Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma β-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-dl-β-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 μg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2 h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8 h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-dl-β-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.     

Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows

Neutrophil function and the severity and incidence of mastitis in dairy cows is related to the intake of many antioxidant nutrients. Because vitamin C is the major water-soluble antioxidant in mammals, we examined the effect of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccahride (LPS) in periparturient dairy cows. At 2 wk before anticipated calving, Holstein cows were fed diets that provided 0 (16 cows) or 30 (15 cows) g/d of supplemental vitamin C (phosphorylated ascorbic acid). Treatments continued until 7 d after cows received an infusion of 10 μg of LPS into one quarter of the mammary gland (on average, this occurred 32 d postcalving). Supplementation of vitamin C increased plasma concentrations of vitamin C at calving, but no differences were observed in samples taken 24 h postinfusion. Concentrations of vitamin C in milk (24 h postinfusion) and in neutrophils (calving and 24 h postinfusion) were not affected by treatment, but vitamin C concentrations in neutrophils isolated from milk were about 3 times greater than concentrations in blood neutrophils. The LPS infusion did not alter concentrations of vitamin C in plasma or milk, suggesting that the LPS model did not produce the same effects as a bacterial infection of the mammary gland with respect to antioxidant effects. Supplemental vitamin C had no effect on neutrophil phagocytosis or bacterial kill. Dietary vitamin C reduced the milk somatic cell count but did not affect the febrile response or milk production following LPS infusion.     

Effects of time and sampling location on concentrations of β-hydroxybutyric acid in dairy cows

Two trials were conducted to examine factors potentially influencing the measurement of blood β-hydroxybutyric acid (BHBA) in dairy cows. The objective of the first trial was to study effects of sampling time on BHBA concentration in continuously fed dairy cows. Furthermore, we determined test characteristics of a single BHBA measurement at a random time of the day to diagnose subclinical ketosis considering commonly used cut-points (1.2 and 1.4 mmol/L). Finally, we set out to evaluate if test characteristics could be enhanced by repeating measurements after different time intervals. During 4 herd visits, a total of 128 cows (8 to 28 d in milk) fed 10 times daily were screened at 0900 h and preselected by BHBA concentration. Blood samples were drawn from the tail vessels and BHBA concentrations were measured using an electronic BHBA meter (Precision Xceed, Abbott Diabetes Care Ltd., Witney, UK). Cows with BHBA concentrations ≥0.8 mmol/L at this time were enrolled in the trial (n = 92). Subsequent BHBA measurements took place every 3 h for a total of 8 measurements during 24 h. The effect of sampling time on BHBA concentrations was tested in a repeated-measures ANOVA repeating sampling time. Sampling time did not affect BHBA concentrations in continuously fed dairy cows. Defining the average daily BHBA concentration calculated from the 8 measurements as the gold standard, a single measurement at a random time of the day to diagnose subclinical ketosis had a sensitivity of 0.90 or 0.89 at the 2 BHBA cut-points (1.2 and 1.4 mmol/L). Specificity was 0.88 or 0.90 using the same cut-points. Repeating measurements after different time intervals improved test characteristics only slightly. In the second experiment, we compared BHBA concentrations of samples drawn from 3 different blood sampling locations (tail vessels, jugular vein, and mammary vein) of 116 lactating dairy cows. Concentrations of BHBA differed in samples from the 3 sampling locations. Mean BHBA concentration was 0.3 mmol/L lower when measured in the mammary vein compared with the jugular vein and 0.4 mmol/L lower in the mammary vein compared with the tail vessels. We conclude that to measure BHBA, blood samples of continuously fed dairy cows can be drawn at any time of the day. A single measurement provides very good test characteristics for on-farm conditions. Blood samples for BHBA measurement should be drawn from the jugular vein or tail vessels; the mammary vein should not be used for this purpose.     

