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.

Interrelations between the rumen microbiota and production, behavioral, rumen fermentation, metabolic, and immunological attributes of dairy cows.

Different studies have shown a strong correlation between the rumen microbiome and a range of production traits (e.g., feed efficiency, milk yield and components) in dairy cows. Underlying dynamics concerning cause and effect are, however, still widely unknown and warrant further investigation. The aim of the current study was to describe possible functional interrelations and pathways using a large set of variables describing the production, the metabolic and immunological state, as well as the rumen microbiome and fermentation characteristics of dairy cows in early lactation (n = 36, 56 ± 3 d in milk). It was further hypothesized that the feed intake-associated behavior may influence the ruminal fermentation pattern, and a set of variables describing these individual animal attributes was included. Principal component analysis as well as Spearman's rank correlations were conducted including a total of 265 variables. The attained plots describe several well-known associations between metabolic, immunological, and production traits. Main drivers of variance within the data set included milk production and efficiency as well as rumen fermentation and microbiome diversity attributes, whereas behavioral, metabolic, and immunological variables did not exhibit any strong interrelations with the other variables. The previously well-documented strong correlation of production traits with distinct prokaryote groups was confirmed. This mainly included a negative correlation of operational taxonomic units ascribed to the Prevotella genus with milk and fat yield and feed efficiency. A central role of the animals' feed intake behavior in this context could not be affirmed. Furthermore, different methodological and interpretability aspects concerning the microbiome analysis by 16S rRNA gene sequencing, such as the discrepancy between taxonomic classification and functional communality, as well as the comparability with other studies, are discussed. We concluded that, to further investigate the driving force that causes the difference between efficient and inefficient animals, studies including more sophisticated methods to describe phenotypical traits of the host (e.g., rumen physiology, metabolic and genetic aspects) as well as the rumen microbiome (e.g., metagenome, metatranscriptome, metaproteome, and metabolome analysis) are needed.

Differential effects of monensin and a blend of essential oils on rumen microbiota composition of transition dairy cows.

In response to oral application, monensin alters the rumen microbiota, increasing ruminal propionate production and energy availability in the animal. Data from different studies indicate that the susceptibility of rumen bacteria to monensin is mainly cell-wall dependent but tracing its activity to specific microbial groups has been challenging. Several studies have shown a similar effect for essential oils but results are inconsistent. To investigate the influence of monensin and a blend of essential oils (BEO, containing thymol, guaiacol, eugenol, vanillin, salicylaldehyde, and limonene) on the rumen microbiome, rumen liquid samples were collected orally on d 56 postpartum from cows that had either received a monensin controlled-release capsule 3 wk antepartum, a diet containing a BEO from 3 wk antepartum onward, or a control diet (n = 12). The samples were analyzed for pH, volatile fatty acid, ammonia, and lipopolysaccharide concentrations and protozoal counts. A 16S rRNA gene fingerprinting analysis (PCR-single-strand conformation polymorphism) and sequencing revealed that the BEO treatment had no effect on the rumen microbiota, whereas monensin decreased bacterial diversity. Twenty-three bacterial species-level operational taxonomic units were identified for which monensin caused a significant decrease in their relative abundance, all belonging to the phyla Bacteroidetes (uncultured BS11 gut group and BS9 gut group) and Firmicutes (Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae). Ten bacterial operational taxonomic units belonging to the phyla Actinobacteria (Coriobacteriaceae), Bacteroidetes (Prevotella), Cyanobacteria (SHA-109), and Firmicutes (Lachnospiraceae and Ruminococcaceae) increased in relative abundance due to the monensin treatment. These results confirm the hypothesis that varying effects depending on cell-wall constitution and thickness might apply for monensin sensitivity rather than a clear-cut difference between gram-negative and gram-positive bacteria. No effect of monensin on the archaea population was observed, confirming the assumption that reported inhibition of methanogenesis is most likely caused through a decrease in substrate availability, rather than by a direct effect on the methanogens. The data support the hypothesis that the observed increase in ruminal molar propionate proportions due to monensin may be caused by a decrease in abundance of non-producers and moderate producers of propionate and an increase in abundance of succinate and propionate producers.

The effects of a ration change from a total mixed ration to pasture on health and production of dairy cows.

