Fatty liver (steatosis) occurs in obese patients, among others, and is related to the development of diabetes type-2. Timely diagnosis of steatosis is therefore of great importance. Steatosis is also the most common liver disease of high-yielding dairy cattle during early lactation. This makes it a suitable animal model for studying liver steatosis. Furthermore, reference of derived ultrasound parameters against a "gold standard" is possible in cattle by taking a liver biopsy for the assessment of fat concentration. The authors undertook this pilot study to investigate the hypothesis that quantitative, computer-aided B-mode ultrasound enables the noninvasive detection of hepatic steatosis. Echographic images were obtained postpartum from dairy cows (n = 12) in transcutaneous and direct (intraoperative) applications using a convex array transducer at 4.2 MHz. During surgery, a biopsy was taken from the caudate lobe to assess the liver fat content (fat score). A custom-designed software package for computer-aided ultrasound diagnosis (CAUS) was developed. After linearizing the post-processing look-up-table (LUT), the image gray levels were transferred into echo levels in decibels relative to the mean echo level in a tissue-mimicking phantom. The quantitative comparison of transcutaneous and intraoperative images enabled the correction for the attenuation effect of skin and subcutaneous fat layer on the mean echo level in the liver, as well as for the effects of the beam formation and attenuation of liver tissue on the echo level vs. depth. The residual attenuation coefficient (dB/cm) in fatty liver vs. normal liver was estimated and compensated for. Finally, echo level was estimated relative to the phantom used for calibration, and echo texture was characterized by the mean axial and lateral speckle size within the regions of interest. In the no fat/low fat group (n = 5) skin plus fat layer attenuation was 3.4 dB/cm. A correlation of skin layer thickness vs. fat score of r = 0.48 was found. The mean transcutaneous liver tissue echo level correlated well with fat score: r = 0.80. A residual liver attenuation coefficient of 0.76 dB/cm and 1.19 dB/cm was found in medium and high fat liver, respectively. In transcutaneous images, correlation of residual attenuation coefficient with fat score was r = 0.69. Axial and lateral speckle sizes were on the order of 0.2 and 1.0 cm, respectively, and no correlation was found with liver fat content. Results for transcutaneous and intraoperative images were similar. The authors conclude that this pilot study shows the feasibility of calibrated, computer-aided ultrasound for noninvasively diagnosing, possibly even screening, steatosis of the liver.

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.

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.

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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.