Urinary metabolomics fingerprinting around parturition identifies metabolites that differentiate lame dairy cows from healthy ones.

Lameness is a very important disorder of periparturient dairy cows with implications on milk production and composition as well as with consequences on reproductive performance. The aetiology of lameness is not clear although there have been various hypotheses suggested over the years. The objective of this study was to metabotype the urine of dairy cows prior to, during and after the onset of lameness by evaluating at weeks -8, -4 pre-calving, the week of lameness diagnosis, and +4 and +8 weeks post-calving. We used a metabolomics approach to analyse urine samples collected from dairy cows around calving (6 cows with lameness v. 20 healthy control cows). A total of 153 metabolites were identified and quantified using an in-house MS library and classified into 6 groups including: 11 amino acids (AAs), 39 acylcarnitines (ACs), 3 biogenic amines (BAs), 84 glycerophospholipids, 15 sphingolipids and hexose. A total of 23, 36, 40, 23 and 49 metabolites were observed to be significantly different between the lame and healthy cows at -8 and -4 weeks pre-calving, week of lameness diagnosis as well as at +4 and +8 weeks post-calving, respectively. It should be noted that most of the identified metabolites were elevated; however, a few of them were also lower in lame cows. Overall, ACs and glycerophospholipids, specifically phosphatidylcholines (PCs), were the metabolite groups displaying the strongest differences in the urine of pre-lame and lame cows. Lysophosphatidylcholines (LysoPCs), although to a lesser extent than PCs, were altered at all time points. Alterations in urinary AA concentrations were also observed during the current study for four time points. During the pre-calving period, there was an observed elevation of arginine (-8 week), tyrosine (-8 week) and aspartate (-4 week), as well as a depression of urinary glutamate (-4 weeks). In the current study, it was additionally observed that concentrations of several sphingomyelins and one BA were altered in pre-lame and lame cows. Symmetric dimethylarginine was elevated at both -8 weeks pre-calving and the week of lameness diagnosis. Data showed that urinary fingerprinting might be a reliable methodology to be used in the future to differentiate lame cows from healthy ones.

Urine metabolic fingerprinting can be used to predict the risk of metritis and highlight the pathobiology of the disease in dairy cows.

INTRODUCTION: Metritis is an uterine pathology that causes economic losses for the dairy industry. It is associated with lower reproductive efficiency, increased culling rates, decreased milk production and increased veterinary costs. OBJECTIVES: To gain a more detailed view of the urine metabolome and to detect metabolite signature in cows with metritis. In addition, we aimed to identify early metabolites which can help to detect cows at risk to develop metritis in the future. METHODS: We used nuclear magnetic resonance spectroscopy starting at 8 and 4 weeks prior to the expected day of parturition, during the week of diagnosis of metritis, and at 4 and 8 weeks after diagnosis of metritis in Holstein dairy cows. RESULTS: At 8 weeks before parturition, pre-metritic cows had a total of 30 altered metabolites. Interestingly, 28 of them increased in urine when compared with control cows (P < 0.05). At 4 weeks before parturition, 34 metabolites were altered. At the week of diagnosis of metritis a total of 20 metabolites were altered (P < 0.05). The alteration continued at 4 and 8 weeks after diagnosis. CONCLUSIONS: The metabolic fingerprints in the urine of pre-metritic and metritic cows point toward excretion of multiple amino acids, tricarboxylic acid cycle metabolites and monosaccharides. Combination of galactose, leucine, lysine and panthotenate at 8 weeks before parturition might serve as predictive biomarkers for metritis.

Targeted metabolomics: new insights into pathobiology of retained placenta in dairy cows and potential risk biomarkers.

A targeted quantitative metabolomics approach was used to study temporal changes of serum metabolites in cows that normally released their fetal membranes and those that retained the placenta. We identified and measured serum concentrations of 128 metabolites including amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids and hexose at -8 and -4 weeks before parturition, during the week of retained placenta (RP) diagnosis, and at +4 and +8 weeks after parturition. In addition, we aimed at identifying metabolite signatures of pre-RP in the serum that might be used as predictive biomarkers for risk of developing RP in dairy cows. Results revealed major alterations in the metabolite fingerprints of pre-RP cows starting as early as -8 weeks before parturition and continuing as far as +8 weeks after calving. Biomarker candidates found in this study are mainly biomarkers of inflammation which might not be specific to RP. Therefore, the relevance of serum Lys, Orn, acetylornithine, lysophophatidylcholine LysoPC a C28:0, Asp, Leu and Ile as potential serum biomarkers for prediction of risk of RP in dairy cows will have to be tested in the future. In addition, lower concentrations of LysoPCs, Trp, and higher kynurenine in the serum during prepartum and the week of occurrence of RP suggest involvement of inflammation in the pathobiology of RP.

