRNA concentration between mixed luteal and CLENDO cell cultures, and TNF had no effect on DRA mRNA concentration in both cell types. Expression of DR by endothelial cells of the midcycle CL may induce anergy of T lymphocytes, or stimulate them to secrete products that enhance normal luteal function.     

Effects of treatment of preweaning dairy calves with recombinant bovine somatotropin on immune responses and somatotropic axis

Weaning may be associated with negative energy balance and body weight loss when calves are still immunologically immature, predisposing them to infectious diseases. The aim of the present experiment was to investigate the effects of treatment of preweaning dairy calves with recombinant bovine somatotropin (rbST) on the somatotropic axis, selected immune parameters, and hematology of calves around weaning. Thirty-six Holstein female calves were randomly assigned to receive 1.5 to 1.8 mg of rbST (Posilac, Elanco Animal Health, Greenfield, IN) per kilogram of body weight or to receive injections of saline (saline solution 0.9%, Valley Vet Supply, Marysville, KS) every 7 d from 21 to 63 d of life. Calves were fed milk replacer ad libitum from birth to 38 d of age (d ?11), when progressive weaning started, and calves were weaned at 49 d of age (d 0). Calves were weighed at birth and weekly from 21 to 63 d of age, when wither height also was measured. Calves were vaccinated with 0.5 mg of ovalbumin on study d ?28 and ?7. Blood samples were collected on d ?28, ?25, ?21, ?11, 0, 3, 7, and 14. Polymorphonuclear leukocytes were isolated and challenged ex vivo with Escherichia coli to determine phagocytosis and oxidative burst capacity. Additionally, expression of cluster of differentiation (CD)62L and CD18 by granulocyte, lymphocyte, and CD14+ monocyte were determined. Blood samples were also used to determine hematological parameters and concentrations of growth hormone, insulin-like growth factor-1, insulin, glucose, fatty acids, β-hydroxybutyrate, haptoglobin, and anti-ovalbumin IgG. Calves treated with rbST had greater concentrations of growth hormone and insulin-like growth factor-1 from d ?25 to 14 than control calves, whereas insulin, fatty acid, and β-hydroxybutyrate concentrations did not differ. On d ?11, glucose concentration was greater for rbST-treated calves. Treatment did not affect polymorphonuclear lymphocyte phagocytosis and oxidative burst, but intensity of expression of CD62L and CD18 by granulocytes tended to be increased by rbST treatment. Treatment did not affect the concentration of anti-ovalbumin IgG in serum. Haptoglobin concentration was reduced in rbST treated calves on d 3 and we noted a tendency for hematocrit to be lower in rbST-treated calves. Treatment did not affect body weight, wither height, and average daily gain, despite the fact that rbST-treated calves had lower daily milk replacer intake. The relatively minor improvements in immune responses resulting from rbST treatment of weaning calves may not be sufficient to reduce the incidence of infectious diseases.     

Decreased insulin response in dairy cows following a four-day fast to induce hepatic lipidosis

Negative energy balance has been implicated in the development of fatty liver, insulin resistance, and impaired health in dairy cows. A 4-d fasting model previously was reported to increase liver triglycerides more than 2.5-fold. The purpose of the present study was to evaluate insulin response in this fasting model. Nonlactating, nonpregnant Holstein cows were fasted for 4 d (6 cows) or fed continuously as control cows (4 cows). Samples were collected 5 d before fasting, during fasting, and immediately after the 4-d fast, 8 d after the fast, and 16 d after the fast. Fasted cows had greater liver triglyceride content (49.4 vs. 16.2 mg/g, wet-weight basis) at the end of the fasting period compared with control cows. Fasted cows also had increased plasma nonesterified fatty acid (NEFA) concentrations (1.24 vs. 0.21 mmol/L) and increased plasma β-hydroxybutyrate (BHBA) concentrations at the end of the fasting period. Liver triglyceride, plasma NEFA, and plasma BHBA in fasted cows returned to prefasting concentrations by the end of the experiment. Plasma glucose concentrations were not affected by fasting. Plasma insulin concentrations were decreased (6.3 vs. 14.1 μU/mL) and insulin-stimulated blood glucose reduction was decreased (24.9 vs. 48.6%) in the fasted cows compared with control cows at the end of the fast, indicating reduced insulin response. Insulin response was negatively correlated with plasma NEFA and liver triglycerides. Decreased insulin response may be an important complication of negative energy balance and hepatic lipidosis.     

