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 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.

Genetic parameters of subclinical macromineral disorders and major clinical diseases in postparturient Holstein cows.

The main objective of this study was to assess the genetic parameters of subclinical disorders associated with subclinical hypocalcemia, hypophosphatemia, subclinical hypomagnesemia, hypokalemia, and hyperphosphatemia, as well as major clinical diseases after calving in Holstein cows. The secondary objective was to estimate the associated genetic and phenotypic correlations among these subclinical and clinical conditions after calving in Holstein cows. The study was conducted in 9dairy herds located in Northern Greece. None of the herds used any kind of preventive measures for milk fever (MF). A total of 1,021 Holstein cows with pedigree information were examined from November 2010 until November 2012. The distribution across parities was 466 (parity 1), 242 (parity 2), 165 (parity 3), and 148 (parity 4 and above) cows. All cows were subjected to a detailed clinical examination and blood was sampled on d 1, 2, 4, and 8 after calving. Serum concentrations of Ca, P, Mg, and K were measured in all samples, whereas ß-hydroxybutyrate (BHB) was measured only for d 8. The final data set included 4,064 clinical and 16,848 biochemical records (4,020 Ca, 4,019 P, 4,020Mg, 3,792K, and 997 BHB). Data of 1,988 observations of body condition score at d 1 and 8 were also available. All health traits were analyzed with a univariate random regression model. The genetic analysis for macromineral-related disorders included 986 cows with no obvious signs of MF (35 cows with MF were excluded). Analysis for other health traits included all 1,021 cows. A similar single record model was used for the analysis of BHB. Genetic correlations among traits were estimated with a series of bivariate analyses. Statistically significant daily heritabilities of subclinical hypocalcemia (0.13-0.25), hypophosphatemia (0.18-0.33), subclinical hypomagnesemia (0.11-0.38), and hyperphosphatemia (0.14-0.22) were low to moderate, whereas that of hypokalemia was low (0.08-0.10). The heritability of body condition score was 0.20±0.10. Statistically significant daily heritabilities of clinical diseases were those of MF (0.07-0.11), left displaced abomasum (0.19-0.31), and mastitis (0.15-0.41). Results suggest that these health disorders are heritable traits and could be minimized with proper genetic selection. Statistically significant phenotypic correlations were estimated for the first time between macromineral concentrations and almost all transition cow metabolic and infectious health disorders.

Incidence and risk factors for canine mastitis and metritis in two guide dog populations.

Canine mastitis and metritis can cause severe illness but the incidence and risk factors have not been well-studied. Goals in the present study were: 1) report the incidence of mastitis and metritis in a large population, and 2) identify potential risk factors that predispose females to those diseases. A retrospective cohort study was conducted using data from two guide dog colonies that was collected for 17 and 10 years, respectively, for the two colonies. A total of 3076 whelpings occurred during the respective study periods and data were analyzed. Clinical mastitis was diagnosed in 13.2 % of whelpings (408 cases) with the average day of diagnosis being 16.7 postpartum. Risk factors for mastitis identified were colony, litter size where bitches that had large litter size of ≥9 pups (16.2 %) were 60 % more likely to develop mastitis compared with bitches that had litters of <9 pups (11.3 %). Bitches with congestion of the mammary gland were 4.8 times more likely to develop mastitis compared with bitches without mammary congestion. Case incidence of metritis was small (0.7 % of whelpings) and occurred on average at day-5 postpartum (range 1-16). There were no significant risk factors identified, and this may be due to the small number of metritis cases (22 cases) in the present study. Interpretations regarding metritis, therefore, should be made with caution. The results from this study provide parameters for breeders and veterinarians to identify bitches that may require close monitoring for mastitis and metritis.

Prediction of Calving to Conception Interval Length Using Algorithmic Analysis of Endometrial mRNA Expression in Bovine.