Intravaginal probiotics modulated metabolic status and improved milk production and composition of transition dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of probiotics (a lactic acid bacteria cocktail) around parturition would influence metabolic status and increase milk production of transition dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of the 3 experimental groups receiving intravaginal infusion of probiotics or carrier (i.e., sterile skim milk) once a week at wk -2, -1, and +1 relative to calving as follows: 2 consecutive probiotics before parturition and 1 carrier dose after parturition (TRT1), 3 consecutive probiotics doses around parturition (TRT2), and 3 consecutive carrier doses around parturition (CTR). The probiotics were a lyophilized culture mixture composed of FUA3089 and FUA3138 and FUA3140 with a cell count of 10 to 10 cfu/dose. Blood was sampled from wk -2 to +3 and milk was sampled on the third day in milk (DIM) and from wk +1 to +5 on a weekly basis. Feed intake and milk production was monitored until wk +8. Results showed that the TRT2 group (366.12 ± 49.77 µmol/L) had a lower ( = 0.01) concentration of NEFA in the serum than the CTR group (550.85 ± 47.16 µmol/L). The concentrations of IgG in the milk were 32.71 ± 3.00 mg/mL in the TRT1 group, 17.47 ± 4.54 mg/mL in the TRT2 group, and 6.73 ± 3.43 mg/mL in the CTR group at 3 DIM ( < 0.01). Meanwhile, both the TRT1 and the TRT2 group had lower haptoglobin in the milk compared with the CTR group at 3 DIM ( < 0.01). The TRT1 group had greater milk protein content than the CTR group (2.99 ± 0.04 vs. 2.82 ± 0.04%; = 0.02), whereas the TRT2 group tended to have greater lactose content compared with the CTR group (4.53 ± 0.03 vs. 4.44 ± 0.03%; = 0.05). The effect of treatment interacted with parity with regards to milk production and feed efficiency. Multiparous cows in the TRT1 and TRT2 groups had greater milk production and feed efficiency than those in the CTR group ( < 0.01 and = 0.02, respectively). Among primiparous cows, those in the TRT2 group had greater milk production ( = 0.04) whereas those in the TRT1 group had lower feed intake ( < 0.01) than those in the CTR group. Both the TRT1 and the TRT2 groups had enhanced feed efficiency compared with the CTR group ( < 0.01). In conclusion, intravaginal infusion of lactic acid bacteria modulated concentrations of selected serum metabolites and milk components and increased milk efficiency of transition dairy cows.

Intravaginally administered lactic acid bacteria expedited uterine involution and modulated hormonal profiles of transition dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of lactic acid bacteria (LAB) around parturition could expedite involution rate of the uterus and improve reproductive performance of postpartum dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of 3 experimental groups: (1) 1 dose of LAB in wk -2 and -1 and 1 dose of carrier in wk 1 relative to the expected day of parturition (TRT1); (2) 1 dose of LAB in wk -2, -1, and 1 (TRT2); and (3) 1 dose of carrier in wk -2, -1, and 1 (CTR). The LAB treatment was a lyophilized mixture of Lactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 with a cell count of 10(8) to 10(9) cfu/dose. Uterine involution and ovarian activity was evaluated by transrectal ultrasonography weekly from d 7 to 49 postpartum. Blood samples were collected from a subset of cows to quantify prostaglandin (PG) F2α metabolite (PGFM), PGE2, and progesterone. Cows treated with LAB had smaller cross-sectional areas of gravid horn and uterine body on d 14 postpartum. Cows in TRT2 resumed ovarian cyclicity earlier, as indicated by increased concentrations of serum progesterone. Cows in TRT1 had fewer days open than those in the CTR (110 vs. 150 d), whereas cows in TRT2 and CTR did not differ in days open. In addition, both TRT1 and TRT2 increased the concentrations of PGFM at calving week, and cows in TRT2 also had greater concentrations of PGE2 on d 14 and d 21 postpartum relative to CTR. Overall, cows treated intravaginally with LAB had smaller gravid horn and uterine body on d 14 postpartum than those in the CTR group. Treatment with LAB also increased concentrations of serum PGFM (3,533±328pg/mL in TRT1, 4,470±372pg/mL in TRT2, and 2,000±328pg/mL in CTR on d 0, respectively), with the TRT1 group having fewer cows that resumed ovarian cyclicity but fewer days open compared with both TRT2 and CTR groups. More research is warranted to better understand the mechanism(s) by which intravaginal LAB expedited uterine involution and affected hormonal profiles.