Use of milk fatty acids to estimate plasma nonesterified fatty acid concentrations as an indicator of animal energy balance

Negative energy balance is an important part of the lactation cycle, and measuring the current energy balance of a cow is useful in both applied and research settings. The objectives of this study were (1) to determine if milk fatty acid (FA) proportions were consistently related to plasma nonesterified fatty acids (NEFA); (2) to determine if an individual cow with a measured milk FA profile is above or below a NEFA concentration, (3) to test the universality of the models developed within the University of Wisconsin and US Dairy Forage Research Center cows. Blood samples were collected on the same day as milk sampling from 105 Holstein cows from 3 studies. Plasma NEFA was quantified and a threshold of 600 µEq/L was applied to classify animals above this concentration as having high NEFA (NEFA_high). Thirty milk FA proportions and 4 milk FA ratios were screened to evaluate their capacity to classify cows with NEFA_high according to determined milk FA threshold. In addition, 6 linear regression models were created using individual milk FA proportions and ratios. To evaluate the universality of the linear relationship between milk FA and plasma NEFA found in the internal data set, 90 treatment means from 21 papers published in the literature were compiled to test the model predictions. From the 30 screened milk FA, the odd short-chain fatty acids (C7:0, C9:0, C11:0, and C13:0) had sensitivity slightly greater than the other short-chain fatty acids (83.3, 94.8, 80.0, and 85.9%, respectively). The sensitivities for milk FA C6:0, C8:0, C10:0, and C12:0 were 78.8, 85.3, 80.1, and 83.9%, respectively. The threshold values to detect NEFA_high cows for the last group of milk FA were ≤2.0, ≤0.94, ≤1.4, and ≤1.8 g/100 g of FA, respectively. The milk FA C14:0 and C15:0 had sensitivities of 88.7 and 85.0% and a threshold of ≤6.8 and ≤0.53 g/100 g of FA, respectively. The linear regressions using the milk FA ratios C18:1 to C15:0 and C17:0 to C15:0 presented lower root mean square error (RMSE = 191 and 179 µEq/L, respectively) in comparison with individual milk FA proportions (RMSE = 194 µEq/L), C18:1 to even short-medium-chain fatty acid (C4:0–C12:0) ratio (RMSE = 220 µEq/L), and C18:1 to C14:0 (RMSE = 199 µEq/L). Models using milk FA ratios C18:1 to C15:0 and C17:0 to C15:0 had a better fit with the external data set in comparison with the other models. Plasma NEFA can be predicted by linear regression models using milk FA ratios.     

车前子多糖对骨髓来源树突状细胞表型和吞噬功能的影响

目的:探讨车前子多糖对小鼠骨髓来源树突状细胞(BMDCs)表型和功能的影响。方法:从小鼠骨髓中分离单核细胞,加入细胞因子rmGM-CSF、rmIL-4诱导分化成未成熟树突状细胞,收集细胞加入促成熟刺激剂LPS(阳性对照组)或车前子多糖。倒置显微镜观察树突状细胞的形态,流式细胞术检测成熟树突状细胞的表面标志CD11c和MHCII的表达及吞噬FITC-dextran的变化。结果:经4种不同的车前子多糖作用40h后,显著促进CD11c+MHCII+双阳性细胞的比率,在浓度(0～50μg/ml)范围内呈现剂量依赖性,当浓度高于50μg/ml时,双阳性细胞表达率呈现降低趋势。经多糖作用后吞噬FITC-dextran的能力降低,其中PS3的作用效果最明显,表达PE-CD11c×FITC-dextran的细胞比率只有11.53%。结论:车前子多糖促进CD11c+MHCII+双阳性细胞的比率,降低对FITC-dextran的吞噬能力,初步表明车前子多糖可以促进树突状细胞的成熟。     

Milk fatty acids as possible biomarkers to early diagnose elevated concentrations of blood plasma nonesterified fatty acids in dairy cows

