Suzanna M. Dunn

Feeding High Proportions of Barley Grain Stimulates an Inflammatory Response in Dairy Cows

The objective of this study was to evaluate effects of feeding increasing proportions of barley grain on acute phase response in lactating dairy cows. Eight cannulated primiparous (60 to 140 d in milk) Holstein dairy cows were assigned to 4 diets in a 4 x 4 Latin square experimental design. The experimental period lasted for 21 d, with 11 d of adaptation and 10 d of measurements. Cows were fed the following diets: 1) no barley grain in the diet, 2) 15% barley grain, 3) 30% barley grain, and 4) 45% barley grain, as well as barley and alfalfa silage and alfalfa hay at 85, 70, 55, and 40% [dry matter (DM) basis]. All cows were supplemented with a 15% concentrate mix. Blood and rumen fluid samples were collected on d 1, 3, 5, 7, and 10 of the measurement period, and pH and endotoxin content were measured in rumen samples. Concentrations of serum amyloid A, lipopolysaccharide-binding protein, haptoglobin, and C-reactive protein in plasma were measured by ELISA. Feeding high proportions of barley grain at 0, 15, 30, and 45% of DM was associated with lower feed intake (32.6, 32.9, 27.34, and 25.18 kg/d +/- 1.30, respectively), lower ruminal pH (6.8, 6.7, 6.7, and 6.5 +/- 0.03, respectively), and higher DM intake (13.33, 15.28, 14.68, and 16.04 +/- 0.63 kg/d, respectively) and milk production (27.2, 28.2, 29.0, and 31.0 +/- 1.2 kg/d, respectively). Ruminal endotoxin increased in cows receiving 30 and 45% barley grain (5,021, and 8,870 +/- 393 ng/mL, respectively) compared with those fed no grain or 15% barley grain (654 and 790 +/- 393 ng/mL, respectively). Plasma concentrations of serum amyloid A, lipopolysaccharide-binding protein, and C-reactive protein increased in cows given higher (30 and 45%) proportions of grain. Plasma haptoglobin was not affected by treatments. In conclusion, feeding dairy cows high proportions (30 and 45% DM basis) of barley grain was associated with lower feed intake and rumen pH, increased endotoxin in the rumen fluid, and stimulation of an inflammatory response.

Metabolomics reveals unhealthy alterations in rumen metabolism with increased proportion of cereal grain in the diet of dairy cows

This study presents the first application of metabolomics to evaluate changes in rumen metabolites of dairy cows fed increasing proportions of barley grain (i.e., 0, 15, 30, and 45% of diet dry matter). 1H-NMR spectroscopy was used to analyze rumen fluid samples representing 4 different diets. Results showed that for cows fed 30 and 45% grain, increases were observed in the concentration of rumen methylamine as well as glucose, alanine, maltose, propionate, uracil, valerate, xanthine, ethanol, and phenylacetate. These studies also revealed lower rumen 3-phenylpropionate in cows fed greater amounts of cereal grain. Furthermore, ANOVA tests showed noteworthy increases in rumen concentrations of N-nitrosodimethylamine, dimethylamine, lysine, leucine, phenylacetylglycine, nicotinate, glycerol, fumarate, butyrate, and valine with an enriched grain diet. Using principal component analysis it was also found that each of the 4 diets could be distinguished on the basis of the measured rumen metabolites. The two closest clusters corresponded to the 0 and 15% grain diets, whereas the 45% barley grain diet was significantly separated from the other clusters. Unhealthly levels of a number of potentially toxic metabolites were found in the rumen of cattle fed 30 and 45% grain diets. These results may have a number of implications regarding the influence of grain on the overall health of dairy cows.