Intravaginal administration of lactic acid bacteria modulated the incidence of purulent vaginal discharges, plasma haptoglobin concentrations, and milk production in dairy cows.

This investigation studied the effects of intravaginal administration of a mixture of lactic acid bacteria (LAB) on the incidence of purulent vaginal discharges (PVD), plasma haptoglobin concentrations, and milk production in dairy cows. A total of 82 pregnant primiparous and multiparous Holstein dairy cows were used in this study. Half of the cows received intravaginally 1mL of LAB at 10(10)-10(12)cfu/mL and the other half 1mL of reconstituted skim milk (i.e., carrier) (controls). Administration of LAB was conducted once per wk during 2 and 1wk before the expected day of calving and at 1, 2, 3, and 4wk postpartum. Data demonstrated that intravaginal administration of LAB decreased the occurrence of PVD at 3wk postpartum (P<0.05). Concentrations of plasma haptoglobin, an acute phase protein often associated with uterine infections, was lower in cows treated with the LAB mixture at 2wk (P<0.001) and 3wk (P<0.05) postpartum. Treatment with LAB did not improve overall pregnancy rate, but the treated multiparous cows produced more milk than their control counterparts (P<0.05), whereas no difference was observed in primiparous cows regarding milk yield (P>0.05). Overall, this is the first study demonstrating that intravaginal LAB administration lowers the incidence of PVD and enhances milk production in dairy cows. Further research is warranted to evaluate the effects of LAB on reproductive performance in a larger cohort of cows.

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.

Intermittently-induced endotoxaemia has no effect on post-challenge plasma metabolites, but increases body temperature and cortisol concentrations in periparturient dairy cows.

This study evaluated the responses of plasma cortisol, metabolites and body temperature to intermittently-induced endotoxaemia in periparturient cows. Sixteen Holstein cows were randomly allocated to one of the two treatment groups. Cows were infused intravenously either with saline solution (control) or with the same solution containing 3 increasing doses of lipopolysaccharide (LPS) for 3 consecutive weeks around parturition as follows: 0.01 µg LPS/kg body weight (BW) on d -14 and -10 prepartum, 0.05 µg LPS/kg BW on d -7 and -3 prepartum, and 0.1 µg LPS/kg BW on d 3 and 7 postpartum. Blood samples were measured shortly before and in 8 time-points after (up to 6h) the challenges on d -14, -7, 3, and 7 to evaluate the post-challenge plasma profile. Results showed greater concentrations of plasma cortisol, in particular after the second and third LPS challenge. An increase in body temperature was recorded after administration of the greatest LPS dose, but this effect diminished during the very last LPS challenge. A biphasic response of glucose was observed; a linear increase up to 60 min after the second LPS challenge followed by a rapid decrease thereafter. Other plasma variables like lactate, cholesterol, non-esterified fatty acids, and beta-hydroxybutyrate were not affected by treatment. In conclusion, LPS administrations did not notably affect post-challenge metabolic responses in periparturient dairy cows but increased the level of plasma cortisol and the body temperature after the highest LPS challenge.

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.

Barley grain-based diet treated with lactic acid and heat modulated plasma metabolites and acute phase response in dairy cows.