Most cows encounter a state of negative energy balance during the periparturient period, which may lead to metabolic disorders and impaired fertility. The aim of this study was to assess the potential of milk fatty acids as diagnostic tools of detrimental levels of blood plasma nonesterified fatty acids (NEFA), defined as NEFA concentrations beyond 0.6 mmol/L, in a data set of 92 early lactating cows fed a glucogenic or lipogenic diet and subjected to 0-, 30-, or 60-d dry period before parturition. Milk was collected in wk 2, 3, 4, and 8 (n = 368) and blood was sampled weekly from wk 2 to 8 after parturition. Milk was analyzed for milk fatty acids and blood plasma for NEFA. Data were classified as “at risk of detrimental blood plasma NEFA” (NEFA ≥0.6 mmol/L) and “not at risk of detrimental blood plasma NEFA” (NEFA <0.6 mmol/L). Concentrations of 45 milk fatty acids and milk fat C18:1 cis-9-to-C15:0 ratio were subjected to a discriminant analysis. Milk fat C18:1 cis-9 revealed the most discriminating variable to identify detrimental blood plasma NEFA. A false positive rate of 10% allowed us to diagnose 46% of the detrimental blood plasma NEFA cases based on a milk fat C18:1 cis-9 concentration of at least 230 g/kg of milk fatty acids. Additionally, it was assessed whether the milk fat C18:1 cis-9 concentrations of wk 2 could be used as an early warning for detrimental blood plasma NEFA risk during the first 8 wk in lactation. Cows with at least 240 g/kg of C18:1 cis-9 in milk fat had about 50% chance to encounter blood plasma NEFA values of 0.6 mmol/L or more during the first 8 wk of lactation, with a false positive rate of 11.4%. Profit simulations were based on costs for cows suffering from detrimental blood plasma NEFA, and costs for preventive treatment based on daily dosing of propylene glycol for 3 wk. Given the relatively low incidence rate (8% of all observations), continuous monitoring of milk fatty acids during the first 8 wk of lactation to diagnose detrimental blood plasma NEFA does not seem cost effective. On the contrary, milk fat C18:1 cis-9 of the second lactation week could be an early warning of cows at risk of detrimental blood NEFA. In this case, selective treatment may be cost effective.     

Short communication: Effect of a stable pen management strategy for precalving cows on dry matter intake, plasma nonesterified fatty acid levels, and milk production

During the close-up transition period, dairy cows are at risk for negative energy balance due to increasing energy demands and decreasing feed intake. This can result in postparturient health problems and decreased milk production after calving. Cows are frequently regrouped during this period, which can negatively affect feeding and resting behavior. The hypothesis was that housing in a stable pen during the close-up transition period should result in a more settled environment resulting in fewer displacements from the feed bunk, which would result in improved feed intake, energy balance [lower nonesterified fatty acid (NEFA) concentrations], and lactation performance. This study addresses precalving pen grouping strategies, which have the potential to affect feed intake and energy balance. A randomized complete block design with pen as the experimental unit was used to compare a stable (S) housing strategy (cows with similar calving dates added to a precalving pen at once) to the more traditional dynamic (D) housing strategy (cows added up to 2 times per week to a precalving pen). Twice-weekly blood samples were collected for NEFA analysis and cow interactions within the pen were observed. Dry matter intake (DMI), milk production, and postparturient health problems were recorded. Mean DMI for the duration of the 28 d of the study was not different (S: 25.5 ± 1.6 vs. D: 25.7 ± 1.0 kg/d), and when examined over time relative to calving, no treatment by time interaction was observed. Concentrations of NEFA were not different when cows initially entered the pens (S: 0.21 ± 0.10 vs. D: 0.18 ± 0.04 mEq/L) and remained not different for the time intervals closer to calving (d −9 to −14: S: 0.28 ± 0.09 vs. D: 0.21 ± 0.04; d −3 to −6: S 0.36 ± 0.10, D 0.32 ± 0.05 mEq/L). Pen grouping strategy did not affect DMI, plasma NEFA concentrations, or milk production.     

Short communication: effects of nonesterified fatty acids on lymphocyte function in dairy heifers

This in vitro study was performed to assess the effects of various concentrations of nonesterified fatty acids (NEFA) on lymphocyte function of heifers. Nine Holstein heifers were studied. Peripheral blood mononuclear cells were incubated with various concentrations of NEFA (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mmol/L). The mixture of NEFA was represented by C16:0 (30%), C16:1 (5%), C18:0 (15%), C18:1 (45%), and C18:2 (5%). The DNA synthesis was diminished at concentrations of NEFA of 2, 1, and 0.5 mmol/L. The IgM secretion was inhibited at concentrations of NEFA of 2, 1, 0.5, and 0.25 mmol/L. Secretion of IFN-gamma was depressed at concentrations of NEFA of 2, 1, 0.5, 0.25, and 0.125 mmol/L. Increases of plasma NEFA might contribute to explain the higher incidence of infections observed in cows suffering from energy deficit.     