A metabolomics approach to uncover the effects of grain diets on rumen health in dairy cows

Dairy cows fed high-grain diets during early lactation have a high incidence of metabolic disorders. However, the precise mechanism(s) of how grain feeding causes disease is not clear. In an effort to understand how this diet transition alters the rumen environment and potentially leads to certain metabolic disorders in dairy cattle, we undertook a comprehensive, quantitative metabolomic analysis of rumen fluid samples from dairy cows fed 4 different diets. Using a combination of proton nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, and direct flow injection tandem mass spectroscopy, we identified and quantified 93 metabolites in rumen samples taken from 8 dairy cows fed graded amounts of barley grain (i.e., 0, 15, 30, and 45% of diet dry matter). We also studied temporal changes in the rumen by studying metabolite concentration differences between the first day and the last day of each diet phase following the diet adaptation period. Multivariate analysis showed that rumen metabolites arising from the diet containing 45% barley grain were clearly different from those containing 0, 15, and 30% barley grain. Likewise, a clear separation of the metabolic composition of the ruminal fluid was evident at the beginning and at the end of each diet phase-contrary to the belief that 11 d are suitable for the adaptation of cows to high-grain diets. High-grain diets (>30%) resulted in increased rumen fluid concentrations of several toxic, inflammatory, and unnatural compounds including putrescine, methylamines, ethanolamine, and short-chain fatty acids. Perturbations in several amino acids (phenylalanine, ornithine, lysine, leucine, arginine, valine, and phenylacetylglycine) were also evident. The present study confirms and greatly extends earlier observations on dietary effects on rumen fluid composition and shows that the use of multiple metabolomic platforms permits a far more detailed understanding of metabolic causes and effects. These results may improve our understanding of diet-related rumen metabolism and the influence of grain on the overall health of dairy cattle.

Feeding barley grain steeped in lactic acid modulates rumen fermentation patterns and increases milk fat content in dairy cows

The objectives of the present in vivo and in situ trials were to evaluate whether feeding barley grain steeped in lactic acid (LA) would affect rumen fermentation patterns, in situ dry matter (DM) degradation kinetics, and milk production and composition in lactating dairy cows. The in vivo trial involved 8 rumen-fistulated Holstein cows fed once daily a total mixed ration containing rolled barley grain (27% in DM) steeped for 48 h in an equal quantity of tap water (CTR) or in 0.5% LA (TRT) in a 2 x 2 crossover design. The in situ trials consisted of incubation of untreated rolled barley grain in cows fed CTR or TRT diets and of incubation of 3 different substrates including CTR or barley grain steeped in 0.5% or 1.0% LA (TRT1 and TRT2, respectively) up to 72 h in the rumen. Results of the in vivo trial indicated that cows fed the TRT diet had greater rumen pH during most intensive fermentation phases at 10 and 12 h post-feeding. The latter effect was associated with a shorter duration in which rumen pH was below 5.8 for cows fed the TRT diet (2.4 h) compared with CTR diet (3.9 h). Furthermore, cows fed the TRT diet had lower concentrations of volatile fatty acids at 2 and 4 h post-feeding. In addition, concentrations of preprandial volatile fatty acids were lower in the rumen fluid of cows fed the TRT diet. Results also showed that molar proportion of acetate was lower, whereas propionate tended to increase by feeding cows the TRT diet. Cows fed the TRT diet demonstrated greater rumen in situ lag time of substrate DM degradation and a tendency to lower the fractional degradation rate. Other in situ results indicated a quadratic effect of LA on the effective rumen degradability of substrates whereby the latter variable was decreased from CTR to TRT1 but increased for TRT2 substrate. Although the diet did not affect actual milk yield, fat-corrected milk, percentages of milk protein, and lactose and concentration of milk urea nitrogen, cows fed the TRT diet increased milk fat content and tended to increase fat:protein ratio in the milk. In conclusion, results demonstrated that treatment of barley grain with LA lowered the risk of subacute rumen acidosis and maintained high milk fat content in late-lactating Holstein cows fed diets based on barley grain.

