A. Starke

No carry over of unmetabolised deoxynivalenol in milk of dairy cows fed high concentrate proportions

To examine the carry over of deoxynivalenol (DON) and its metabolite de-epoxy DON (DOM-1) in milk, lactating German Holstein cows (n = 13) were fed an isoenergetic total mixed ration in Period 1 with 50% concentrates and 5.3 mg DON/kg dry matter (DM) over 11 wk and were compared with control cows (n = 14). In Period 2 (18 wk), an elevated concentrate proportion was compared to a low concentrate ration by dividing the cows into four Groups (n = 8): Control-30 (30% concentrates), Myco-30 (30% concentrates, 4.4 mg DON/kg DM), Control-60 (60% concentrates) and Myco-60 (60% concentrates, 4.6 mg DON/kg DM). Taken both periods together, no unmetabolised DON was detected in milk samples using the HPLC-UV method. DOM-1 concentrations ranged between below the LOD and 3.2 microg/kg milk in mycotoxin fed cows, while control cows did not excrete any measurable amounts of DOM-1. Regarding the concentrate effects, the carry over of DON as DOM-1 in milk was negligible (between 0.0001 and 0.0011) but significantly higher in Group Myco-30 than in Group Myco-60. This effect may result from an altered bioavailability of DON from maize silage which made up a higher proportion of the daily ration.

Technical note: Analysis of total lipid and triacylglycerol content in small liver biopsy samples in cattle

A procedure is described for analyzing total lipid (TL) and triacylglycerol (TAG) in 2 sequential steps using small amounts (<100 mg) of bovine liver tissue. The TL was measured gravimetrically and TAG was measured enzymatically in the TL extract, using an automated analyzer. For gravimetric TL determination in milligrams per gram of liver fresh weight (FW), TL was extracted from homogenized tissue samples with hexane:isopropanol (at 20 degrees C, 24 h, constant agitation). The routine method was modified by adding a second hexane extraction step to optimize lipid extraction. The dry lipid extract was dissolved in hexane and aliquoted according to TL content for TAG analysis. An extra incubation period of 16 h was included for complete hydrolysis of TAG, using microbial lipase and nonaethylene glycol monododecyl ether detergent, before TAG was measured enzymatically using commercial test kits. Triolein was used as an internal standard. Repeated TL analysis (n = 3) of liver specimens from 10 cows (range, 40 to 314 mg/g of FW) yielded a mean CV of 2.2%, whereas repeated TAG analysis (range, 4 to 260 mg/g of FW) yielded a mean intraday CV of 2.5% (n = 5) and a mean interday CV of 3.4% (n = 4). Intraday (n = 5) and interday (n = 4) CV for repeated TAG analysis in triolein standards were <1 and <3%, respectively. Recovery of TAG in triolein standards varied between 99 and 103%. In part 2 of the experiment, hepatic TL and TAG were measured in 150 German Holstein cows to verify the test method in a large sample size. For repeated hepatic TL (n = 3) and TAG (n = 5) determination, mean CV of <2.8 and <1.5%, respectively, were found. The proportion of TAG relative to TL increased linearly to a breakpoint of approximately 100 mg TL/g of FW, at which point it reached a plateau at approximately 68%, indicating an accumulation of other lipid fractions in hepatic tissue with hepatic TL above the breakpoint. Calculation of hepatic TAG from TL was reasonably accurate when a 2-slope linear broken-line model (r(2) = 0.98) was used. Above a TL of approximately 40 mg/g of FW, calculated TAG values deviated by only +/-15% from measured hepatic TAG.

Noninvasive detection of hepatic lipidosis in dairy cows with calibrated ultrasonographic image analysis.

The aim was to test the accuracy of calibrated digital analysis of ultrasonographic hepatic images for diagnosing fatty liver in dairy cows. Digital analysis was performed by means of a novel method, computer-aided ultrasound diagnosis (CAUS), previously published by the authors. This method implies a set of pre- and postprocessing steps to normalize and correct the transcutaneous ultrasonographic images. Transcutaneous hepatic ultrasonography was performed before surgical correction on 151 German Holstein dairy cows (mean +/- standard error of the means; body weight: 571+/-7 kg; age: 4.9+/-0.2 yr; DIM: 35+/-5) with left-sided abomasal displacement. Concentration of triacylglycerol (TAG) was biochemically determined in liver samples collected via biopsy and values were considered the gold standard to which ultrasound estimates were compared. According to histopathologic examination of biopsies, none of the cows suffered from hepatic disorders other than hepatic lipidosis. Hepatic TAG concentrations ranged from 4.6 to 292.4 mg/g of liver fresh weight (FW). High correlations were found between the hepatic TAG and mean echo level (r=0.59) and residual attenuation (ResAtt; r=0.80) obtained in ultrasonographic imaging. High correlation existed between ResAtt and mean echo level (r=0.76). The 151 studied cows were split randomly into a training set of 76 cows and a test set of 75 cows. Based on the data from the training set, ResAtt was statistically selected by means of stepwise multiple regression analysis for hepatic TAG prediction (R(2)=0.69). Then, using the predicted TAG data of the test set, receiver operating characteristic analysis was performed to summarize the accuracy and predictive potential of the differentiation between various measured hepatic TAG values, based on TAG predicted from the regression formula. The area under the curve values of the receiver operating characteristic based on the regression equation were 0.94 (<50 vs. >or=50mg of TAG/g of FW), 0.83 (<100 vs. >or=100mg of TAG/g of FW), and 0.97 (<50 vs. >or=100mg of TAG/g of FW). The CAUS methodology and software for digitally analyzing liver ultrasonographic images is considered feasible for noninvasive screening of fatty liver in dairy herd health programs. Using the single parameter linear regression equation might be ideal for practical applications.