This study investigated the effects of feeding barley grain treated with lactic acid (LA) and heat on the profile of plasma metabolites related to carbohydrate and lipid metabolism and variables related to rumen health and acute phase response. Eight primiparous rumen-fistulated lactating Holstein cows were randomly assigned, in a crossover design, to 1 of the 2 dietary treatments consisting of 32% (DM basis) rolled barley grain steeped in an equal quantity of either tap water alone (CTR) or a 1.0% LA solution and heated at 55°C for 48 h (LAH). Each experimental period was 21 d, with the last 10 d used for measurements. Blood samples were collected on d 1, 3, 5, 7, 9, and 11 before the morning feeding and on the last day of each period at 0, 2, 4, 6, 8, 10, and 12 h postfeeding to measure glucose, lactate, cholesterol, beta-hydroxybutyrate (BHBA), NEFA, haptoglobin (Hp), serum amyloid A (SAA), and tumor necrosis factor-alpha (TNF-α). Also, rumen samples were collected on d 1, 5, and 11 as well as at 0, 4, 8, and 12 h postfeeding on the last day of each period for measuring the concentration of rumen endotoxin. Results of the day-to-day analysis indicated that cows fed the LAH diet had reduced preprandial concentrations of rumen endotoxin (472 vs. 793 ng/mL; P < 0.01) and cholesterol and greater lactate in the plasma; however, treatment had no effect on plasma Hp and TNF-α (P > 0.10). Postprandial responses showed that the LAH diet tended to decrease the concentration of SAA (4.67 vs. 8.50 µg/mL; P = 0.06). Also, there was a treatment by time interaction for rumen endotoxin (P < 0.01), suggesting a role for both the treatment and the time of sampling on this variable. Furthermore, greater concentration of BHBA and a tendency for greater NEFA and reduced concentrations of plasma glucose were observed in cows fed the LAH diet. In conclusion, results indicated that feeding dairy cows barley grain steeped in 1.0% LA and treated with heat modulated the profile of plasma metabolites and acute phase response.

Meta-analysis reveals threshold level of rapidly fermentable dietary concentrate that triggers systemic inflammation in cattle.

This study examined the extent by which changes in the concentrate level and neutral detergent fiber (NDF) content in the diet as well as the severity of acidotic insult, measured as the duration time of rumen pH below 6.0 and daily mean rumen pH, and the concentration of endotoxin in the rumen fluid are involved in the development of inflammatory conditions in cattle. A meta-analytical approach accounting for inter- and intraexperimental variation was used to generate prediction models, and data from recent studies were used to parameterize these models. A total of 10 recently conducted experiments with 43 different dietary treatments fulfilled the criteria for inclusion in this study. Diets of all of the experiments included in this meta-analysis were based on rapidly degradable grain sources, such as barley and wheat, and the findings of this study apply only to these kinds of diets. Data indicated that greater levels of concentrate in the diet were associated with increased concentrations of rumen endotoxin (R(2)=0.27), plasma haptoglobin (R(2)=0.19), and serum amyloid A (SAA) level (R(2)=0.46). Similar correlations, but in opposite directions, were observed between dietary NDF content and rumen endotoxin (R(2)=0.39) and plasma SAA concentrations (R(2)=0.22). The meta-analysis revealed that the relationships between those variables were not linear. Additionally, the breakpoint model fitted to the data of rumen endotoxin, plasma haptoglobin, and SAA indicated the presence of a threshold level of dietary concentrate and NDF, above which those responses became linear to increasing amounts of concentrate or decreasing contents of NDF in the diet. Also, feeding cattle more than 44.1% concentrate or less than 39.2% NDF in the diet was associated with a linear increase in the risk of systemic inflammation. Low daily mean rumen pH (R(2)=0.38) and duration of rumen pH <6.0 (R(2)=0.59) were associated with increased concentrations of endotoxin in the rumen fluid; although those events were not always associated with systemic inflammation. Accordingly, only 15 to 21% of the overall variation in the responses of SAA was explained by variables of rumen pH, whereas the concentrate level in the diet accounted for 46% of this variation. In conclusion, data from this study indicated the presence of thresholds of dietary concentrate and NDF levels in the diets based on rapidly fermentable grains beyond which the risk of systemic inflammation in cattle increases linearly.

Invited review: Role of physically effective fiber and estimation of dietary fiber adequacy in high-producing dairy cattle.

