Effects of abomasal infusion of essential fatty acids together with conjugated linoleic acid in late and early lactation on performance, milk and body composition, and plasma metabolites in dairy cows.

Rations including high amounts of corn silage are currently very common in dairy production. Diets with corn silage as forage source result in a low supply of essential fatty acids, such as α-linolenic acid, and may lead to low conjugated linoleic acid (CLA) production. The present study investigated the effects of abomasal infusion of essential fatty acids, especially α-linolenic acid, and CLA in dairy cows fed a corn silage-based diet on performance, milk composition, including fatty acid (FA) pattern, and lipid metabolism from late to early lactation. Rumen-cannulated Holstein cows (n = 40) were studied from wk 9 antepartum to wk 9 postpartum and dried off 6 wk before calving. The cows were assigned to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 and 4 g/d; linseed/safflower oil ratio = 19.5:1; n-6/n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; BASF SE, Ludwigshafen, Germany; isomers cis-9,trans-11 and trans-10,cis-12 each 10 g/d) or EFA+CLA. Milk composition was analyzed weekly, and blood samples were taken several times before and after parturition to determine plasma concentrations of metabolites related to lipid metabolism. Liver samples were obtained by biopsy on d 63 and 21 antepartum and on d 1, 28, and 63 postpartum to measure triglyceride concentration. Body composition was determined after slaughter. Supplementation of CLA reduced milk fat concentration, increased body fat mass, and improved energy balance (EB) in late and early lactation, but EB was lowest during late lactation in the EFA group. Cows with CLA treatment alone showed an elevated milk citrate concentration in early lactation, whereas EFA+CLA did not reveal higher milk citrate but did have increased acetone. Milk protein was increased in late lactation but was decreased in wk 1 postpartum in CLA and EFA+CLA. Milk urea was reduced by CLA treatment during the whole period. After calving, the increase of nonesterified fatty acids in plasma was less in CLA groups; liver triglycerides were raised lowest at d 28 in CLA groups. Our data confirm an improved metabolic status with CLA but not with exclusive EFA supplementation during early lactation. Increased milk citrate concentration in CLA cows points to reduced de novo FA synthesis in the mammary gland, but milk citrate was less affected in EFA+CLA cows, indicating that EFA supplementation may influence changes in mammary gland FA metabolism achieved by CLA.

Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) supplementation on fatty acid (FA) composition, performance, and systemic and hepatic antioxidative and inflammatory responses in dairy cows. Four cows (126 ± 4 d in milk) were investigated in a 4 × 4 Latin square and were abomasally infused with 1 of the following for 6 wk: (1) coconut oil (control treatment, CTRL; 38.3 g/d; providing saturated FA), (2) linseed and safflower oil (EFA treatment; 39.1 and 1.6 g/d, respectively; providing mainly α-linolenic acid), (3) Lutalin (BASF, Ludwigshafen, Germany; CLA treatment; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d each), (4) or EFA+CLA. The initial dosage was doubled every 2 wk, resulting in 3 dosages (dosage 1, 2, and 3). Cows were fed a corn silage-based total mixed ration with a high n-6/n-3 FA ratio. Dry matter intake and milk yield were recorded daily, and milk composition was measured weekly. The FA compositions of milk fat and blood plasma were analyzed at wk 0, 2, 4, and 6. The plasma concentration and hepatic mRNA abundance of parameters linked to the antioxidative and inflammatory response were analyzed at wk 0 and 6 of each treatment period. Infused FA increased in blood plasma and milk of the respective treatment groups in a dose-dependent manner. The n-6/n-3 FA ratio in milk fat was higher in CTRL and CLA than in EFA and EFA+CLA. The sum of FA

Analysis of alternariol and alternariol monomethyl ether in foodstuffs by molecularly imprinted solid-phase extraction and ultra-high-performance liquid chromatography tandem mass spectrometry.

Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6H-dibenzo[b,d]pyran-6-one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (λex=258nm; λem=440nm) or MS/MS analysis. The MISPE method was validated by UPLC-MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1-2.8µgkg-1 range in both samples.

Assessment and introduction of quantitative resistance to Fusarium head blight in elite spring barley.

Breeding for resistance is a key task to control Fusarium head blight (FHB), a devastating disease of small cereals leading to economic losses and grain contamination with mycotoxins harmful for humans and animals. In the present work, FHB resistance of the six-rowed spring barley 'Chevron' to FHB in Germany was compared with those of adapted German spring barley cultivars. Both under natural infection conditions and after spray inoculation with conidia of Fusarium culmorum, F. sporotrichioides, and F. avenaceum under field conditions, Chevron showed a high level of quantitative resistance to the infection and contamination of grain with diverse mycotoxins. This indicates that Chevron is not only a little susceptible to deoxynivalenol-producing Fusarium spp. but also to Fusarium spp. producing type A trichothecenes and enniatins. Monitoring the initial infection course of F. culmorum on barley lemma tissue by confocal laser-scanning microscopy provided evidence that FHB resistance of Chevron is partially mediated by a preformed penetration resistance, because direct penetration of floral tissue by F. culmorum was observed rarely on Chevron but was common on susceptible genotypes. Alternatively, F. culmorum penetrated Chevron lemma tissue via stomata, which was unusual for susceptible genotypes. We generated double-haploid barley populations segregating for the major FHB resistance quantitative trait loci (QTL) Qrgz-2H-8 of Chevron. Subsequently, we characterized these populations by spray inoculation with conidia of F. culmorum and F. sporotrichioides. This suggested that Qrgz-2H-8 was functional in the genetic background of European elite barley cultivars. However, the degree of achieved resistance was very low when compared with quantitative resistance of the QTL donor Chevron, and the introgression of Qrgz-2H-8 was not sufficient to mediate the cellular resistance phenotype of Chevron in the European backgrounds.

Studies on accuracy of trichothecene multitoxin analysis using stable isotope dilution assays.

Critical parameters in mycotoxin analysis were examined by using stable isotope-labelled tricho-thecenes. Sample weight was downsized to 1 g without loosing precision when sufficiently homogenized samples were taken for analysis. Complete extraction of trichothecenes could be achieved with a solvent mixture of acetonitrile+water (84+16; v+v) even without the use of stable isotope labelled standards. However, in particular for the analysis of deoxynivalenol the absolute amount of water in the solvent volume used for extraction appeared critical. Depending on the matrix a low water amount resulted in too low quantitative values when no stable isotope-labelled standards are applied to correct for incomplete extraction. In this case the used extraction volume had to be at least 10 ml for 1 g sample when acetonitrile + water (84+16; v+v) was used as extraction solvent.Losses during sample preparation using two different clean-up columns were not observed. On the contrary, matrix suppression in the ESI-interface of the LC-MS equipment was found to be a serious problem. Depending on the matrix, the latter effect resulted in considerably lower values for trichothecenes when no stable isotope-labelled standards were used to counterbalance this suppression.

Pulmonary function between 40 and 80 years of age.