Identification of predictive biomarkers of disease state in transition dairy cows

In dairy cows, periparturient disease states, such as metritis, mastitis, and laminitis, are leading to increasingly significant economic losses for the dairy industry. Treatments for these pathologies are often expensive, ineffective, or not cost-efficient, leading to production losses, high veterinary bills, or early culling of the cows. Early diagnosis or detection of these conditions before they manifest themselves could lower their incidence, level of morbidity, and the associated economic losses. In an effort to identify predictive biomarkers for postpartum or periparturient disease states in dairy cows, we undertook a cross-sectional and longitudinal metabolomics study to look at plasma metabolite levels of dairy cows during the transition period, before and after becoming ill with postpartum diseases. Specifically we employed a targeted quantitative metabolomics approach that uses direct flow injection mass spectrometry to track the metabolite changes in 120 different plasma metabolites. Blood plasma samples were collected from 12 dairy cows at 4 time points during the transition period (-4 and -1 wk before and 1 and 4 wk after parturition). Out of the 12 cows studied, 6 developed multiple periparturient disorders in the postcalving period, whereas the other 6 remained healthy during the entire experimental period. Multivariate data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation between healthy controls and diseased cows at all 4 time points. This analysis allowed us to identify several metabolites most responsible for separating the 2 groups, especially before parturition and the start of any postpartum disease. Three metabolites, carnitine, propionyl carnitine, and lysophosphatidylcholine acyl C14:0, were significantly elevated in diseased cows as compared with healthy controls as early as 4 wk before parturition, whereas 2 metabolites, phosphatidylcholine acyl-alkyl C42:4 and phosphatidylcholine diacyl C42:6, could be used to discriminate healthy controls from diseased cows 1 wk before parturition. A 3-metabolite plasma biomarker profile was developed that could predict which cows would develop periparturient diseases, up to 4 wk before clinical symptoms appearing, with a sensitivity of 87% and a specificity of 85%. This is the first report showing that periparturient diseases can be predicted in dairy cattle before their development using a multimetabolite biomarker model. Further research is warranted to validate these potential predictive biomarkers.

Backgrounding and finishing diets are associated with inflammatory responses in feedlot steers

The objective of this investigation was to study the effects of feeding backgrounding and finishing diets on selected acute phase proteins in the plasma of feedlot steers. Two groups of 12 steers each, at the backgrounding and finishing stages, were offered either a backgrounding (45% barley grain-based concentrate and 55% barley silage on a DM basis) or a finishing (91% barley grain-based concentrate and 9% barley silage) diet for 12 and 15 wk, respectively. Steers at the backgrounding and finishing stages had initial BW of approximately 250 and 380 kg, respectively, at the beginning of the experiment. Blood samples were obtained from a jugular vein at 3-wk intervals during the experimental period beginning at wk 3 or 0 for the backgrounding and finishing periods, respectively. Plasma samples were analyzed for serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP), haptoglobin, and alpha(1)-acid glycoprotein. Steers fed the finishing diet showed peak plasma SAA, LBP, and haptoglobin within 3 wk from the initiation of the diet (20, 23, and 1,940 microg/mL for SAA, LBP, and haptoglobin, respectively). Although plasma alpha(1)-acid glycoprotein reached a peak concentration (449 microg/mL) at the beginning of the finishing phase, no diet effect was obtained for this variable. Steers fed the backgrounding diet showed less variation in the concentrations of plasma acute phase proteins measured; plasma haptoglobin reached a peak concentration (1,720 microg/mL) 9 wk after the beginning of this diet. In conclusion, feeding feedlot steers the backgrounding or finishing diet was associated with increased peak concentrations of acute phase proteins in the plasma. More research is warranted to elucidate the mechanisms behind the inflammatory responses observed in feedlot steers and their implications for health issues and the production efficiency of feedlot operations.