Transcutaneous vs. Intraoperative Quantitative Ultrasound for Staging Bovine Hepatic Steatosis

The aim of this study was to test the hypothesis that the Computer Aided UltraSound (CAUS) method developed by the authors [1-4] for the estimation of UltraSound Tissue Characteristics (UTC) parameters on transcutaneous (Transc) ultrasound (US) images can predict the liver fat content with similar accuracy and precision as with intraoperative (Intraop) US. A large animal study in post partum dairy cows (N=151) was performed to test these hypotheses. Five Transc B-Mode US liver image were acquired before surgery. During abomasal displacement surgery five Intraop US B-Mode liver images and a liver biopsy was taken. In liver tissue samples, triacylglycerol (TAG) content was measured by biochemical analysis. Firstly the equipment preset, which was kept fixed during whole study time, was carefully calibrated[5]. For the echo level calibration a TMP was used, and all UTC parameters were expressed relatively to those of the phantom. Prior to UTC parameters estimation several pre-processing steps were performed: Back-Scan Conversion (BSC); Look Up Table (LUT) correction; superficial tissue layers (Fat layer) attenuation correction and Automatic Gain Correction (AGC) were performed. Also several postprocessing steps were incorporated like: Automatic segmentation and residual attenuation correction were performed. Stepwise multiple linear regression analysis on a training set (N=76) was performed. In all cases the Residual Attenuation coefficient (ResAtt, R=0.81) was the only selected parameter. The results were tested on the residual cows (test set N=75) to predict the TAG content in the liver. Receiver Operating Characteristics (ROC) analysis then was applied to estimate the Area Under the Curve (AUC) and the sensitivity and specificity of the CAUS method. Equivalent high predictive values for AUC (95%), sensitivity(87%) and specificity (83%) for Intraop and Transc applications were found. Consequently, it can be concluded, applied Fat layer attenuation correction to Transc US images was performed adequately.

Evaluation of portal blood flow using transcutaneous and intraoperative Doppler ultrasonography in dairy cows with fatty liver

The objective of the study was to investigate portal blood flow (PBF) in dairy cows with fatty liver by means of Doppler ultrasonography. Eighty lactating German Holstein cows less than 100 d in milk were used (mean ± standard error of the mean; body weight: 583 ± 9 kg, age: 5 ± 0.2 yr, withers height: 145.4 ± 0.5 cm, milk yield: 9 ± 0.6 kg). All cows had left abomasal displacement and underwent omentopexy via right flank laparotomy. The size of the liver and the thickness over the portal vein were determined ultrasonographically. Doppler ultrasonographic examinations of PBF were carried out transcutaneously and intraoperatively directly via liver surface. The PBF velocities [peak maximum (v(max)), peak minimum (v(min)), and mean maximum (v(mean)) velocity] were recorded. Venous pulsatility index (VPI) was calculated. Because transcutaneous Doppler ultrasonography revealed images of very poor quality in 58 of the 80 cows, only data obtained intraoperatively were presented. Liver biopsies were used for hepatic triacylglycerol (TAG) determination and histological examination. Based on histopathologic and ultrasonographic examinations, none of the cows suffered from hepatic disorders other than hepatic lipidosis. Hepatic TAG content ranged from 5 to 292 mg/g of liver fresh weight (FW). Cows were allocated to 1 of 4 groups according to their hepatic TAG content (very severe: TAG >150 mg/g of FW, n=27; severe: >100-150 mg/g of FW, n=18; moderate: ≥ 50-100mg/g of FW, n=19; mild: <50mg/g of FW, n=16). The VPI decreased with increasing TAG content (r=-0.55). The VPI did not differ between cows with severe and very severe fatty liver but it differed between cows of these 2 groups and cows with mild and moderate fatty liver. Velocities of PBF (v(mean), v(min), v(max)) correlated negatively with hepatic TAG content (r=-0.26 to -0.37). Mean PBF velocity of the cows with very severe fatty liver differed from cows with severe, moderate, and mild fatty liver. Variables of PBF were inversely related to hepatic size and thickness (r=-0.06 to -0.35). In conclusion, the lower VPI and PBF velocities in cows with fatty liver and the negative correlations with the degree of hepatosteatosis may be explained by a reduction of vascular compliance in the liver because of fatty infiltration. These changes, which are believed to result from parenchymal swelling, were particularly pronounced when hepatic TAG content exceeded 150 mg/g FW.