Highly fermentable diets require the inclusion of adequate amounts of fiber to reduce the risk of subacute rumen acidosis (SARA). To assess the adequacy of dietary fiber in dairy cattle, the concept of physically effective neutral detergent fiber (peNDF) has received increasing attention because it amalgamates information on both chemical fiber content and particle size (PS) of the feedstuffs. The nutritional effects of dietary PS and peNDF are complex and involve feed intake behavior (absolute intake and sorting behavior), ruminal mat formation, rumination and salivation, and ruminal motility. Other effects include fermentation characteristics, digesta passage, and nutrient intake and absorption. Moreover, peNDF requirements depend on the fermentability of the starch source (i.e., starch type and endosperm structure). To date, the incomplete understanding of these complex interactions has prevented the establishment of peNDF as a routine method to determine dietary fiber adequacy so far. Therefore, this review is intended to analyze the quantitative effects of and interactions among forage PS, peNDF, and diet fermentability with regard to rumen metabolism and prevention of SARA, and aims to give an overview of the latest achievements in the estimation of dietary fiber adequacy in high-producing dairy cattle. Recently developed models that synthesize the effects of both peNDF and fermentable starch on rumen metabolism appear to provide an appropriate basis for estimation of dietary fiber adequacy in high-producing dairy cows. Data suggest that a period lasting more than 5 to 6h/d during which ruminal pH is <5.8 should be avoided to minimize health disturbances due to SARA. The knowledge generated from these modeling approaches recommends that average amounts of 31.2% peNDF inclusive particles >1.18mm (i.e., peNDF(>1.18)) or 18.5% peNDF inclusive particles >8mm (i.e., peNDF(>8)) in the diet (DM basis) are required. However, inclusion of a concentration of peNDF(>8) in the diet beyond 14.9% of diet DM may lower DM intake level. As such, more research is warranted to develop efficient feeding strategies that encourage inclusion of energy-dense diets without the need to increase their content in peNDF above the threshold that leads to lower DM intake. The latter would require strategies that modulate the fermentability characteristics of the diet and promote absorption and metabolic capacity of ruminal epithelia 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, ß-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, ß-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.

Feeding rolled barley grain steeped in lactic acid modulated energy status and innate immunity in dairy cows.

Feeding dairy cows large proportions of cereal grain is commonly associated with rumen acidosis, activation of innate immunity, and perturbation of intermediary metabolism. We previously showed that steeping barley grain in 0.5% lactic acid (LA) decreased the rate of starch degradation, lowered the risk of subacute rumen acidosis, modulated rumen fermentation profile, and increased milk fat content in dairy cows. This study sought to investigate whether feeding of LA-treated barley grain would affect carbohydrate and lipid metabolism as well as innate immunity. Eight rumen-fistulated late-lactation (approximately 217 d in milk, DIM) Holstein cows were randomly assigned, in a 2 × 2 crossover design, to 1 of the 2 dietary treatments consisting of 27% (dry matter basis) rolled barley grain steeped for 48 h in an equal volume (wt/vol) of tap water (CTR) or 0.5% LA (TRT). Each experimental period lasted 21 d, with the first 11 d for diet adaptation. Blood and rumen samples were collected on d 12, 15, 17, and 21 of the experimental period before the morning feeding to evaluate the effects of dietary treatment on preprandial day-to-day variation of plasma and rumen variables. To establish the effect of treatment on diurnal variation of plasma variables, blood samples were collected on the last day of each period at 0, 2, 4, 6, 8, 10, and 12h after the morning feeding (i.e., 0800 h). Results of the day-to-day study showed that cows fed the TRT diet had greater overall preprandial concentrations of glucose, cholesterol, and insulin, and a lower concentration of haptoglobin in plasma. Diurnal data indicated lower concentrations of haptoglobin and serum amyloid A and a tendency for greater plasma lactate in cows fed the TRT diet. A treatment by time interaction was observed for glucose, lactate, insulin, haptoglobin, and lipopolysaccharide-binding protein, suggesting a role for both the processing of grain and the time of sampling on those variables. No effect of diet on plasma concentrations of cortisol, ß-hydroxybutyrate, and nonesterified fatty acids or rumen endotoxin was evidenced. Taken together, our results demonstrated that feeding barley grain steeped in 0.5% LA modulated both energy status and innate immunity of dairy cows fed relatively high levels (45% of dry matter) of dietary concentrate.

Dietary supplementation of n-3 PUFA reduces weight gain and improves postprandial lipaemia and the associated inflammatory response in the obese JCR:LA-cp rat.

