Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

The effects of incomplete milking and increased milking frequency on milk production rate and milk composition1.

Increased milking frequency and incomplete milking have differential effects on milk yield and mammary gland physiology that are important for optimization of milking practices in dairy herds. The objectives of this experiment were to determine the effects of increased milking frequency and incomplete milking on milk production rate (MPR) and milk composition and to determine if milking 3 times daily (3×) could rescue the negative production effects of incomplete milking. Twenty-two multiparous cows were enrolled onto this experiment beginning at 5 days in milk (DIM) and continuing through 47 DIM. A split-plot design was used to randomize the 2 treatments, which were milking frequency and incomplete milking. Eleven cows were randomly assigned to be milked 2 times (2×) daily and 11 cows were randomly assigned to be milked 3×. Within each cow, a contralateral half-udder was randomly assigned to be incompletely milked (30% milk remaining in the gland; IM), and the other half-udder was randomly assigned to be milked completely (CM). Quarter-level milk yields were recorded at each milking session. Milk samples from all quarters were collected twice weekly at the beginning of the morning milking for analysis. Cows milked 2× tended to have reduced MPR compared with 3× milked cows (1.81 ± 0.06 vs. 1.97 ± 0.06 kg milk/h; P = 0.06). Half-udders that were CM and IM produced 1.09 ± 0.03 and 0.80 ± 0.03 kg milk/h, respectively. There was an interaction between incomplete milking treatment and week of lactation (P = 0.04). No interaction was detected between milking frequency and incomplete milking for MPR or milk components. Cows milked 3× had increased milk fat percent (1.93 ± 0.09% vs. 1.65 ± 0.09%, P = 0.047), decreased milk lactose percent (4.80 ± 0.04% vs. 4.93 ± 0.04%, P = 0.04), and exhibited no differences in milk protein percent or milk somatic cell count (SCC) compared with cows milked 2×. Half-udders that were IM had increased milk fat percent (2.15 ± 0.07% vs. 1.43 ± 0.07%, P < 0.0001), decreased lactose percent (4.75 ± 0.03% vs. 4.99 ± 0.03%, P < 0.0001), increased milk log10SCC (4.22 ± 0.05 vs. 4.41 ± 0.05, P = 0.0004), and no differences in milk protein percent compared with CM half-udders. These results indicate that a 3× milking frequency in IM half-udders was not able to improve milk production compared with IM half-udders milked 2×. Our results indicate that 30% milk remaining in the gland had an irreversible impact on milk yield as increased milking frequency was not able to reverse the milk yield lost.

Effect of induced hypocalcemia in nonlactating, nonpregnant Holstein cows fed negative DCAD with low, medium, or high concentrations of calcium.

The main objective of this study was to determine how feeding different dietary calcium (Ca) concentrations in combination with a negative dietary cation-anion difference (DCAD) would affect the cow's response to induced hypocalcemia. We conducted an experiment with multiparous, nonlactating, nonpregnant Holstein cows fed a negative DCAD (average -18.2 across all diets) for 21 d with low (LC; 0.45% Ca; n = 5), medium (MC; 1.13% Ca; n = 6), or high (HC; 2.02% Ca; n = 6) concentrations of dietary Ca. Urine and blood samples were collected and urine pH measured daily during the 21-d feeding period prior to hypocalcemia challenge. Cows were then subjected to a controlled induction of hypocalcemia to determine how dietary Ca intake affected the response to a hypocalcemia challenge. On days 22, 23, and 24, hypocalcemia was induced with an intravenous infusion of 5% EGTA in 2 different cows from each treatment daily. During infusion, blood samples were collected every 15 min until 60% of prechallenge ionized calcium (iCa) concentrations were achieved. Samples were collected postinfusion at 0, 2.5, 5, 10, 15, 30, and every 30 min thereafter until 90% of prechallenge iCa was reached. Blood pH, hematocrit, and serum total Ca (tCa), sodium (Na), potassium (K), phosphorous (P), magnesium (Mg), and serotonin did not differ (P > 0.05) among treatments during the feeding period. Blood iCa (P = 0.04) and glucose (P = 0.03) were significantly elevated in HC compared with LC and MC cows during the feeding period. Urine pH was less than 6.0 in all cows, but was lowest in LC (P = 0.02) compared with MC and HC cows during the feeding period. Urine Ca, P, Mg, and deoxypyridinoline did not differ among treatments (P > 0.05). Cows fed HC maintained higher concentrations of iCa (P = 0.03) during the challenge period than MC (P = 0.04), and LC (P = 0.004), and required a longer time to reach 60% of whole blood iCa, and required more EGTA to reach 60% iCa than MC or LC cows (P = 0.01). Serum tCa decreased in all cows during infusion (P < 0.0001) but did not differ among treatments. Serotonin concentrations were elevated in MC cows compared with HC and LC cows during EGTA infusion (P = 0.05), suggesting an interdependent relationship between iCa and serotonin. Cows fed HC had a slower rate of decrease in iCa, but not tCa, when induced with hypocalcemia, indicating potential metabolic benefits of feeding higher dietary Ca in combination with a negative DCAD.

