Perceived usefulness and ease of use of fundoscopy by medical students: a randomised crossover trial of six technologies (eFOCUS 1).

BACKGROUND: Fundoscopy outside ophthalmology is in decline, and the technical demands of the traditional direct ophthalmoscope examination are likely contributing. Alternative fundoscopy technologies are increasingly available, yet valid comparisons between fundoscopy technologies are lacking. We aimed to assess medical students' perceptions of usefulness and ease of use of traditional and contemporary fundus-viewing technologies including smartphone fundoscopy. METHODS: One hundred forty-six second-year medical students participated in a cross-sectional, randomised, cross-over study of fundoscopy methods. Medical students completed small group training sessions using six current fundoscopy technologies including: a non-mydriatic fundus camera; two types of direct fundoscopy; and three types of smartphone fundoscopy. A novel survey of perceived usefulness and ease of use was then completed by students. RESULTS: Repeated-measures ANOVA found students rated both the perceived usefulness (p< 0.001) and ease of use (p< 0.001) of smartphone fundoscopy significantly higher than both the non-mydriatic camera and direct fundoscopy. CONCLUSIONS: Smartphone fundoscopy was found to be significantly more useful and easier to use than other modalities. Educators should optimise student access to novel fundoscopy technologies such as smartphone fundoscopy which may mitigate the technical challenges of fundoscopy and reinvigorate use of this valuable clinical examination.

177Lu radiolabeling and preclinical theranostic study of 1C1m-Fc: an anti-TEM-1 scFv-Fc fusion protein in soft tissue sarcoma.

PURPOSE: TEM-1 (tumor endothelial marker-1) is a single-pass transmembrane cell surface glycoprotein expressed at high levels by tumor vasculature and malignant cells. We aimed to perform a preclinical investigation of a novel anti-TEM-1 scFv-Fc fusion antibody, 1C1m-Fc, which was radiolabeled with 177Lu for use in soft tissue sarcomas models. METHODS: 1C1m-Fc was first conjugated to p-SCN-Bn-DOTA using different excess molar ratios and labeled with 177Lu. To determine radiolabeled antibody immunoreactivity, Lindmo assays were performed. The in vivo behavior of [177Lu]Lu-1C1m-Fc was characterized in mice bearing TEM-1 positive (SK-N-AS) and negative (HT-1080) tumors by biodistribution and single-photon emission SPECT/CT imaging studies. Estimated organ absorbed doses were obtained based on biodistribution results. RESULTS: The DOTA conjugation and the labeling with 177Lu were successful with a radiochemical purity of up to 95%. Immunoreactivity after radiolabeling was 86% ± 4%. Biodistribution showed a specific uptake in TEM-1 positive tumor versus liver as critical non-specific healthy organ, and this specificity is correlated to the number of chelates per antibody. A 1.9-fold higher signal at 72 h was observed in SPECT/CT imaging in TEM-1 positive tumors versus control tumors. CONCLUSION: TEM-1 is a promising target that could allow a theranostic approach to soft-tissue sarcoma, and 1C1m-Fc appears to be a suitable targeting candidate. In this study, we observed the influence of the ratio DOTA/antibody on the biodistribution. The next step will be to investigate the best conjugation to achieve an optimal tumor-to-organ radioactivity ratio and to perform therapy in murine xenograft models as a prelude to future translation in patients.

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.

Recent trends in water quality in an agricultural catchment in Eastern Scotland: elucidating the roles of hydrology and land use.

Across the EU, programmes of measures have been introduced as part of river basin management planning as a means of tackling problems of diffuse pollution from agriculture. Evidence is required to demonstrate the effectiveness of these measures and with this overarching objective, monitoring of an agricultural catchment in Eastern Scotland was initiated in 2007. As a precursor to evaluating the effect of new management measures it is essential to understand how other factors, including hydrology and land use changes, could have influenced water quality. This study undertook an analysis of the trends in concentrations and loads of nitrate, soluble reactive phosphorus (SRP), suspended solids (SS) and turbidity measured at six points in the catchment over a six year period. The results identified both differing trends between determinands and differing trends occurring over varying spatial scales. The only direct relationships between land use and water quality that could be identified based on annual data was a positive link between arable cropping and nitrate concentrations. At the sub-catchment scale some temporal changes in land use and management explained short-term trends in nitrate but not in SRP. Lags in the system were identified due to soil adsorption, in-stream/loch processing and groundwater transport making the identification of cause and effect problematic. The results have implications for the demonstration of effectiveness of measures over the shorter term and the timescales of recovery from diffuse pollution. Longer term monitoring at small scales will be important in this regard.