BACKGROUND: Postprandial dyslipidaemia occurs in obesity and insulin resistance (IR), and is associated with an increased risk of developing cardiovascular disease. We have recently established that the JCR:LA-cp rodent model develops postprandial dyslipidaemia concomitant with complications of the metabolic syndrome. Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) are proposed to modulate plasma lipids, serum hormone levels, lipoprotein metabolism and the inflammatory state; however, results remain inconsistent during conditions of IR. AIM: To assess the acute metabolic and inflammatory effects of dietary fish oil supplementation on existing postprandial dyslipidaemia in the JCR:LA-cp model. METHODS: JCR:LA-cp rats (14 weeks of age) were fed either a control, isocaloric, lipid balanced diet (15% w/w total fat, 1.0% cholesterol, P:S ratio 0.4), a lipid balanced diet with 5% n-3 PUFA [fish oil derived eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA)] or a lipid balanced diet with 10% n-3 PUFA for 3 weeks. Fasting plasma lipid, cytokine levels, postprandial chylomicron (apoB48) metabolism and the postprandial inflammatory response [haptoglobin and lipopolysaccharide binding protein (LBP)] were assessed following a standardized 'oral fat challenge'. RESULTS: n-3 PUFA treatment resulted in a significant improvement (i.e. decrease) in the postprandial response for triglyceride (45%) (p < 0.05), apoB48 (45%) (p < 0.03) and LBP (33%) (p < 0.05) compared to controls (measured as area under the clearance curve). In contrast, we observed a significant elevation in postprandial haptoglobin (165%) (p < 0.001) in obese rats supplemented with 10% n-3 PUFA. Treatment with 5% n-3 PUFA in the JCR:LA-cp obese animals resulted in a complementary decrease in total body weight gain (6%) (p < 0.001) and an increase (i.e. improvement) in adiponectin (33%) (p < 0.05) compared to controls, without a concomitant reduction in food intake. CONCLUSION: Acute dietary n-3 PUFA dietary supplementation can improve fasting as well as postprandial lipid metabolism and components of the associated inflammatory response in the JCR:LA-cp rat. Further, moderate dose n-3 PUFA supplementation may reduce corresponding body weight during conditions of hypercholesterolaemia and/or modulate inflammation associated with obesity and the metabolic syndrome.

Dose response to cinnamaldehyde supplementation in growing beef heifers: ruminal and intestinal digestion.

The objective of this study was to determine if cinnamaldehyde (CIN) could be used to improve feed intake, digestion, and immune status in growing beef heifers fed high-concentrate diets. The experiment was designed as a 4 x 4 Latin square using 4 ruminally and duodenally cannulated beef heifers with 4 treatments: control (no CIN added), 400 mg/d of CIN (low), 800 mg/d of CIN (medium), and 1,600 mg/d of CIN (high), and four 21-d periods. Feed intake, rumen pH and fermentation characteristics, site and extent of digestion, microbial N synthesis, blood metabolites, and acute phase protein response were measured. The diets consisted of 15% barley silage, 80% dry-rolled barley grain, and 5% supplement (DM basis). Intakes (kg/d) of DM, OM, NDF, starch, and N were quadratically (P = 0.04) changed with increasing CIN supplementation. The amount of OM fermented in the rumen quadratically (P = 0.02) decreased with increasing CIN. Digestibilities (% of intake) of OM, NDF, and N in the rumen were not affected by supplementing with low and medium CIN, but they were reduced by 8% (P = 0.10), 31% (P = 0.05), and 17% (P = 0.05), respectively, with high CIN. Similarly, digestibilities of OM and NDF in the total tract also tended to be reduced by 7% (P = 0.10) and 20% (P = 0.10), respectively, with high CIN because supplementation of CIN had minimal effects on intestinal digestibility. Flows (g/d) of microbial N and other nutrients to the duodenum were not affected by CIN supplementation, even though the amount of ruminal fermented OM varied with level of CIN supplementation. Rumen pH, total VFA concentration, and molar proportions of individual VFA were not affected by CIN. Although concentrations of NEFA (P = 0.06) and triglyceride (P = 0.01) were quadratically changed with increasing CIN supplementation, blood concentrations of glucose and urea N, white blood cell counts, serum amyloid A, and lipopolysaccharide in plasma were not affected by CIN. Plasma haptoglobin numerically (P = 0.11) decreased with the medium dose of CIN fed compared with control. The results indicate that supplementation of a high-concentrate diet with a low dose of CIN resulted in small increases in nutrient availability in the rumen due to increased feed intake and greater ruminal digestion of OM. However, feed intake and ruminal digestion of feeds were adversely affected when a high dose of CIN was used.

