Aspects of transition cow metabolomics-Part II: Histomorphologic changes in the liver parenchyma throughout the transition period, in cows with different liver metabotypes and effects of a metaphylactic butaphosphan and cyanocobalamin treatment.

The aims of this study were to evaluate histopathologic changes during the transition period, describe the histopathological features of the metabotypes identified in Part I (Schären et al., 2021b), and investigate effects of a metaphylactic treatment with butaphosphan and cyanocobalamin (BCC) on the liver parenchyma. Eighty German Holstein cows (mean 305-d production: 10,957 kg, range: 6,480-15,193 kg; mean lactation number: 3.9, range: 2-9) from a commercial dairy farm in Saxony, Germany, were enrolled in a randomized, prospective, triple-blinded study. Two groups received a treatment with BCC (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin, Catosal, Bayer Animal Health, n = 20 each) and one group a placebo treatment (NaCl 0.9%, n = 40). Liver biopsy specimens were collected 14 d antepartum (AP) and 7, 28, and 42 d postpartum (PP), routinely processed for histologic examination, and stained with hematoxylin and eosin, Sudan III, periodic acid-Schiff, and picrosirius red stains. The sections were assessed for fat and glycogen content and degenerative, inflammatory, fibrotic, and proliferative changes. The statistical analysis included the effects of the sampling day, the lactation number, the treatment, and the metabotype (A = medium, B = minor, C = large alterations in the liver metabolome profile between AP and PP status). There was mild to moderate fat infiltration in the liver of 37% of cows in the last 2 wk AP, and moderate to severe fat infiltration in 66% of cows in the first days PP. The degree of fat infiltration increased from 2 wk AP until the end of the first week PP, and then decreased until the end of the study period, at which time about 25% of cows had moderate to severe fatty infiltration. Lipidosis was positively correlated with the severity of liver cell degeneration, and negatively correlated with the degree of glycogen deposits. Complete glycogen depletion of hepatocytes was not observed in cows, even in the presence of severe hepatic lipidosis. Moderate to severe lymphocytic hepatitis was seen in 39% of cows throughout the study period, and cows with lactation numbers 5 or greater had perisinusoidal fibrosis more often than younger cows. Severe fibrosis and cirrhosis of the liver did not occur. Metabotype B animals exhibited a higher chance of fatty infiltration, lower glycogen storage, and perisinusoidal fibrosis and for this metabotype positive correlations were calculated between increased fat deposition in the liver and marked glycogen depletion, and increased degenerative, inflammatory, fibrotic, and proliferative changes of hepatic tissue. For the treatment with BCC, no significant effect was observed. In summary, during the transition period, the liver of dairy cows is characterized by fat accumulation and glycogen depletion and histologic signs of hepatitis and hepatocyte degeneration. These histomorphologic changes were accentuated in animals exhibiting little alterations in their liver metabolome profile across the transition period (metabotype B) and support the assumption of a decreased grass silage quality as a causative factor.

Blood lactate response, oxygen consumption, and muscle activity during treadmill running with constraint.

The induction of self-organization during running with a special harness may lead to reduced energy requirements. This experiment was designed to investigate the effect of practicing with a rubber tubing constraint attached between the heel and the hip for 7 wk. (18 treadmill running sessions) on oxygen consumption, caloric unit cost, blood lactate concentration, and muscle activity. 18 male recreational runners (M age = 26.3 yr.) were assigned to either an intervention or a control group. The intervention group trained with the constraint and the control group trained without it. Test 1 was conducted before the intervention, Test 2 after the intervention, and Test 3 7 wk. after Test 2 (no training between Tests 2 and 3). At Test 1, lactate and muscle activity were significantly increased during constrained running. For lactate, a significant decrease was found in the intervention group for running with the constraint; at Test 3, lactate returned to Test 1 level. No notable changes occurred in the physiological parameters. Furthermore, there was no observed transfer effect on normal running.

Minimally-invasive catheterization of the portal, hepatic and cranial mesenteric veins and the abdominal aorta for quantitative determination of hepatic metabolism in dairy cows.

