Aspects of transition cow metabolomics-Part I: Effects of a metaphylactic butaphosphan and cyanocobalamin treatment on the metabolome in liver, blood, and urine in cows with different liver metabotypes.

Dairy cows in modern production systems are at risk to develop metabolic disorders during the transition period. Reasons for individual differences in susceptibility, as well as the underlying pathomechanisms, are still only partially understood. The development of metaphylactic treatment protocols is needed. In this context, an on-farm prospective 3-fold blinded randomized study involving 80 German Holstein cows was performed throughout 1 yr. The trial involved a thorough recording of the production and clinical traits, clinical chemistry, and liver biopsies and blood and urine sampling at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), and d 7 (7, 4-13) and 28 (28, 23-34) postpartum (PP) for metabolomics analyses. Two groups received a treatment with butaphosphan and cyanocobalamin (BCC) at either the dosage recommended by the manufacturer or the double dosage (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin (Catosal, Bayer Animal Health), n = 20 in each group, parity: 4.2 ± 2.0 and 3.4 ± 1.3, respectively (mean ± SD)] and one group a placebo treatment (NaCl 0.9%, n = 40, parity: 4.0 ± 1.9). The animals were treated at 6 time points (7, 6, and 5 d AP, and 1, 2, and 3 d PP) via intravenous injection. Mass spectroscopy-based targeted metabolomics analysis of blood plasma and liver samples were performed using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), whereas the urine samples were analyzed by nuclear magnetic resonance spectroscopy. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). Multivariate data analysis (PLS-DA plots) of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in liver metabolome profile between AP and PP status). Metabotype B animals were characterized by higher PP lipomobilization (stronger PP body condition decrease and higher blood bilirubin, fatty acids, gamma-glutamyltransferase, and triglyceride levels) and a higher occurrence of transition cow diseases, compared with the animals in metabotype C. Analysis of the feeding data showed that the period of metabotype B animals (calving in a distinct time frame) was characterized by a decreased grass silage quality. The PP liver metabolome of the metabotype C animals was characterized by higher concentrations of AA, acylcarnitines, lysoPC and sphingomyelins compared with metabotype B. For the metaphylactic treatment with BCC a dose-dependent effect was confirmed, differing between the metabotypes. In all matrices and metabotypes at various time points significant treatment effects were observed, with different profiles in clinical chemistry and as well in metabolomics data. The most clear-cut treatment effect was observed in metabotype B in the liver at 7 d PP, characterized by an increase in several acylcarnitines and phosphatidylcholines, indicating a more efficient influx and oxidation of fatty acids in mitochondria and thereby an increase in energy supply and more efficient triglyceride export in the liver. The results from the liver metabolomics analysis support the application of an indication-based metaphylactic treatment with BCC.

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.

Aspects of transition cow metabolomics-Part III: Alterations in the metabolome of liver and blood throughout the transition period in cows with different liver metabotypes.

The liver plays a central role in the postpartum (PP) energy metabolism of the transition dairy cow; however, studies describing the liver metabolome during this period were lacking. The aim of the presented study was therefore to compare the alterations in the liver and blood metabolome of transition dairy cows. For this purpose, an on-farm trial with 80 German Holstein cows (mean lactation number: 3.9; range: 2-9) was performed, with thorough documentation of clinical traits and clinical chemistry, as well as production data. Liver biopsies and blood samples were collected at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), d 7 (7, 4-13) and 28 (28, 23-34; mean, earliest-latest) PP for targeted mass spectroscopy-based metabolomics analysis using the AbsoluteIDQ p180 kit (Biocrates Life Sciences). Statistical analysis was performed using multivariate (partial least squares discriminant analysis) as well as univariate methods (linear mixed model). Multivariate data analysis of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in the liver metabolome profile between AP and PP). In metabotype C, an increase of almost all acylcarnitines, lysophosphatidylcholines (lysoPC), sphingomyelins, and some phosphatidylcholines (PC, mainly at 7 d PP) was observed after calving. In contrast to metabotype C, the clinical data of the metabotype B animals indicated a higher PP lipomobilization and occurrence of transition cow diseases. The liver metabolome profile of these animals most likely mirrors a failure of adaptation to the PP state. This strong occurrence of metabotypes was much less pronounced in the blood metabolome. Additionally, differences in metabolic patterns were observed across the transition period when comparing liver and blood matrices (e.g., in different biogenic amines, acylcarnitines and sphingolipids). In summary, the blood samples at 7 d PP showed lower acylcarnitines and PC, with minor alterations and a heterogeneous pattern in AA, biogenic amines, and sphingomyelins compared with 14 d AP. In contrast to 7 d PP, the blood samples at 28 PP revealed an increase in several AA, lysoPC, PC, and sphingomyelins in comparison to the AP state, irrespective of the metabotype. In the liver biopsies metabotype B differed from metabotype C animals ante partum by following metabolites: higher α aminoadipic acid, lower AA, serotonin, taurine, and symmetric dimethylarginine levels, lower or higher concentrations of certain acylcarnitines (higher: C2, C3, C5, C4:1; lower: C12:1, C14:1-OH, C16:2), and lower lysoPC (a C16:0, C18:0, C20:3, C20:4) and hexose levels. In blood samples, fewer differences were observed, with lower serotonin, acylcarnitine C16:2, lysoPC (a C16:0, C17:0, C18:0 and C18:1), PC aa C38:0, and PC ae C42:2. The results show that the use of only the blood metabolome to assess liver metabolism may be hampered by the fact that blood profiles are influenced by the metabolism of many organs, and metabolomics analysis from liver biopsies is a more suitable method to identify distinct metabotypes. Future studies should investigate the stability and reproducibility of the metabotype and phenotypes observed, and the possible predictive value of the metabolites already differing AP between metabotype B and C.