Effects of grain processing and bovine somatotropin on metabolism and ovarian activity of dairy cows during early lactation

This study compared the effects of exogenous bovine somatotropin (bST) on the metabolism and ovarian activity of cows fed diets differing in ruminally degradable starch. Twenty-four multiparous and eight primiparous Holstein cows in early lactation were divided into four groups and fed diets containing 39% grain as steam-flaked sorghum or steam-rolled corn with or without bST for 90 d in a 2 x 2 factorial design. Flaked sorghum improved energy status of cows during early lactation, tending to increase plasma glucose and insulin. Administration of bST decreased plasma urea nitrogen and increased nonesterified fatty acids (NEFA). Plasma levels of beta-hydroxybutyrate (BHBA) and hepatic concentrations of triglycerides were not altered by treatments. Temporal changes in plasma glucose, urea nitrogen, NEFA, and BHBA were detected in a quadratic manner and insulin increased linearly with time, but treatments did not affect postpartum changes in these metabolites. There were greater decreases in body weight and net energy balance in cows on bST during the first 7 wk of treatment. Cows receiving bST took longer to reach the nadir of negative energy balance, and bST tended to delay the period to reach a positive energy balance. Follicular populations and incidence of cystic ovaries were not affected by treatments, but cows receiving bST had fewer double ovulations. Flaked sorghum increased plasma progesterone during the early luteal phase of the first two postpartum estrous cycles. Feeding more ruminally degradable starch improved the energy status and luteal activity of cows in early lactation.     

Dietary energy source in dairy cows in early lactation: metabolites and metabolic hormones

Negative energy balance-related metabolic disorders suggest that the balance between available lipogenic and glucogenic nutrients is important. The objectives of this study were to compare the effects of a glucogenic or a lipogenic diet on liver triacylglycerides (TAG), metabolites, and metabolic hormones in dairy cows in early lactation and to relate metabolite concentrations to the determined energy retention in body mass (ER). Sixteen dairy cows were fed either a lipogenic or glucogenic diet from wk 3 prepartum to wk 9 postpartum (pp) and were housed in climate respiration chambers from wk 2 to 9 pp. Diets were isocaloric (net energy basis). Postpartum, cows fed a lipogenic diet tended to have higher nonesterified fatty acid concentration (NEFA; 0.46 ± 0.04 vs. 0.37 ± 0.04 mmol/L) and lower insulin concentration (4.0 ± 0.5 vs. 5.5 ± 0.6 μIU/mL). No difference was found in plasma glucose, β-hydroxybutyrate, insulin-like growth factor-I, and thyroid hormones. Liver TAG was equal between both diets in wk −2 and 2 pp. In wk 4 pp cows fed the glucogenic diet had numerically lower TAG levels, although there was no significant dietary effect. Negative relationships were detected between ER and milk fat and between ER and NEFA. A positive relationship was detected between ER and insulin concentration. Overall, results suggest that insulin plays a regulating role in altering energy partitioning between milk and body tissue. Feeding lactating dairy cows a glucogenic diet decreased mobilization of body fat compared with a lipogenic diet. The relative abundance of lipogenic nutrients, when feeding a more lipogenic diet, is related to more secretion of lipogenic nutrients in milk, lower plasma insulin, and higher plasma NEFA concentration.     

Relationship of plasma nonesterified fatty acids and walking activity in postpartum dairy cows

To survive and produce milk, postpartum dairy cows use their reserves through lipolysis. If the negative energy balance is severe, nonesterified fatty acids (NEFA) are formed that can impair several physiological processes. A pilot study suggested that increased walking activity after calving may be related to a reduced serum concentration of NEFA. The objective of this study was to determine the relationship between plasma concentrations of NEFA and walking activity in dairy cattle during the postpartum period. Data were collected from 33 multiparous Holstein-Friesian dairy cows. Walking activities were quantified using pedometry, and blood samples were collected for determination of NEFA. Results of this study indicated that a negative relationship existed between walking activity and plasma NEFA concentrations in postpartum dairy cows.     