Baseline Assessment of 25-Hydroxyvitamin D Reference Material and Proficiency Testing/External Quality Assurance Material Commutability: A Vitamin D Standardization Program Study.

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials were analyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of American Pathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing to choose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.

Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

Elevation of circulating serotonin improves calcium dynamics in the peripartum dairy cow.

Hypocalcemia is a metabolic disorder that affects dairy cows during the transition from pregnancy to lactation. Twelve multiparous Holstein cows and twelve multiparous Jersey cows were intravenously infused daily for approximately 7 days prepartum with either saline or 1.0mg/kg bodyweight of the immediate precursor to serotonin synthesis, 5hydroxy-l-tryptophan (5-HTP). On infusion days, blood was collected before, after, and at 2, 4, and 8h postinfusion. Blood and urine were collected daily before the infusion period, for 14 days postpartum and on day 30 postpartum. Milk was collected daily during the postpartum period. Feed intake and milk yield were unaffected by 5-HTP infusion postpartum. Cows infused with 5-HTP had elevated circulating serotonin concentrations prepartum. Infusion with 5-HTP induced a transient hypocalcemia in Jersey cows prepartum, but not in any other treatment. Holstein cows infused with saline had the highest milk calcium on the day of and day after parturition. Postpartum, circulating total calcium tended to be elevated, and urine deoxypyridinoline (DPD) concentrations were elevated in Holstein cows infused with 5-HTP. Overall, Jerseys had higher urine DPD concentrations postpartum when compared with Holsteins. Taken together, these data warrant further investigation of the potential therapeutic benefit of 5-HTP administration prepartum for prevention of hypocalcemia. Further research should focus on delineation of mechanisms associated with 5-HTP infusion that control calcium homeostasis during the peripartum period in Holstein and Jersey cows.

Low Dietary c9t11-Conjugated Linoleic Acid Intake from Dairy Fat or Supplements Reduces Inflammation in Collagen-Induced Arthritis.

Dietary cis-9,trans-11 (c9t11) conjugated linoleic acid (CLA) fed at 0.5 % w/w was previously shown to attenuate inflammation in the murine collagen-induced (CA) arthritis model, and growing evidence implicates c9t11-CLA as a major anti-inflammatory component of dairy fat. To understand c9t11-CLA's contribution to dairy fat's anti-inflammatory action, the minimum amount of dietary c9t11-CLA needed to reduce inflammation must be determined. This study had two objectives: (1) determine the minimum dietary anti-inflammatory c9t11-CLA intake level in the CA model, and (2) compare this to anti-inflammatory effects of dairy fat (non-enriched, naturally c9t11-CLA-enriched, or c9t11-CLA-supplemented). Mice received the following dietary fat treatments (w/w) post arthritis onset: corn oil (6 % CO), 0.125, 0.25, 0.375, and 0.5 % c9t11-CLA, control butter (6 % CB), c9t11-enriched butter (6 % EB), or c9t11-CLA-supplemented butter (6 % SB, containing 0.2 % c9t11-CLA). Paw arthritic severity and pad swelling were scored and measured, respectively, over an 84-day study period. All c9t11-CLA and butter diets decreased the arthritic score (25-51 %, P < 0.01) and paw swelling (8-11 %, P < 0.01). Throughout the study, plasma tumor necrosis factor (TNFα) was elevated in CO-fed arthritic mice compared to non-arthritic (NA) mice but was reduced in 0.5 % c9t11-CLA- and EB-fed mice. Interleukin-1ß and IL-6 were increased in arthritic CO-fed mice compared to NA mice but were reduced in 0.5 % c9t11-CLA- and EB-fed mice through day 42. In conclusion, 0.125 % c9t11-CLA reduced clinical arthritis as effectively as higher doses, and decreased arthritis in CB-fed mice suggested that the minimal anti-inflammatory levels of c9t11-CLA might be below 0.125 %.

Maternal iron deficiency worsens the associative learning deficits and hippocampal and cerebellar losses in a rat model of fetal alcohol spectrum disorders.