Effects of different nutrient supply on metabolism and mammary immune response to an LPS challenge in early lactation of dairy cows

Energy and nutrient deficiency in dairy cows in early lactation is considered to contribute to their increased susceptibility to mastitis. We have tested the hypothesis that feeding diets with high contents of either nitrogenic, glucogenic, or lipogenic components in early lactation affects both the endocrine and metabolic status, as well as the mammary immune competence. After calving, cows were fed increasing amounts of concentrate up to 10 kg/d rich in crude protein (nitrogenic, n = 10), glucogenic precursors (glucogenic, n = 11), or lipids (lipogenic, n = 11). In wk 3, one udder quarter was challenged with lipopolysaccharide (LPS) from Escherichia coli. Blood and milk were sampled on the day before LPS challenge (d −1), and on d 0, 1, 2, 3, and 9 after LPS challenge. On the day of LPS challenge additional samples were taken hourly for quarter milk and every 3 h for blood. Urea concentrations were higher in plasma and milk of cows fed the nitrogenic diet. However, plasma concentrations of glucose, cholesterol, triglycerides, β-hydroxybutyrate, nonesterified fatty acids, as well as insulin, glucagon, and insulin-like growth factor-1 were not affected by the different diets. The mammary immune challenge induced a substantial increase of somatic cell count (SCC) in the treated quarter, and a transient decrease of total milk yield and white blood cells similar in all diet groups for one day. The absolute phagocytosis of blood leukocytes was decreased; however, the phagocytosis per cell was increased in glucogenic-fed cows at 6 h after LPS challenge. During mammary inflammation an insulin resistance, shown by increased plasma glucose, insulin, and glucagon, developed similarly in all diet groups. β-hydroxybutyrate and nonesterified fatty acids were decreased at 1 d after LPS challenge in glucogenic-fed cows only. Cholesterol did not change, and triglycerides only decreased significantly in lipogenic-fed cows 6 h after challenge. On d 9 after LPS challenge, SCC and milk yield and metabolic factors were recovered in all groups. In conclusion, the endocrine and metabolic situation, and the immune response to intramammary LPS of dairy cows during early lactation was not substantially influenced by the elevated supply of nitrogenic, glucogenic, or lipogenic components due to the provided feed in this study.     

Acute experimental mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows

Twenty Holstein cows in early lactation (7 d in milk) were administered 100 μg of Escherichia coli lipopolysaccharide (LPS) dissolved in 10 mL of sterile 0.9% NaCl saline (treatment; TRT) or 10 mL of sterile saline (control) into both right mammary quarters to test the hypothesis that acute experimental mastitis would have negative impacts on aspects of energy metabolism that might lead to the development of metabolic disorders. A primed continuous intravenous infusion (14-μmol/kg of BW priming dose; 11.5-μmol/kg of BW per h continuous infusion) of 6,6-dideuterated glucose was used to determine pre- and posttreatment glucose kinetics using steady-state tracer methodologies. The LPS-treated cows displayed productive, clinical, and physiological signs of moderate to severe inflammation; control cows displayed no signs of immune activation. Pretreatment glucose rates of appearance (Ra) into plasma were similar (715 and 662 ± 33 mmol/h for TRT and control, respectively) between treatment groups. Intramammary LPS infusion into TRT cows resulted in increased glucose Ra relative to control cows (mean glucose Ra from 150 through 270 min after intramammary infusion were 815 and 674 ± 21 mmol/h for TRT and control cows, respectively). Furthermore, plasma concentrations of glucose increased, whereas plasma nonesterified fatty acids, glycerol, and β-hydroxybutyrate concentrations decreased, in TRT relative to control cows. Interestingly, plasma insulin concentration increased dramatically in TRT cows and occurred prior to the small increase in plasma glucose concentration. Although these results only represent the early stages of inflammation, they are not consistent with a causal relationship between mastitis and energy-related metabolic disorders and instead suggest a coordinated protective effect by the immune system on metabolism during the early stages of mammary insult.     

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.     