Spirometry is the most frequently performed lung function test. To determine a normal range of spirometry results, reference formulas are used. Predicted values play an important role in establishing whether the volumes measured in an individual fall within a range to be expected in a healthy person of the same gender, height, and age. Such standards enable to assess the development of the respiratory system in the youth, the early recognition of the influence of a disease on the respiratory system and the influence of environmental factors on lung function. The objective of the present study was to estimate lung function prediction equations and to identify appropriate normal reference values for the Lublin Region local population of adults. We addressed the issue by analyzing the data from a lung function screening program conducted in the Lublin Region of Poland. Pulmonary function of adults aged 40-80 years was assessed from the measurements of forced vital capacity (FVC) and forced expired volume in the first second (FEV(1)) in 136 adults. Reference values of FVC and FEV(1) for females and males were calculated by linear multiple regressions with age and height used as predictors. Different equations were compared to show their reliability when applied to the local population. The results were as follows. In females, the mean FEV(1) was 2.856 +/-0.534 (L) (113.7 +/-14.3%) and the mean FVC was 3.517 +/-0.662 (L) (118.5 +/-14.1%), in males, 3.913 +/-0.773 (L) (110.9 +/-15.1%), 4.922 +/-0.941 (L) (112.1 +/-14.1%), respectively. The estimated prediction equations were: for the FVC - for females - FVC (L) = 0.0528 (height) - 0.0262 (age) - 3.676 and for males - FVC = 0.0756 (height) - 0.0649 (age) - 4.904; and for the FEV(1) - for females - FEV(1) (L) = 0.0378 (height) - 0.0282 (age) - 1.799 and for males - FEV(1) (L) = 0.0553 (height) - 0.0553 (age) - 2.874. Units are years for age and centimeters for height. In conclusion, the analysis of the lung function data showed that there were significant difficulties in determining the appropriate reference values of FEV(1) and FVC. The predicted FEV(1) and FVC values derived from equations based on the ECSC (1) reference populations are considerably lower than those calculated in the present study, re-emphasizing the need to be cautious when applying the ECSC reference values for the local Lublin population. There seems to be a need for a constant refinement of spirometric standards.

Quantification of the mycotoxins patulin and ochratoxin A by stable isotope dilution assays.

For analysis of trace compounds, stable isotope dilution assays (SIDAs) have gained increasing importance in the past years. This methodology is based on the use of stable isotopically labelled analogues of the analytes as internal standards (IS). To take the mycotoxins patulin and ochratoxin A as examples, the benefits of SIDAs were demonstrated both for foods and for clinical analyses.Regarding PAT, an isotopomer labelled with(13)C was used as IS and enabled quantitation of the mycotoxin in tissues and blood. By applying this technology, a fast passive diffusion into tissue was proven with the model of the perfused rat stomach. Furthermore, rapid degradation of PAT was observed when it was reacted with blood, which was attributed to the formation of PAT-GSH adducts detected by LC-MS/MS.For OTA, a SIDA was based on the use of [(2)H5]-OTA as the IS and proved to be more accurate when compared to alternative methods such as HPLC-FD or ELISA. In contrast to PAT, OTA was detectable in human blood and urine samples. Under the assumption that the majority of OTA is circulating in blood, an urinary excretion rate of about 1% of the whole body content per day was calculated.

Mass spectrometric studies of trimethylsilylpantothenic acid and related substances.

The characteristic fragment of trimethylsilylated pantothenic acid (TMS-PA) at m/z 291 upon electron ionization was shown to originate from the molecular ion by a McLafferty rearrangement instead of by ejection of 1,1,3,3-tetramethyl-1,3-disilacyclobutane. The verification consisted of labelling experiments and high-resolution mass spectrometry of the fragment and studies on its isotopic distribution. The remaining fragmentation pathways of TMS-PA were clarified by B/E-linked scans and collision-induced dissociation.

Quantification of free and bound pantothenic acid in foods and blood plasma by a stable isotope dilution assay.

A stable isotope dilution assay for quantification of pantothenic acid in food and blood plasma uses a 4-fold labeled isotopomer of the vitamin as an internal standard. Pantothenic acid and its labeled analogue were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry, showing a minimized spectral overlap. In starch a detection limit of 44 microg/kg, an intrasample relative standard deviation of 6.7%, and recovery values ranging between 97.5 and 99.4% were determined. Total pantothenic acid content was determined in rice, milk powder, apple juice, and blood plasma after enzymatic hydrolysis of the vitamin's conjugates; free pantothenic acid was quantified prior to enzyme treatment. Almost all results were found to be in good agreement with literature data.

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.