Metabolic and hormonal control of energy utilization and partitioning from early to mid lactation in Sarda ewes and Saanen goats

In a recent study, we observed that starch-rich diets used in mid lactation induced lower milk production persistency and higher body fat accumulation in dairy ewes compared with dairy goats. Because these species differences could be linked to hormonal mechanisms that drive energy partitioning, in the same experiment, we explored the evolution of metabolic and hormonal status during lactation to test this hypothesis. Twenty mature Sarda dairy ewes and 20 mature Saanen goats [15–134 ± 11 d in milk (DIM), mean ± SD] were compared simultaneously. In early lactation, each species was allocated to one dietary treatment: high-starch diet [HS: 20.4% starch, on dry matter (DM) basis], whereas from 92 ± 11 DIM, each species was allocated to 1 of 2 dietary treatments: HS (20.0% starch, on DM basis) and low-starch (LS: 7.8% starch, on DM basis) diets. Blood samples were collected in the morning to analyze glucose, nonesterified fatty acids (NEFA), growth hormone (GH), insulin, and insulin-like growth factor I (IGF-I). Data were analyzed using the PROC MIXED procedure of SAS with repeated measurements (SAS Version 9.0). The HS and LS diets applied in mid lactation did not affect metabolic status of the animal within species; thus, only a comparison between species was carried out. From early to mid lactation, plasma glucose concentration was higher in ewes than in goats (54.57 vs. 48.35 ± 1.18 mg/dL), whereas plasma NEFA concentration was greater in goats than in ewes (0.31 vs. 0.25 ± 0.03 mmol/L). Goats had higher plasma GH concentration and lower plasma insulin content than ewes (4.78 vs. 1.31 ng/mL ± 0.47; 0.11 vs. 0.26 μg/L ± 0.02). Plasma IGF-I concentration did not vary between species. The comparison of metabolic and hormonal status of lactating Sarda dairy ewes and Saanen goats, carried out by studying simultaneously the 2 species in the same stage of lactation and experimental conditions, suggests that the higher insulin and glucose concentration observed in Sarda ewes explains why they partitioned more energy toward body reserves than to the mammary gland, especially in mid lactation. This can justify the negative effect of high-starch diets in mid-lactating Sarda ewes. Conversely, the highest GH and NEFA concentration observed in Saanen goats explain why they partitioned more energy of starch diets toward the mammary gland than to body reserves and justify the positive effect of high-starch diet in mid lactation. Together, these different responses contribute to explain why specialized dairy goats, such as the Saanen breed, have a higher milk production persistency than specialized dairy sheep breeds, such as the Sarda.     

Effects of plane of nutrition and feed deprivation on insulin responses in dairy cattle during late gestation

Nonlactating Holstein cows (n=12) in late pregnancy were used to determine effects of plane of nutrition followed by feed deprivation on metabolic responses to insulin. Beginning 48 d before expected parturition, cows were fed to either a high plane (HP) or a low plane (LP) of nutrition (162 and 90% of calculated energy requirements, respectively). Cows were subjected to an intravenous glucose tolerance test [GTT; 0.25 g of dextrose/kg of body weight (BW)] on d 14 of treatment and a hyperinsulinemic-euglycemic clamp (HEC; 1 μg/kg of BW/h) on d 15. Following 24 h of feed removal, cows were subjected to a second GTT on d 17 and a second HEC on d 18 after 48 h of feed removal. During the feeding period, plasma nonesterified fatty acid (NEFA) concentrations were higher for cows fed the LP diet compared with those fed the HP diet (163.6 vs. 73.1 μEq/L), whereas plasma insulin was higher for cows fed the HP diet during the feeding period (11.1 vs. 5.2 μIU/mL). Glucose areas under the curve during both GTT were higher for cows fed the LP diet than for those fed the HP diet (4,213 vs. 3,750 mg/dL × 60 min) and was higher during the GTT in the feed-deprived state (4,878 vs. 3,085 mg/dL × 60 min) than in the GTT during the fed state, suggesting slower clearance of glucose during negative energy balance either pre-or post-feed deprivation. This corresponded with a higher dextrose infusion rate during the fed-state HEC than during the feed-deprived-state HEC (203.3 vs. 90.1 mL/h). Plasma NEFA decreased at a faster rate following GTT during feed deprivation compared with that during the fed state (8.7 vs. 2.9%/min). Suppression of NEFA was highest for cows fed the HP diet during the GTT conducted during feed deprivation, and lowest for cows fed the HP diet during the fed-state GTT (68.6 vs. 50.3% decrease from basal). Plasma insulin responses to GTT were affected by feed deprivation such that cows had a much lower insulin response to GTT by 24 h after feed removal (995 vs. 3,957 μIU/mL × 60 min). During the fed-state HEC, circulating concentrations of NEFA were 21% below basal for cows fed the HP diet and 62% below basal for cows fed the LP diet; during feed deprivation, NEFA were 79 and 59% below basal for the HP and LP diets, respectively (diet × HEC). Cows that are fed below energy requirements or are feed deprived have slower clearance of glucose and greater NEFA responses to glucose challenge. Additionally, feed deprivation had a large effect on insulin secretion. Overall, effects of feed deprivation were larger than effects of plane of nutrition.     