The objective of this study was to establish a minimally-invasive, ultrasound (US)-guided technique for the placement of indwelling catheters into the portal, hepatic, and cranial mesenteric veins as well as the abdominal aorta. Catheters were placed in eight healthy dairy cows on day 1. The patency of catheters was tested daily until day 14 when a necropsy was carried out. On day 6, energy intake and hepatic net output of glucose, removal of lactate, and oxygen were determined in seven cows. Post mortem examination revealed that all implanted catheters were in the intended locations. Loss of patency in one portal vein catheter on day 9 was attributable to a fibrin clot. Significant correlations were found between mean energy intake and mean hepatic plasma flow (r=0.91; P=0.004), hepatic glucose output (r=0.81; P=0.027) and hepatic removal of lactate (r=-0.70; P=0.08) and oxygen (r=-0.77; P=0.039), as well as between hepatic glucose net output and removal of lactate (r=-0.92; P=0.004). Minimally-invasive, US-guided transcutaneous catheter placement into the cranial mesenteric, portal and hepatic veins as well as the technique for catheterization of the abdominal aorta appear to be safe, and suitable for studies of quantitative hepatic metabolism in cattle.

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.

Ultrasonographic assessment of liver dimensions in dairy cows with different hepatic triacylglycerol content.

The objective of this study was to investigate the relationship between hepatic triacylglycerol content (TAG) and hepatic size and shape assessed by transcutaneous ultrasonography in dairy cows. In a second part, the usefulness of ultrasonographic measurements pertaining to the size and shape of the liver for the diagnosis of fatty liver was evaluated. Transcutaneous ultrasonographic examination of the liver was carried out in 133 German Holstein cows (BW: 578 ± 6 kg; age: 5 ± 0.2 yr; withers height: 145 ± 0.3 cm). The size of the liver, angle of the ventral liver margin, and thickness of the liver over the portal vein and caudal vena cava were measured in the 10th and 11th intercostal spaces via ultrasonography. As the gold standard, TAG was determined enzymatically in liver biopsies. Hepatic TAG ranged from 5 to 292 mg/g of liver fresh weight (FW). Cows were allocated to 4 groups according to their hepatic TAG content (TAG ≥150 mg/g of FW, n = 32; ≥100 to 150 mg/g of FW, n = 34; ≥50 to 100 mg/g of FW, n = 38; <50 mg/g of FW, n = 29). Weak to moderate correlations (r = 0.26 to 0.49) between the ultrasonographic liver measurements and hepatic TAG indicated an increased size and angle of the liver with increasing hepatic fat accumulation. All mean liver ultrasonographic measurements revealed differences between TAG classes (P < 0.001), with the greatest values in cows with hepatic TAG concentrations ≥150 mg/g of FW. Stepwise multiple linear regression for hepatic TAG prediction (r(2) = 0.34, P < 0.001) and stepwise discriminant analysis used only ultrasonographic measurements obtained via the 10th intercostal space and age. Regression analysis revealed that TAG prediction widely overestimated measured TAG in the low TAG range and underestimated TAG in the high TAG range. The sensitivity and specificity based on discriminant analysis for differentiation of the TAG groups were 0.61 and 0.61 (<50 vs. ≥50 mg/g of FW), 0.67 and 0.71 (<50 vs. ≥100 mg/g of FW), 0.83 and 0.82 (<50 vs. ≥150 mg/g of FW), 0.70 and 0.77 (<100 vs. ≥100 mg/g of FW), and 0.83 and 0.85 (<100 vs. ≥150 mg/g of FW), respectively. Results revealed that with a hepatic TAG content of approximately 100 mg/g of FW, the liver increased in size and its margins became rounded. Pronounced increases in the size and thickness of the liver and in the angle of the liver occurred only in cows with very severe fatty liver (TAG ≥150 mg/g of FW). However, the determination of ultrasonographic measurements of liver size and shape appeared to be of limited diagnostic value in the recognition of fatty liver because of increased interindividual variance in dairy cows.

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.

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.