Interactive vs. automatic ultrasound image segmentation methods for staging hepatic lipidosis.

The aim of this study was to test the hypothesis that automatic segmentation of vessels in ultrasound (US) images can produce similar or better results in grading fatty livers than interactive segmentation. A study was performed in postpartum dairy cows (N= 151), as an animal model of human fatty liver disease, to test this hypothesis. Five transcutaneous and five intraoperative US liver images were acquired in each animal and a liver biopsy was taken. In liver tissue samples, triacylglycerol (TAG) was measured by biochemical analysis and hepatic diseases other than hepatic lipidosis were excluded by histopathologic examination. Ultrasonic tissue characterization (UTC) parameters — Mean echo level, standard deviation (SD) of echo level, signal-to-noise ratio (SNR), residual attenuation coefficient (ResAtt) and axial and lateral speckle size—were derived using a computer-aided US (CAUS) protocol and software package. First, the liver tissue was interactively segmented by two observers. With increasing fat content, fewer hepatic vessels were visible in the ultrasound images and, therefore, a smaller proportion of the liver needed to be excluded from these images. Automatic-segmentation algorithms were implemented and it was investigated whether better results could be achieved than with the subjective and time-consuming interactive-segmentation procedure. The automatic-segmentation algorithms were based on both fixed and adaptive thresholding techniques in combination with a ‘speckle’-shaped moving-window exclusion technique. All data were analyzed with and without postprocessing as contained in CAUS and with different automated-segmentation techniques. This enabled us to study the effect of the applied postprocessing steps on single and multiple linear regressions of the various UTC parameters with TAG. Improved correlations for all US parameters were found by using automatic-segmentation techniques. Stepwise multiple linear-regression formulas where derived and used to predict TAG level in the liver. Receiver-operating-characteristics (ROC) analysis was applied to assess the performance and area under the curve (AUC) of predicting TAG and to compare the sensitivity and specificity of the methods. Best speckle-size estimates and overall performance (R 2 = 0.71, AUC = 0.94) were achieved by using an SNR-based adaptive automatic-segmentation method (used TAG threshold: 50 mg/g liver wet weight). Automatic segmentation is thus feasible and profitable.

Zearalenone (ZEN) metabolism and residue concentrations in physiological specimens of dairy cows exposed long-term to ZEN-contaminated diets differing in concentrate feed proportions.

A long-term feeding experiment with dairy cows was performed to investigate the effects of feeding a Fusarium toxin contaminated (FUS) and a background-contaminated control (CON) ration with a mean concentrate feed proportion of 50% during the first 11 weeks after parturition (Groups FUS-50, CON-50, Period 1), and with concentrate feed proportions of 30% or 60% during the remaining 17 weeks (Groups CON-30, CON-60, FUS-30 and FUS-60, Period 2), on zearalenone (ZEN) residue levels in blood serum, milk, urine and bile. ZEN, α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), zearalanone (ZAL), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) were determined by HPLC with fluorescence detection. The ZEN concentrations of the rations fed to Groups CON-50, FUS-50 (Period 1), CON-30, CON-60, FUS-30 and FUS-60 (Period 2) amounted to 53.1, 112.7, 35.0, 24.4, 73.8 and 72.5 µg/kg dry matter, respectively. The concentrations of ZEN, α-ZEL, β-ZEL, ZAN, α-ZAL and β-ZAL in serum, urine and milk were lower than 1, 1, 4, 100, 50 and 200 ng/g, respectively, while ZEN, α-ZEL and β-ZEL were detected in bile. Their levels changed with oral ZEN exposure in the course of the experiment and in a similar direction with concentrate feed proportion (Period 2 only). Thus the proportions of the individual β-ZEL, α-ZEL and ZEN concentrations of their sum varied only in narrow ranges of 68–76%, 6–13% and 12–20%, respectively. Interestingly, the bile concentrations of β-ZEL, α-ZEL and ZEN of Groups CON-60 and FUS-60 amounted to only approximately 50%, 45% and 62%, respectively, of those of Groups CON-30 and FUS-30 despite a similar or even lower ZEN exposure. The results indicate that conversion of ZEN to its detectable metabolites was not changed by different dietary concentrate feed proportions while their absolute levels were decreased. These findings might suggest concentrate feed proportion-dependent and rumen fermentation-mediated alterations in ZEN/metabolite degradation, and/or liver associated alterations in bile formation and turnover.