Feeding high proportions of barley grain in a total mixed ration perturbs diurnal patterns of plasma metabolites in lactating dairy cows

The aim of this study was to evaluate effects of feeding increasing proportions of barley grain in a total mixed ration (TMR) on diurnal plasma metabolite fluctuations in high-producing dairy cows. Eight early- to mid-lactation (60 to 140 d in milk) primiparous Holstein cows were assigned to a double 4 x 4 Latin square experimental design. Each experimental period lasted 21 d with the first 11 d used for diet adaptation. Cows were fed a TMR once daily at 0800 h containing no barley grain (control diet), or 15, 30, and 45% (dry matter basis) barley grain as well as barley silage. Blood samples were collected from the tail vein on the last day of each period shortly before (i.e., 0 h) and at 2, 4, 6, 8, 10, and 12 h after the morning feeding. Concentrations of glucose, nonesterified fatty acids (NEFA), beta-hydroxybutyric acid, cholesterol, and lactate in plasma were measured. Results of this study showed that feeding increasing proportions of barley grain affected concentrations of glucose and lactate in plasma with greater plasma glucose and lactate in cows fed the highest amount of grain; however, the amount of grain in the diet did not have an effect on diurnal patterns of plasma glucose. Additionally, the concentration of NEFA in plasma was greater in cows fed the higher grain diets and was greater in the hours following the morning meal than later in the day. The amount of grain in the diet was associated with lower plasma beta-hydroxybutyric acid, which increased particularly after the morning meal. Interestingly, cows fed the most barley grain had the lowest plasma cholesterol and this decreased during the day. In conclusion, the concomitant increase of glucose, lactate, and NEFA as well as the decrease of plasma cholesterol in cows fed high proportions of barley grain suggest that high inclusion of barley grain in the diet played a role in the diurnal patterns of plasma metabolites in lactating dairy cows. However, further research is warranted to understand involvement of these metabolic changes on the long-term health and productivity of dairy cows.

Intermittent parenteral administration of endotoxin triggers metabolic and immunological alterations typically associated with displaced abomasum and retained placenta in periparturient dairy cows

This study sought to investigate the effects of induced intermittent endotoxemia on plasma mediators of carbohydrate and lipid metabolism, humoral immunity, and clinical health status in periparturient dairy cows. Sixteen pregnant Holstein cows were blocked by parity and day of calving, and were randomly allocated to 1 of 2 different treatment groups. Eight cows were infused intravenously (i.v.) with 100mL of sterile saline and served as the control group (CON). The other 8 cows were infused i.v. with 100mL of sterile saline containing 3 increasing doses of lipopolysaccharide (LPS), from Escherichia coli O111:B4, for 3 consecutive weeks during the 2 wk before and 1 wk after parturition as follows: (1) 0.01 μg of LPS/kg of body weight (BW) on d -14 and -10; (2) 0.05 μg of LPS/kg of BW on d -7 and -3; and (3) 0.1 μg of LPS/kg of BW on d 3 and 7 postpartum. Nine blood samples were collected during the experimental period (i.e., from -14 to 28 d postpartum) and analyzed for calcium, zinc, iron, copper, glucose, lactate, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), cholesterol, insulin, cortisol, serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), and anti-LPS IgA, IgG, and IgM. Results showed that intermittently induced endotoxemia decreased feed intake and milk production and triggered alterations in plasma cholesterol, BHBA, Hp, Ca, Cu, and anti-LPS IgG and IgM. All of these changes were associated with a greater number of cows affected by metabolic disorders such as left displaced abomasum (LDA, 2 from 8 LPS cows vs. 0 from 8 CON cows) and retained placenta (RP; 4 from 8 LPS cows vs. 0 from 8 CON cows). In addition, the discriminant analysis differently clustered the cow responses within LPS group, each corresponding to LDA, RP, and the cows displaying no clinical health problems (LPS-NO). The stepwise selection procedure of the best discriminant variables revealed that plasma Ca and anti-LPS IgG, as well as glucose and cortisol, were the best discriminating variables for cows affected by LDA, whereas NEFA and cholesterol better discriminated for cows affected by RP. This analysis also revealed that the cluster of plasma variables including plasma Cu, SAA, BHBA, and anti-LPS IgA were the best discrimination for the LPS-NO group. In conclusion, our results indicate a role of endotoxemia, during the periparturient period, in development of metabolic and immune disturbances, as well as in the etiopathology of displaced abomasum and retained placenta in dairy cows.