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.

Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors.

BACKGROUND AND PURPOSE: It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3 A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3 AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. EXPERIMENTAL APPROACH: Mutations were introduced into the portal region of the human 5-HT3 A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. KEY RESULTS: Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3 A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. CONCLUSIONS AND IMPLICATIONS: These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy.

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.

Importance of recognition loops B and D in the activation of human 5-HT3 receptors by 5-HT and meta-chlorophenylbiguanide.

The 5-HT3 receptor is a cation selective member of the pentameric Cys-loop ligand-gated ion channels. While five subunits are known to exist, only two receptor subtypes have been significantly characterized: the homomeric receptor consisting of five A subunits and the heteromeric receptor containing both A and B subunits. The agonist recognition and activation of these receptors is orchestrated by six recognition loops three, A-C, on the principal subunit, and three, D-F, on the complementary subunit. In this study we have focused on the B loop of the principal subunit and loop D of the complementary subunit where aligned amino acids differ between the two subunits. A mutational analysis has been carried out using both 5-HT and m-chlorophenylbiguanide (mCPBG) to characterize receptor activation in the mutant receptors using two-electrode voltage clamp in Xenopus oocytes. The results show that the B loop W178I mutation of the 5-HT3A subunit markedly reduces the efficacy of mCPBG in both the homomeric and heteromeric receptors, while activation by 5-HT remains intact. Replacement of the D loop amino acid triplet RQY of the 5-HT3A subunit, with the aligned residues from the 5-HT3B subunit, QEV, converts 5-HT to a weak partial agonist in both the homomer and heteromer, but does not compromise activation by mCPBG. Exchange of the RQY triplet for the 5-HT3B subunit homologue, QEV, increases the Hill coefficient and decreases the EC50 of this mutant when expressed with the wild type 5-HT3A subunit.

Antibodies to heteromeric glycolipid complexes in multifocal motor neuropathy.

BACKGROUND: Measurement of anti-GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. METHODS: A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme-linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. RESULTS: By ELISA, 22/33 MMN cases had detectable anti-GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti-GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA-negative samples. CONCLUSIONS: The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti-glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening.

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.

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.

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.

Long-term trends in hydro-climatology of a major Scottish mountain river.

The River Dee, in North East Scotland, is a mountainous river strongly influenced by patterns of snow accumulation and melt from the Cairngorm Mountains. Analysis of this river's flow record from 1929-2004, the longest in Scotland, supports anecdotal evidence that river extreme flows are increasing. There was no detectable change in the overall annual flow patterns. However, an analysis of seasonal data suggested a shift towards increased flows in spring (March-May) and decreased flows in summer (June-August) over the 75 years of the record. Flows in spring exceeded 29 m(3) s(-1) for 50% of the time over the earliest part of the record (1930 to 1954), whereas in the last 25 years of the record (1979 to 2004) 50% of the flows exceeded 35 m(3) s(-1). Precipitation is increasing in the spring and decreasing in July and August. If these trends continue they have important implications for water management in the Dee, with a potential increase in flood risk in spring and the increased possibility of drought in summer. Combined with this increase in flows the river appears to be more responsive to precipitation events in the catchment. In large heterogeneous catchments with a marginal alpine/high latitude climate it is difficult to assess the amount of precipitation falling as snow and its relative accumulation and ablation dynamics on daily to seasonal time scales. Changes in the temporal pattern of coherence between flow and precipitation are thought to be linked to changing snow patterns in the upland part of the catchment. A decreased amount of precipitation occurring as snow has led to higher coherence. We also show that in responsive systems it is important to record river flows at an hourly rather than daily time step in order to characterise peak flow events.

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.