Intramammary challenge of lipopolysaccharide stimulates secretion of lingual antimicrobial peptide into milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is found in bovine milk. However, it is unclear whether LAP is involved in the early immune response to mammary infection. The aim of the study was to investigate the changes of LAP concentration in milk after intramammary challenge with lipopolysaccharide (LPS), the gram-negative bacteria cell membrane component, in dairy cows. Milk was collected before and after LPS or phosphate-buffered saline (control) challenge every hour for 12 h on d 0 and twice daily from d 1 to 7. Somatic cell count (SCC), LAP concentration, and lactoperoxidase (LPO) activity in the milk were measured. Somatic cell count started to increase at 2 h postchallenge and remained high until d 5 (694 ± 187 × 10³ to >1,000 ± 0 × 10³ cells/mL at d 0; >1,000 ± 0 × 10³ cells/mL at d 1 to 3; 684 ± 194 × 10³ to 829 ± 108 × 10³ cells/mL at d 4; 527 ± 197 × 10³ to 656 ± 145 × 10³ cells/mL at d 5). Somatic cell count increased in the control cows, although the levels were lower compared with those in the LPS challenge group. The LAP concentration in milk increased significantly at 2 h post-LPS-challenge and was maintained at high levels until d 2 (8.6 ± 0.6 to 17.5 ± 2.3 nM). In the control cow infused with phosphate-buffered saline, there was no increase of LAP concentration in milk (5.1 ± 0.6 to 7.2 ± 0.8 nM). Increase of LPO activity in the milk was observed at 6 h after LPS challenge and continued until d 3 (4.7 ± 0.3 to 9.4 ± 1.1 U). No increase of LPO activity was observed in the milk of control cows. The increase and subsequent decrease in LAP concentration after LPS challenge occurred earlier than those of LPO activity. In multiparous cows with LPS infusion, there was a significantly negative relationship between the days leading to the basal levels in LAP concentration and LPO activity (r = −0.75). These results suggest that LPS induces secretion of LAP into milk within hours and that LPO may have a synergistic antimicrobial function with LAP in mammary glands of dairy cows.     

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24 h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24 h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.     

Effects of supplementation with a phytobiotics-rich herbal mixture on performance,udder health,and metabolic status of Holstein cows with various levels of milk somatic cell counts

This study evaluated the effects of dietary supplementation of a novel phytobiotics-rich herbal mixture (PRHM) on feed intake, performance, udder health, ruminal fermentation, and plasma metabolites in cows with moderate or high somatic cell counts (SCC) in the milk. Twenty-four Holstein dairy cows (117 ± 26 d in milk and 46.3 ± 4.7 kg of milk/d at the start of the experiment) were blocked by parity and days in milk and split into 2 groups, based on SCC in the milk; 12 cows were with moderate SCC (260,000 < SCC <500,000 cells/mL), whereas 12 other cows had high levels of SCC (>500,000 cells/mL) in the milk. Within each SCC group, cows were blocked by milk yield and parity, and were randomly assigned to 2 different feeding regimens. Half of the cows in each SCC group (n = 6) were supplemented with PRHM (185 g/cow per day, providing 12.4 g of phenolic compounds per day), and the other half (n = 6) were not supplemented in their diets. The experiment lasted 36 d, whereby the first 24 d were used for adaptation to the diets and the last 12 d for sampling. Data showed that supplementation of PRHM decreased somatic cell score in the milk, indicating improved udder health of cows with high initial SCC, but not in cows with moderate SCC. Also, cows supplemented with PRHM consumed more feed DM, produced greater amounts of milk, and showed an improvement of feed utilization efficiency. However, these cows also lost more back-fat thickness during the experiment. Supplementation of PRHM increased fat- and energy-corrected milk yields in cows with high initial SCC, but not in cows with moderate SCC. Supplementation of PRHM decreased milk fat content, whereas other milk components were not affected by PRHM feeding. The PRHM supplementation decreased the acetate-to-propionate ratio in the rumen fluid, but increased β-hydroxybutyrate and cholesterol concentration in the plasma, irrespective of the initial SCC level in the milk. Other plasma metabolites and liver enzymes were not affected by PRHM supplementation. Apparent nutrient digestibility did not differ among treatments. Overall, supplementation of PRHM seems to be an effective strategy to enhance performance and lower SCC, particularly in cows having high SCC levels in the milk. Further research is warranted to evaluate long-term effects of PRHM supplementation, especially with regard to metabolic health status and reproduction.     

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.     