In pasture-based dairy production systems, dairy cows often receive a silage- and concentrate-based ration [total mixed ration (TMR)] during wintertime and are gradually introduced to fresh herbage in spring. The present study aimed to investigate how the transition to this new nutritional situation influenced different production and health indicators. A 10-wk trial was performed in spring 2014, including 60 dairy cows of the German Holstein breed (166±23 d in milk, 23.5±3.7 kg of milk/d; means ± SD). The cows were divided into a pasture and a confinement group (PG and CG, respectively). The CG stayed on a TMR-based diet (35% corn silage, 35% grass silage, 30% concentrate; DM basis), whereas the PG was gradually transitioned from a TMR- to a pasture-based ration (wk 1=TMR-only, wk 2=3 h/d on pasture, wk 3 and 4=12 h/d on pasture, wk 5-10=pasture-only). A continuous grazing system was implemented on a ryegrass dominated pasture and temperature humidity indices were assessed based on continuous recording of temperature and humidity indoors as well as outdoors. Dry matter intake (DMI) from TMR, milk production, body weight (BW), and body condition score decreased as soon as the PG had partial access to pasture. Milk production and BW decreased even further in the first week on a full grazing ration, but thereafter BW increased again and milk production stabilized. The DMI estimation using the n-alkane method in wk 7 and 9 revealed an increase in DMI from pasture between the 2 time points and indicates an adaptation of grazing behavior and metabolism over several weeks. Increased serum ß-hydroxybutyrate and fatty acids concentrations at several time points, as well as a continuous body condition score decrease during the whole course of the trial, indicate an energy deficit in the PG. A significant correlation between serum glucose concentrations and the temperature humidity indices was observed. An increase in serum and milk urea concentrations as well as an increase in the urine total N to creatinine ratio occurred in the PG. To assess possible negative effects of the ration change on metabolic and liver health, different clinical chemistry variables and complete blood counts were assessed. No biologically relevant changes were observed for serum albumin, total protein, cholesterol, aspartate transaminase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, as well as for white and red blood cell counts.

The effects of a ration change from a total mixed ration to pasture on rumen fermentation, volatile fatty acid absorption characteristics, and morphology of dairy cows.

To investigate the effect of the change from a concentrate and silage-based ration (total mixed ration, TMR) to a pasture-based ration, a 10-wk trial (wk 1-10) was performed, including 10 rumen- and duodenum-fistulated German Holstein dairy cows (182±24 d in milk, 23.5±3.5kg of milk/d; mean ± standard deviation). The cows were divided in either a pasture group (PG, n=5) or a confinement group (CG, n=5). The CG stayed on a TMR-based ration (35% corn silage, 35% grass silage, 30% concentrate; dry matter basis), whereas the PG was gradually transitioned from a TMR to a pasture-based ration (wk 1: TMR only; wk 2: 3 h/d on pasture wk 3 and 4: 12 h/d on pasture wk 5-10: pasture only). Ruminal pH, volatile fatty acids (VFA), NH3-N, and lipopolysaccharide (LPS) concentrations were measured in rumen fluid samples collected medially and ventrally on a weekly basis. Ruminal pH was continuously recorded during 1 to 4 consecutive days each week using ruminal pH measuring devices. In wk 1, 5, and 10, rumen contents were evacuated and weighed, papillae were collected from 3 locations in the rumen, and subsequently a VFA absorption test was performed. In the PG, mean rumen pH and molar acetate proportions decreased, and molar butyrate proportions increased continuously over the course of the trial, which can most likely be ascribed to an increased intake of rapidly fermentable carbohydrates. During the first weeks on a full grazing ration (wk 5-7), variation of rumen pH decreased, and in wk 5 a lower rumen content, papillae surface area, and potential for VFA absorption were observed. In wk 8 to 10, variation of rumen pH and total VFA concentrations increased again, and acetate/propionate ratio decreased. In wk-10 rumen content, papillae area and VFA absorption characteristics similar to initial levels were observed. Although continuous rumen pH assessments and LPS concentrations did not reveal an increased risk for subacute rumen acidosis (SARA) during the adaption period, histopathology of rumen papillae and potential for VFA absorption indicated a possible risk for rumen health. An increased risk for SARA was observed in wk 9 and 10 in the PG, but rumen LPS concentrations and histopathology were not adversely affected. Results of the present study suggest that after behavioral and metabolic adaptation to the transition from a TMR to a pasture-based ration, no adverse effects on rumen morphology and absorption capacity occurred, although rumen pH after adaptation to pasture indicated increased risk of SARA.