Cinnamaldehyde in feedlot cattle diets: intake, growth performance, carcass characteristics, and blood metabolites.

Cinnamaldehyde (CIN), a natural chemical compound found in the bark of cinnamon trees, can alter rumen fermentation by inhibiting selected ruminal microbes, and consequently, may improve growth performance and feed efficiency of animals. The objective of this study was to evaluate the effects of supplementing the diet of feedlot cattle with CIN on intake, growth performance, carcass characteristics, and blood metabolites. Seventy yearling steers (BW = 390 +/- 25.2 kg) were assigned to a randomized complete block design with 5 treatments: control (no additive), monensin (MO; 330 mg*steer(-1)*d(-1)), and 400, 800, or 1,600 mg of CIN*steer(-1)*d(-1). At the start of the experiment, steers were blocked according to BW and assigned to 14 blocks of 5 cattle, with cattle within block assigned to treatments. The diets consisted of 9% barley silage, 86% dry-rolled barley grain, and 5% supplement (DM basis). Dry matter intake responded quadratically (P = 0.03) to CIN supplementation with 13% more feed consumed for steers fed CIN (mean of 3 CIN levels) compared with those fed control during the first 28 d of the experiment, and with a tendency of 4% increase over the entire experiment. The ADG (kg/d) tended to respond quadratically (P = 0.08) to CIN supplementation during the first 28 d, but was not affected over the entire experiment (112 d). Feed efficiency (G:F) linearly declined (P = 0.03) during the first 28 d with CIN supplementation and was quadratically affected between d 29 to 56 and d 85 to 112 by CIN dose. Supplementation of MO did not affect (P > 0.15) DMI or growth performance at any time during the experiment. Serum NEFA concentrations were reduced (P = 0.05) by 35, 29, 30, and 22%, respectively, on d 56, 84, 112, and overall with CIN supplementation. Concentrations of serum amyloid A were reduced on d 28 by 56, 60, or 56% for 800 mg of CIN, 1,600 mg of CIN, and MO, respectively, compared with control. Plasma concentrations of lipopolysaccharide binding protein were linearly decreased (P = 0.05) with increasing CIN supplementation on d 28. Results indicate that supplementing a feedlot finishing diet with a small dose of CIN ameliorated feed intake during the initial month but had minimal effects on ADG, feed efficiency, and carcass traits over the entire experiment. Including CIN in the diet of feedlot cattle, particularly early in the feeding period, may help promote intake and reduce the effects of stress.

Strong associations among rumen endotoxin and acute phase proteins with plasma minerals in lactating cows fed graded amounts of concentrate.

The objective of this investigation was to determine associations among rumen endotoxin, plasma serum amyloid A (SAA), and C-reactive protein (CRP) with plasma Ca, Fe, Zn, and Cu in lactating cows challenged with graded amounts of rolled barley grain in the diet (i.e., 0, 15, 30, and 45% of DMI). Correlative relationships among variables were determined by linear and nonlinear regression procedures adjusted for the effects of day, animal, and experimental period. Increasing the amount of grain in the diet was successful in inducing an acute phase response, as assessed by augmentation of rumen endotoxin and plasma CRP and SAA (P < 0.01). The correlative analysis revealed inverse, nonlinear relationships of rumen endotoxin and plasma SAA with circulating Ca. Interestingly, plasma Ca reached the asymptotic plateau at 10.6 mg/dL. The increase in rumen endotoxin was associated with an abrupt decrease in plasma Fe (R(2) = 0.91; P < 0.001). A similar relationship, although at a reduced estimation accuracy (R(2) = 0.21; P < 0.01), was observed between rumen endotoxin and plasma Zn. Augmentation of rumen endotoxin and plasma CRP resulted in a positive, biphasic response of plasma Cu. In conclusion, the increase in rumen endotoxin in response to high-grain diets, and the resulting increases in plasma SAA and CRP, were strongly correlated with fluctuations of plasma minerals. Results suggest that new feeding strategies should be developed to curb the release of endotoxin in the rumen fluid to prevent perturbing minerals in the plasma.

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.