Potential risk indicators of retained placenta and other diseases in multiparous cows

Retained placenta (RP), defined as fetal membranes not being expelled within 24 h after calving, is a costly disease in multiparous dairy cows that has been linked to immune suppression, infections, elevated lipid mobilization, and depleted status of antioxidants including α-tocopherol, and that increases the risk of other diseases (OD) in early lactation. Early detection of cows at increased risk of developing RP, OD, or both in early lactation could improve treatment success and result in improved milk production and reproductive performance. To identify risk indicators of RP, OD, or both, we used a nested case-control design and compared multiparous dairy cows that developed RP (n = 32) with cows that remained healthy (H; n = 32) or cows that developed OD (n = 32) in early lactation. We compared peripartal body condition score (BCS) as well as serum concentrations of α-tocopherol, metabolites [β-hydroxybutyrate (BHBA), cholesterol, glucose, nonesterified fatty acids (NEFA), and urea N], haptoglobin, and macrominerals (i.e., calcium, magnesium, and phosphorus) on d −21, −14, −7, −3, −1, 0, 1, 3, 7, 14, 21, 28, 35, 42, and 49 postpartum. In addition, average serum concentrations were calculated for each cow for the last 3 wk prepartum, for 3 and 2 wk prepartum combined, for the last week prepartum, and for the morning after calving and compared between groups. The RP cows had lower BCS than the H or OD cows until 2 wk postpartum. During the prepartal periods, RP and OD cows had lower α-tocopherol concentrations (corrected or not for cholesterol concentration) and higher NEFA and BHBA concentrations than H cows. Thus, lower prepartal BCS could be an early predictor for RP risk, and lower α-tocopherol concentrations and higher NEFA and BHBA concentrations could be early predictors for disease.     

Effect of postruminal glutamine supplementation on immune response and milk production in dairy cows

Seventeen multiparous Holstein cows were used to examine the effect of an increased duodenal supply of Gln on immune function and production. Cows received continuous abomasal infusions of water (control: n = 8) or 300 g/d of Gln (n = 9) for 21 d starting within 48 h of calving. There were nonsignificant increases in milk and milk protein yields in response to Gln supplementation. Glutamine treatment had no effect on plasma glucose, nonesterified fatty acids (NEFA), or β-hydroxybutyrate (BHBA) concentrations but did tend to increase plasma urea N concentration. The Gln treatment resulted in an increase of 108 μM in the plasma Gln concentration. Total essential AA concentrations decreased with the Gln treatment, whereas total nonessential AA concentrations were unaffected. T Lymphocyte proliferation did not differ between the control and Gln-treated cows. Treatment had no effect on the relative abundance of CD8 T cells but did increase the abundance of CD4 T cells. Cytokine production, as measured by IFN-γ concentration determined in vitro in concanavalin-A-stimulated peripheral blood mononuclear cells, was similar between the treatments. Over the first 3 wk following calving, Gln supplementation had limited effects on milk production, metabolic parameters, and immune function.     

Short communication: Glucose infusion into early postpartum cows defines an upper physiological set point for blood glucose and causes rapid and reversible changes in blood hormones and metabolites

Low blood glucose concentrations after calving are associated with infertility in postpartum dairy cows perhaps because glucose is a master regulator of hormones and metabolites that control reproductive processes. The hypothesis was that low blood glucose postpartum is caused by inadequate glucose entry rate relative to whole-body demand as opposed to the alternative possibility that postpartum cows have a lower regulatory set point for blood glucose. Eight early postpartum (10 to 25 d) dairy cows (5 Holstein and 3 Guernsey) were jugular catheterized. During the first 24 h, cows were infused with physiological saline at 83.3 mL/h. After 24 h, the infusion solution was switched to 50% dextrose that was infused at a rate of 41.7 mL/h (total daily glucose dose = 500 g). On d 3 and d 4, the rate of glucose infusion was increased to 83.3 mL/h (daily dose = 1,000 g) and 125 mL/h (daily dose = 1,500 g), respectively. On d 5, physiological saline was infused at 83.3 mL/h. Blood was sampled hourly through a second jugular catheter (contralateral side) and analyzed for glucose, nonesterified fatty acids, β-hydroxybutyrate, insulin-like growth factor 1, and insulin. Blood glucose concentrations on d 1 (saline infusion) averaged 53.4 ± 1.7 mg/dL. Blood glucose concentrations increased on d 2 when cows were infused with 500 g/d and increased further on d 3 when cows were infused with 1,000 g of glucose/d. Increasing the infusion rate to 1,500 g/d on d 4 did not cause a further increase in blood glucose concentrations. Based on a segmented regression analysis, the upper physiological set point for blood glucose was 72.1 mg/dL. Both insulin and insulin-like growth factor 1 concentrations increased in response to glucose infusion and decreased when cows were infused with saline on d 5. Serum nonesterified fatty acids and β-hydroxybutyrate concentrations decreased in response to glucose infusion and rebounded upward on d 5 (saline infusion). In conclusion, early postpartum cows had circulating blood glucose concentrations that were well below the upper set point defined in this study (72.1 mg/dL). Infusing approximately 1,000 g of glucose daily increased blood glucose to the physiological set point and rapidly changed the hormonal and metabolic profile that typifies postpartum cows. The inability of the early postpartum cow to achieve an adequate entry rate for glucose relative to whole-body demand is a possible mechanism that links postpartum physiology and nutrition to reproduction in dairy cows.     