Perturbations of plasma metabolites correlated with the rise of rumen endotoxin in dairy cows fed diets rich in easily degradable carbohydrates

Feeding dairy cows diets high in easily degradable carbohydrates increases the incidence of rumen and systemic metabolic disorders; however, the triggering factor is not well understood. In this study, dairy cows were fed 4 different amounts of barley grain-based concentrate at 15, 30, 45, and 60% (dry matter basis) of a total mixed ration to determine whether alterations in the rumen environment would be associated with perturbations of the plasma profile of selected metabolites. In addition, associations among free rumen endotoxin and several plasma metabolites were determined. The study was a replicated 4 × 4 Latin square design with 8 rumen-cannulated lactating dairy cows (60 ± 15 d in milk). Multiple rumen fluid and blood plasma samples were collected and analyzed for pH and rumen fluid endotoxin and for concentrations of glucose, insulin, cholesterol, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), and lactate in the plasma. Rumen pH decreased below 6.0, from 8 to 12h after the morning feeding, with the augmentation of the proportion of concentrate in the diet of ≥ 30%. Feeding diets with >30% concentrate resulted in a rise of free endotoxin in the rumen fluid (8.87 ± 0.39 μg/mL). Inclusion of 60% concentrate in the total mixed ration was associated with enhanced concentrations of glucose (64.5 ± 1.0 mg/dL) and lactate (540.9 ± 36.5 μmol/L) and lowered cholesterol (265.5 ± 13.7 mg/dL), BHBA (449.1 ± 47.4 μmol/L), and NEFA (138.8 ± 19.1 μEq/L) in the blood plasma. The regression analysis revealed that greater concentrations of plasma lactate and lower concentrations of cholesterol, BHBA, and NEFA were related to the rise of rumen endotoxin. Interestingly, 93% of the increase in the plasma lactate was explained by the rise of rumen endotoxin. Moreover, the analysis revealed inverse relationships of rumen endotoxin with plasma cholesterol (R(2)=0.47), BHBA (R(2)=0.37), and NEFA (R(2)=0.50) and a biphasic response of plasma insulin (R(2)=0.58). Taken together, feeding dairy cows diets rich in rumen-degradable carbohydrates and low in fiber led to lower rumen pH and a large accumulation of rumen endotoxin; the latter was correlated with perturbations of plasma metabolites allied to carbohydrate and lipid metabolism.

The Bovine Ruminal Fluid Metabolome

The rumen is a unique organ that serves as the primary site for microbial fermentation of ingested plant material for domestic livestock such as cattle, sheep and goats. The chemical composition of ruminal fluid is thought to closely reflect the healthy/unhealthy interaction between rumen microflora and diet. Just as diet and feed quality is important for livestock production, rumen health is also critical to the growth and production of high quality milk and meat. Therefore a detailed understanding of the chemical composition of ruminal fluid and the influence of diet on its composition could help improve the efficiency and effectiveness of farming and veterinary practices. Consequently we have undertaken an effort to comprehensively characterize the bovine ruminal fluid metabolome. In doing so, we combined NMR spectroscopy, inductively coupled plasma mass-spectroscopy (ICP-MS), gas chromatography-mass spectrometry (GC-MS), direct flow injection (DFI) mass spectrometry and lipidomics with computer-aided literature mining to identify and quantify essentially all of the metabolites in bovine ruminal fluid that can be routinely detected (with today’s technology). The use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these techniques. Tables containing the set of 246 ruminal fluid metabolites or metabolite species, their concentrations, related literature reference and links to their known diet associations for the bovine rumen metabolome are freely available at http://www.rumendb.ca.