Effect of intravenous lipid infusion on biomarkers of insulin resistance and immune functions of dry and nonpregnant dairy cows

During the transition period, dairy cows often experience negative energy balance, which can induce metabolic and immunological disturbances. Previous work has shown that there is a relationship between the dysfunction of immune cells and the increase in blood nonesterified fatty acid (NEFA) concentration. Nevertheless, it is difficult to determine the exact effect of NEFA on the immune system, as other metabolic and hormonal perturbations occur simultaneously during the transition period. In the present study, we have determined the effect of NEFA on immune functions using an experimental model designed to assess the effects independently of energy balance, as well as hormonal and metabolic changes due to parturition. Six dry and nonpregnant cows were infused with either sterile water (control treatment) or a lipid emulsion (Intralipid 20%, Frenesius Kabi, lipid treatment) at a rate of 1 mL/kg per hour for 6 h according to a crossover design. Blood concentrations of NEFA, β-hydroxybutyrate (BHB), and glucose were measured every hour throughout the infusion period, and 1 and 18 h after the end of infusion. Proliferation and interferon-γ secretion of lymphocytes, phagocytosis, and oxidative burst of neutrophils and blood insulin concentration were evaluated before, during, and at the end of the infusion. For NEFA, BHB, and glucose, treatment × time interactions were present. When compared with the control condition, NEFA and BHB levels were greater in the plasma of cows infused with lipids from 1 h after the start of infusion until 1 h after the end of infusion. Glucose level also increased in response to lipid infusion from 2 h of infusion until 1 h after the end of treatment. For sterile water and lipid infusions, respectively, maximal concentrations were 0.06 ± 0.10 mM and 1.39 ± 0.10 mM for NEFA, 0.70 ± 0.05 mM and 1.06 ± 0.05 mM for BHB, and 4.56 ± 0.27 mM and 6.90 ± 0.27 mM for glucose. For all blood metabolites, there were no differences between treatments 18 h postinfusion. Lipid infusion significantly increased blood insulin concentration at 3 and 6 h of infusion. However, it returned to its basal concentration 18 h after the end of the infusion. Lymphoproliferation declined as early as 3 h after the start of the lipid infusion. At 3 and 6 h of infusion, lipid treatment significantly reduced INF-γ concentration in the culture cell supernatant. The lipid infusion did not affect neutrophil phagocytosis. Nevertheless, the efficacy of the response was affected by a reduction of neutrophils' oxidative burst. These results confirm that NEFA inhibits immune functions independently of energy balance and other changes that occur during the transition period. They also indicate that high blood lipid concentration causes insulin resistance.     

Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro

In this study concentration and composition of non-esterified fatty acids (NEFA) in follicular fluid (FF) of high-yielding dairy cows were determined during the period of negative energy balance (NEB) early post partum. NEFA were then added during in vitro maturation at concentrations measured previously in FF to evaluate their effect on the oocyte's developmental competence. At 16 and 44 days post partum, FF of the dominant follicle and blood were collected from nine high-yielding dairy cows. Samples were analysed for NEFA concentration and composition. NEFA concentrations in FF (0.2-0.6 mmol/l) during NEB remained +/- 40% lower compared with serum (0.4-1.2 mmol/l). The NEFA composition differed significantly between serum and FF with oleic acid (OA), palmitic acid (PA) and stearic acid (SA) being the predominant fatty acids in FF. Based on these results, 5115 oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of 0.200 mmol/l OA, 0.133 mmol/l PA or 0.067 mmol/l SA dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in SOF medium. Addition of PA or SA during oocyte maturation had negative effects on maturation, fertilization and cleavage rate and blastocyst yield. More (late) apoptotic cumulus cells were observed in cumulus-oocyte complexes matured in the presence of SA or PA. Ethanol or OA had no effect. These in vitro results suggest that NEB may hamper fertility of high-yielding dairy cows through increased NEFA concentrations in FF affecting oocyte quality.     