Effects of Fusarium mycotoxins in rations with different concentrate proportions on serum haptoglobin and hepatocellular integrity in lactating dairy cows

It was hypothesized that long-term intake of a diet contaminated with deoxynivalenol (DON) and differing in the proportion of concentrate might affect hepatocellular integrity and function as well as biomarkers of systemic inflammation in lactating dairy cows. In Period 1 (11 weeks), 26 lactating German Holstein cows (13 primiparous and 13 pluriparous, 31 days in milk, 522 kg body weight, on average) were divided into two groups and fed diets (50% concentrate) with (MYC, n = 12; on average 5.3 mg DON/kg DM) or without (CON, n = 14) DON contaminations. In Period 2 (16 weeks), each group was further divided into two groups to test whether elevated concentrate proportion as additional burden might enhance the toxicity of DON. The cows in MYC60 (n = 6; 4.6 mg DON/kg DM) and CON60 (n = 7) received the diet with 60% concentrate, while cows in MYC30 (n = 6; 4.4 mg DON/kg DM) and CON30 (n = 7) received the diet with 30% concentrate. Blood samples were taken in biweekly intervals for activities of aspartate amino transferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase as well as for concentration of total bilirubin and haptoglobin. Biopsies from liver were collected in week 27 for morphological analyses. No DON effect was found for the variables assessed in blood. The diet with 60% concentrate led to higher activities of AST and GLDH in Period 2. No morphological change was found by both light and electron microscopic analyses of liver samples. Results indicated that long-term intake of DON-contaminated diet over 27 weeks led to neither relevant damages of hepatocytes nor systemic inflammatory responses in lactating dairy cows, even if the dietary concentrate proportion was increased to 60%.

Computer-aided ultrasound diagnosis of hepatic steatosis

In this study, a Computer-Aided UltraSound (CAUS) diagnosis method for the detection and staging of hepatic steatosis (non-alcoholic fatty liver disease, NAFLD) is investigated using a bovine model (n=151). In humans as well as in cows, hepatic steatosis increases the risk of comorbidity [1,2,3,4]. Assessment of liver fat content is mostly done by taking liver biopsies [2,4,5]. The authors’ goal was to estimate the liver fat content using on non-invasive, echographic parameters. Since skin and subcutaneous fat layer influence the characteristics of echographic B-mode liver-images, both transcutaneous and intraoperative images were acquired to study this effect. During image acquisition a fixed preset of the ultrasound equipment controls was used. One liver biopsy was taken from each animal to assess the liver fat weight percentage (fat score). The software package CAUS was developed to perform objective and unambiguous analysis on echographic B-mode images by estimating several echographic parameters. Prior to the analysis of the images certain preprocessing steps were performed in order to achieve relative echo strength in decibel (dB), rather than image gray level, as a quantitative parameter. Furthermore, corrections were made for ultrasound propagation through skin/fat layer and through an average “healthy liver”. After these corrections, the biopsy results were correlated with the estimated echographic parameters and ROC curves and parameters were estimated to achieve the goal. High correlations with the liver fat score were found for several echographic parameters. ROC curve analysis show promising results for sensitivity (0.93), specificity (0.86) and area under the curve (0.93) in distinguishing fatty livers from healthy livers. This study showed the feasibility of generic computer-aided ultrasound for non-invasively diagnosing, maybe even screening, of liver steatosis.

Chronic Effects of Fusarium Mycotoxins in Rations with or without Increased Concentrate Proportion on the Insulin Sensitivity in Lactating Dairy Cows

The objective of this study was to investigate the effect of long-term exposure to a Fusarium toxin deoxynivalenol (DON, 5 mg/kg DM) on the energy metabolism in lactating cows fed diets with different amounts of concentrate. In Period 1 27 German Holstein cows were assigned to two groups and fed a control or mycotoxin-contaminated diet with 50% concentrate for 11 weeks. In Period 2 each group was further divided and fed either a diet containing 30% or 60% concentrate for 16 weeks. Blood samples were collected in week 0, 4, 8, 15, 21, and 27 for calculation of the Revised Quantitative Insulin Sensitivity Check Index and biopsy samples of skeletal muscle and the liver in w 0, 15, and 27 for analysis by real-time RT-qPCR. The DON-fed groups presented lower insulin sensitivities than controls at week 27. Concomitantly, muscular mRNA expression of insulin receptors and hepatic mRNA expression of glucose transporter 2 and key enzymes for gluconeogenesis and fatty acid metabolism were lower in DON-fed cows compared to the control. The study revealed no consistent evidence that DON effects were modified by dietary concentrate levels. In conclusion, long-term dietary DON intake appears to have mild effects on energy metabolism in lactating dairy cows.