Changes in measures of biotin status do not reflect milk yield responses when dairy cows are fed supplemental biotin

A sensitive indicator of biotin status for lactating dairy cows is necessary to understand factors that affect milk yield responses to biotin supplementation. 3-Hydroxyisovaleric acid (3HIA) is an alternative metabolite in the pathway of Leu catabolism when the biotin-dependent enzyme methylcrotonyl-coenzyme A carboxylase is limiting. We evaluated urinary excretion of 3HIA as a determinant of biotin status in lactating dairy cows. We hypothesized that high-producing cows would have a greater biotin requirement and excrete more 3HIA than low-producing cows and that biotin supplementation would decrease 3HIA excretion. Twenty high-producing and 20 low-producing Holstein cows (43 ± 5 and 23 ± 4 kg/d of milk, respectively) were fed diets that contained either 0 or 0.96 mg/kg of supplemental biotin. On d 16 cows were given an intraruminal infusion of 1.4 mol of isovaleric acid and urine was sampled. Biotin supplementation did not affect basal urinary excretion of 3HIA. The infusion of isovaleric acid increased urinary excretion of 3HIA (maximum at 8 h after infusion), but biotin supplementation did not attenuate this increase. The increase in urinary 3HIA excretion was less for low-producing cows than for high-producing cows. Biotin increased yields of milk and milk components in high-producing cows but had no effect in low-producing cows. However, potential measures of biotin status (concentrations of avidin-binding substances in the plasma, milk, and urine, and urinary 3HIA excretion) responded similarly to biotin supplementation for both high- and low-producing cows. A sensitive indicator of biotin status for lactating dairy cows is still needed.     

The effect of injectable butaphosphan and cyanocobalamin on postpartum serum β-hydroxybutyrate, calcium, and phosphorus concentrations in dairy cattle

The objective of this study was to determine the effect of an injection of 10% butaphosphan and cyanocobalamin (Catosal, Bayer, Shawnee Mission, KS) on the day of calving and 1 d later on the prevalence of subclinical ketosis in dairy cattle in the early postpartum period. Cows from 4 herds (n = 1,122) were randomized to receive either 25 mL of 10% butaphosphan and cyanocobalamin or 25 mL of sterile water subcutaneously on both days. Each milliliter of Catosal contained 0.05 mg of cyanocobalamin and 100 mg of butaphosphan, which provided 17.3 mg of P in the form of [1-(butylamino)-1-methylethyl]-phosphonic acid. Serum was sampled for β-hydroxybutyrate (BHBA) concentration at calving (pretreatment) and again between 3 and 10 d in milk. A subset of samples from mature cows was also evaluated for serum Ca and P concentrations. When cows from all age groups were included in the analysis, there was no difference between the median serum BHBA concentrations of cows in the 2 treatment groups, and no difference in the proportion of hyperketonemic cows (serum BHBA ≥1,200 μmol/L) during the first week postpartum. When the analysis was restricted to mature cows (lactation ≥3), both the median BHBA concentration and the proportion of hyperketonemic cows were significantly lower in the treatment group than in the placebo group. Serum Ca and P concentrations did not differ between treatment groups. Our results suggest that injection of butaphosphan and cyanocobalamin on the day of calving and 1 d later may decrease the prevalence of subclinical ketosis during the week after calving in mature dairy cows, but not in first- and second-lactation animals.     