Test accuracy of metabolic indicators in predicting decreased fertility in dairy cows

Negative energy balance is a known risk factor for decreased fertility in dairy cows. This study evaluated the accuracy of plasma concentrations of nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and insulin-like growth factor 1 (IGF-1)—factors related to negative energy balance—in predicting decreased fertility. One plasma sample per cow was collected from 480 cows in 12 herds during the period from d 4 to 21 in milk and analyzed for NEFA, BHBA, and IGF-1. For each cow, data on breed, parity, calving date, gynecological examinations, and insemination dates were obtained. Milk samples from 241 cows in 7 of the participating herds were analyzed for progesterone concentration to define the first day of luteal activity. The diagnostic sensitivity (Se) and specificity (Sp) at different cut-off concentrations of NEFA, BHBA, or IGF-1 were calculated and related to individual cow fertility status, measured as anestrus (ANEST), delayed first artificial insemination (DFAI), or delayed conception. Positive and negative predictive values (PV+; PV−) were calculated considering different levels of (within-herd) prevalence. Strata (i.e., subgroup)-specific Se and Sp and associations between test results and fertility parameters were investigated using logistic regression. The NEFA and BHBA tests for ANEST and DFAI had the highest combined Se and Sp and were thus evaluated further. Cut-off values with Sp around 80% were used in this step to provide a reasonable number of test-positive cows, representing a practical situation. This corresponded to a cut-off value for the NEFA test of 400 µEq/L (Se 0.27–0.45) and for the BHBA test of 1.8 mM (Se 0.15–0.30) across all cows included in the study. The estimated Sp was generally higher than the original 80%, but the corresponding Se was further decreased when the test was used in heifers compared with older cows. The true prevalence of ANEST in the study population was 27%, which gave a PV+ of 0.36 to 0.45 and a PV− of 0.76 to 0.79. With 35% true prevalence of DFAI, PV+ was 0.29 to 0.38 and PV− was 0.64 to 0.66. Thus, overall test performance was low when metabolic indicators measured as single values in early lactation were used to predict fertility in dairy cows, but accuracy was influenced by cow-level factors such as parity. The prevalence of the target condition (in this case, decreased fertility) also influences test usefulness and should be considered when planning test systems and interpreting test results.     

Short communication: Effect of sampling time relative to the first daily feeding on interpretation of serum fatty acid and β-hydroxybutyrate concentrations in dairy cattle

Serum nonesterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) concentrations are used to evaluate energy status in peripartum dairy cows. Blood samples from 37 cows in the week before parturition and 47 cows in the first week after parturition from 3 dairy herds were taken 1 h before the first feeding (−1 h) as well as 4 and 10 h after the first feeding. Nonesterified fatty acid concentrations were measured in samples from cows before calving and BHBA was measured in samples from lactating cows. Mean NEFA concentrations in the prepartum cows were significantly higher at −1 h (0.20 mmol/L) than at 4 h (0.14 mmol/L), but were not different between 4 and 10 h (0.17 mmol/L). Using a cutpoint of NEFA ≥0.4 mmol/L, 32% of cows had high concentrations at −1 h compared with 16% of the same cows at 4 and 10 h. There were no differences in mean BHBA between −1 h (646 μmol/L) and 4 h (596 μmol/L), but mean BHBA was higher at 10 h (711 μmol/L) than at −1 h. Using a cutpoint of BHBA ≥1,400 μmol/L, there were no differences in the proportions of high BHBA, which were 9, 11, and 13% of cows at −1, 4, and 10 h, respectively. Prandial effects on serum NEFA may affect interpretation of this analyte. In order not to misclassify cows when assessing energy status, samples for NEFA must at least be taken at a consistent time relative to feeding within a given herd. When sampling cows to monitor elements of energy metabolism in the prepartum period, there was twice the probability of detecting animals with NEFA values ≥0.4 mmol/L if they were sampled 1 h before the first feed delivery compared with sampling the same cows 4 or 10 h after feeding.