BACKGROUND: Gestational alcohol exposure causes lifelong physical and neurocognitive deficits collectively referred to as fetal alcohol spectrum disorders (FASDs). Micronutrient deficiencies are common in pregnancies of alcohol-abusing women. Here we show the most common micronutrient deficiency of pregnancy-iron deficiency without anemia-significantly worsens neurocognitive outcomes following perinatal alcohol exposure. METHODS: Pregnant rats were fed iron-deficient (ID) or iron-sufficient diets from gestational day 13 to postnatal day (P) 7. Pups received alcohol (0, 3.5, 5.0 g/kg) from P 4 to P 9, targeting the brain growth spurt. At P 32, learning was assessed using delay or trace eyeblink classical conditioning (ECC). Cerebellar interpositus nucleus (IPN) and hippocampal CA1 cellularity was quantified using unbiased stereology. RESULTS: Global analysis of variance revealed that ID and alcohol separately and significantly reduced ECC learning with respect to amplitude (ps ≤ 0.001) and conditioned response [CR] percentage (ps ≤ 0.001). Iron and alcohol interacted to reduce CR percentage in the trace ECC task (p = 0.013). Both ID and alcohol significantly reduced IPN (ps < 0.001) and CA1 cellularity (ps < 0.005). CR amplitude correlated with IPN cellularity (delay: r = 0.871, trace: r = 0.703, ps < 0.001) and CA1 cellularity (delay: r = 0.792, trace: r = 0.846, ps < 0.001) across both tasks. The learning impairments persisted even though the offsprings' iron status had normalized. CONCLUSIONS: Supporting our previous work, gestational ID exacerbates the associative learning deficits in this rat model of FASD. This is strongly associated with cellular reductions within the ECC neurocircuitry. Significant learning impairments in FASD could be the consequence, in part, of pregnancies in which the mother was also iron inadequate.

Dietary trans-10,cis-12 CLA reduces murine collagen-induced arthritis in a dose-dependent manner.

Dietary trans-10,cis-12 (t10c12) conjugated linoleic acid (CLA) has been shown to reduce inflammation in a murine collagen-induced arthritis (CA) model. To understand the anti-inflammatory potential of t10c12-CLA in the diet, the minimum dose of pure dietary t10c12-CLA capable of reducing CA was investigated. Because plasma inflammatory cytokines often do not reflect the progression of late-stage arthritis, inflamed tissue cytokine concentrations were also investigated in relation to increasing dietary t10c12-CLA amounts. Mice were randomly assigned to the following dietary treatments upon the establishment of arthritis: corn oil (CO) or 0.125%, 0.25%, 0.375%, or 0.5% t10c12-CLA (wt:wt) for 84 d. Sham mice (no arthritis) were fed CO and served as controls. Arthritic paw score, based on subjective assessment of arthritic severity, and paw thickness decreased linearly overall [16-65% (P < 0.001) and 0.5-12% (P < 0.001), respectively] as dietary t10c12-CLA increased (P < 0.001, R(2) < 0.81). Increasing dietary t10c12-CLA was associated with a decrease in plasma interleukin (IL)-1ß at days 21 and 42 compared with CO-fed arthritic mice, such that mice fed ≥0.25% t10c12-CLA had IL-1ß concentrations that were similar to sham mice. Plasma cytokines returned to sham mice concentrations by day 63 regardless of treatment; however, an arthritis-induced elevation in paw IL-1ß decreased linearly as dietary t10c12-CLA concentrations increased at day 84 (P = 0.007, R(2) = 0.92). Similarly, increasing dietary t10c12-CLA linearly decreased paw tumor necrosis factor (TNF)-α (P = 0.05, R(2) = 0.70). In conclusion, ≥0.125% t10c12-CLA dose-dependently reduced inflammation in a murine CA model.

Cardiac isoenzymes in healthy Holstein calves and calves with experimentally induced endotoxemia.

This paper describes a controlled study designed to establish normal values for cardiac troponins I and T (cTnI and cTnT) and CK-MB mass in healthy newborn Holstein calves, and to compare values for cTnI, cTnT, CK-MB and total creatine kinase (CK) with age-matched calves experiencing experimentally induced endotoxemia. Nineteen healthy Holstein bull calves, 48 to 72 h of age were used. Baseline cTnI, cTnT, CK-MB and total CK measurements were obtained from control (n = 9) and experimental (n = 10) calves. Controls then received physiological saline and experimental calves received endotoxin (O55:B5 Escherichia coli LPS) intravenously after which cardiac biomarkers and total CK were measured at 3 h, 6 h, 12 h, and 24 h post-initiation of infusion. Measured values were analyzed and compared using analysis of variance (ANOVA) by repeated measure design, with statistical significance set at P < 0.05. The cardiac biomarker cTnT was not detected in any calf at any time point, and CK-MB was only detected in 5 of 95 samples. The cTnI was significantly increased compared to baseline and controls, 3 h post lipopolysaccharide (LPS) infusion. Total CK was significantly increased in LPS administered calves at 18 and 24 h post infusion. The mean, standard deviation, and range for cTnI in healthy controls were 0.023 ng/mL (s = 0.01), and 0.01 to 0.05 ng/mL, respectively. In conclusion, LPS administration was associated with rapid and significant increases in cTnI but CK-MB and cTnT were not detected in the plasma of healthy calves. Total CK values increased significantly following LPS administration. Biochemical evidence of myocardial injury occurs within 3 h following LPS administration to neonatal Holstein calves.