Using behavioral observations in freestalls and at milking to improve pain detection in dairy cows after lipopolysaccharide-induced clinical mastitis

This study aimed to determine the effect of lipopolysaccharide (LPS)-induced mastitis with or without nonsteroidal anti-inflammatory drug (NSAID) on dairy cows' clinical, physiological, and behavioral responses in the milking parlor and freestalls as well as the specificity (Sp) and sensitivity (Se) of behavioral responses in detecting cows with LPS-induced mastitis. Twenty-seven cows received an intramammary infusion of 25 µg of Escherichia coli LPS in 1 healthy quarter. Following LPS infusion, 14 cows received a placebo (LPS cows), and 13 cows received 3 mg/kg of body weight of ketoprofen i.m. (LPS+NSAID cows). Cow response to the challenge was monitored at regular intervals from 24 h before to 48 h postinfusion (hpi) through direct clinical observations, markers of inflammation in milk, and via point-in-time direct behavioral observations in the barn and at milking. In LPS cows, infusion induced a significant increase of plasma cortisol levels at 3 and 8 hpi, milk cortisol levels at 8 hpi, somatic cell counts from 8 to 48 hpi, IL-6 and IL-8 at 8 hpi, milk amyloid A (mAA) and haptoglobin at 8 and 24 hpi, rectal temperature at 8 hpi, and respiratory rate at 8 hpi. Their rumen motility rate decreased at 8 and 32 hpi. Compared with before the challenge, significantly more LPS cows stopped feeding/ruminating and pressed their tail between their legs at 3 and 5 hpi, increased feeding/ruminating at 24 hpi, and had the tendency to be less responsive, dropping their head, and dropping their ears at 5 hpi. At milking, compared with before challenge, significantly more LPS cows lifted their hooves at forestripping at 8 hpi. The 2 groups showed similar patterns of response for milk cortisol, somatic cell count, respiratory rate, mAA, haptoglobin, and IL-6, IL-1β, and IL-8. Compared with LPS cows, LPS+NSAID cows had significantly lower plasma cortisol levels at 3 hpi, their rectal temperature decreased at 8 hpi, their rumen motility rate increased at 8 and 32 hpi, and their heart rate increased at 32 hpi. Compared with LPS cows, a significantly larger proportion of LPS+NSAID cows were feeding/ruminating, a lower proportion had ears down at 5 hpi, and a larger proportion lied down at 24 hpi. At milking, whatever the phase of milking, for “hoof to belly,” 9 out of 14 cows did not show this behavior before infusion (Sp = 64%) and 14/14 did not kick during pre-infusion milking (Sp = 100%). Regarding sensitivity, at maximum, 5 cows out of 14 (Se = 36%) displayed “hoof to belly” after infusion. For “lifting hoof,” 14/14 did not show hoof-lifting before infusion (Sp = 100%) and 6/14 displayed it after infusion (Se = 43%) at forestripping only. In the freestall barn, 9 behaviors had a Sp >75% (at minimum, 10/14 did not show the behavior) whatever the time point but Se < 60% (at maximum, 8/14 displayed the behavior). Finally, “absence of feeding and ruminating” had Sp of 86% (12/14 ate/ruminated) and Se of 71% (10/14 did not eat/ruminate) at 5 hpi. This study shows that feeding/ruminating, tail position, and reactivity at forestripping could be used as behavioral indictors for early detection of mastitis-related pain in dairy cows.     

Accuracy of milk ketone bodies from flow-injection analysis for the diagnosis of hyperketonemia in dairy cows

The objectives of this study were (1) to determine the correlations between blood β-hydroxybutyrate (BHBA) and milk components [BHBA, acetone, fat, protein, and fat:protein (F:P) ratio], and (2) to establish optimal thresholds for milk components to predict hyperketonemia in dairy cows. Data on 163 cows from 37 herds were used in this cross-sectional study. Herds were visited once during the study period, and cows between 2 and 90 d in milk were blood sampled within 4 h of milk sampling for the Dairy Herd Improvement test. Blood BHBA concentrations were measured using a cow-side electronic meter, Precision Xtra, which was considered the gold standard test in this study. Milk BHBA and acetone concentrations were measured in Dairy Herd Improvement milk samples by flow-injection analysis; whereas, milk fat and protein were tested using Fourier transform infrared spectroscopy. Hyperketonemia was defined by a blood BHBA concentration ≥1.4 mmol/L. The prevalence of hyperketonemia (based on blood BHBA values) in this study population was 21.0%. Pearson correlation coefficients between blood BHBA and milk BHBA, acetone, fat, protein, and F:P ratio were 0.89, 0.73, 0.21, 0.04, and 0.17, respectively. Receiver operating characteristic curves were generated and thresholds for each individual milk component were determined based on the maximal sum of sensitivity and specificity. Optimal threshold values for hyperketonemia were milk BHBA ≥0.20 mmol/L, acetone ≥0.08 mmol/L, fat ≥4.2%, and F:P ratio ≥1.3. Based on these thresholds, milk BHBA and acetone had greater sensitivity (84 and 87%, respectively) and greater specificity (96 and 95%, respectively) than the other milk components (fat, protein, and F:P). Series and parallel testing slightly improved the accuracy of milk BHBA and acetone values to predict hyperketonemia. A multivariable model that accounted for milk BHBA and milk acetone values simultaneously had the highest accuracy of all tested models for predicting hyperketonemia. These results support that milk BHBA and milk acetone values from flow-injection analysis are accurate diagnostic tools for hyperketonemia in dairy cows and could potentially be used for herd-level hyperketonemia surveillance programs.     

Effect of time of artificial insemination and supplemental estradiol on reproduction of lactating dairy cows

Our objectives were to compare reproductive responses of dairy cows receiving timed artificial insemination (AI) either at 48 or 72 h after induction of luteolysis and supplemented or not with estradiol cypionate (ECP). Holstein cows (971) had their estrous cycles presynchronized with injections of PGF_2α at 37 and 51 d in milk (DIM) and then received an injection of GnRH at 64 DIM and an injection of PGF_2α at 71 DIM. Cows were then assigned to a 2 × 2 factorial randomized block experiment; cows in the CoSynch 48 h (CoS48) received a final injection of GnRH concurrent with timed AI 48 h after PGF_2α, whereas cows in the CoSynch 72 h (CoS72) received GnRH and timed AI 72 h after PGF_2α. Half of the cows in each CoSynch protocol received an injection of 1 mg of ECP 24 h after PGF_2α. Therefore, the 4 treatments were as follows: CoS48-NECP (n = 240), CoS72-NECP (n = 246), CoS48-ECP (n = 245), and CoS72-ECP (n = 240). Blood was sampled at 7 d before and at the first GnRH of the CoSynch from all cows for analysis of progesterone concentration in plasma. Cows were classified as anovular when progesterone was less than 1.0 ng/mL in both samples. Blood was also sampled during proestrus from a subset of 123 cows to measure concentrations of estradiol and at 7 d after timed AI to measure concentrations of progesterone. Ovaries from the same subset of 123 cows were examined by ultrasonography to determine ovulatory follicle diameter and incidence of ovulation. Pregnancy was diagnosed at 40 and 68 d after AI. Prevalence of cyclic cows was 72.4% and was similar among treatments. Concentrations of estradiol increased after ECP treatment and at 72 h of proestrus with CoS72. Pregnancy at 40 and 68 d after AI and pregnancy loss were not affected by timing of AI or supplemental ECP. Delaying timed AI to 72 h and supplementation with ECP increased the proportion of cows displaying estrus at AI, and cows detected in estrus had increased pregnancy per AI associated with improved ovulation and increased postovulatory progesterone concentration. These results indicate that extending the proestrus by delaying timed AI from 48 to 72 h plus supplemental ECP, despite increased expression of estrus at timed AI, did not improve reproductive performance of lactating dairy cows at first AI.