After parturition, the uterus undergoes significant reconstruction, allows the endometrium to create an environment for subsequent embryo development. Here, we used an unsupervised algorithmic approach to select characteristic endometrial mRNA expression patterns of proposed markers and investigate each marker's role as an individual indicator of reproductive success. Clinically healthy cows at a sixth week postpartum were examined, the percentage of neutrophils (PMNs%) in the cytological smear was calculated, and an endometrial biopsy was taken for qPCR. Based on pregnancy examination, cows were divided into three groups: Pregnant before 100 days postpartum (P100, n = 11), pregnant between 100-200-day (P200, n = 14), and culled (C, n = 10). Animals were also classified based on two PMNs% thresholds > 5% PMNs and > 10% PMNs. The expression of IL1B, IL6, CXCL8, and IL17A was higher in >10%PMNs. The expression of PTGS1 was higher in the P200 compared to P100. Upregulation of inhibin A subunit (INHA) and downregulation of inhibin ß A subunit (INHBA) were observed in the P100. INHBA/INHA ratio was the most accurate linear predictor of the calving-to-conception interval. The application of the k-means algorithm allowed the identification of five unique expression patterns. The sensitivity and specificity of predicting allocation to P100 were 81% and 79%. We also documented the low efficiency of genes associated with subclinical endometritis and PMNs% in determining reproductive capability. These results suggested the presence of distinctive expression patterns in 6 weeks postpartum, correlated with cows' reproductive capacity. Furthermore, we proposed the INHBA/INHA ratio as an indicator of calving-to-conception interval length.

Evaluation of Statistical Process Control Techniques in Monitoring Weekly Body Condition Scores as an Early Warning System for Predicting Subclinical Ketosis in Dry Cows.

The management of body condition score (BCS) during the dry period is associated with the postpartum health outcomes of dairy cows. However, the difference between the actual BCS and the fixed ideal value is not able to accurately predict the occurrence of postpartum diseases. This study aimed to use statistical process control (SPC) technology to monitor the BCS of dry cows, to evaluate the effect of control charts on nutritional strategies, and to explore the utility of SPC in predicting the incidence of postpartum subclinical ketosis (SCK). The BCS and SCK data of 286 cows from the dry off period to 60 days postpartum were collected to set up the early warning function. Three control charts, including a control chart for the average BCS of the herds, for the BCS of each dry cow, and for individual BCS, were established. The early warning signs for postpartum SCK development were: (1) an individual BCS more than 3.5 that remained unchanged for six weeks; (2) a capability index (CPK), an SPC tool, greater than -0.52. Using these parameters, the early warning signs of SCK development were verified in 429 dry cows. The results showed that the accuracy of early warning signal was 0.64 and the precision was 0.26. The control chart showed that the average BCS of dry cows was consistently higher than the expected upper limit of BCS during the experimental period, and that the addition of new cows to the herds increased the average BCS. In summary, the application of SPC technology to monitor the BCS of dry cows was not a good tool for the prediction of postpartum SCK occurrence but was an appropriate tool for guiding positive nutrition strategies.

Milk Beta-Hydroxybutyrate and Fat to Protein Ratio Patterns during the First Five Months of Lactation in Holstein Dairy Cows Presenting Treated Left Displaced Abomasum and Other Post-Partum Diseases.

The main objective of the present study was to evaluate the beta-hydroxybutyrate (BHB) and fat to protein content (F:P) ratio patterns in the milk of Holstein cows with postpartum diseases throughout the first five months of lactation. This prospective study was performed at Vestjyske Dyrlaeger ApS (Nørre Nebel, Denmark). The milk fat, protein, and BHB were evaluated in the Danish Eurofins laboratory according to the monthly averaged days in milk (DIM1 to 5). According to clinical records, five groups were formed: A (control group; cows without diseases; n = 32), B (cows with left displaced abomasum -LDA- and concomitant diseases; n = 25); C (cows with other diseases up to DIM3; n = 13); D (cows with foot disorders up to DIM3; n = 26); and E (cows with disease manifestations in DIM4 and DIM5; n = 26). All the sick cows were treated after diagnosis, and laparoscopy was performed on cows with LDA. In group B, a higher concentration of BHB (0.18 ± 0.02 mmol/L; p < 0.001) was observed than in the control group (0.07 ± 0.02 mmol/L; p < 0.001) in DIM1, presenting an odds ratio (OR) = 8.9. In all groups, BHB decreased to 0.03-0.05 mmol/L (p < 0.05) since DIM3. The F:P ratio was higher in group B (1.77 ± 0.07) than in group A (1.32 ± 0.06; p < 0.05) in DIM1. A similar profile is observed in DIM2. It was observed that animals in group B were four to six times more likely to have a F:P ratio ≥1.29 during DIM1 (OR = 4.0; 95% CI:1.3-14.4; p = 0.01) and DIM2 (OR = 5.9; 95% CI %:1.9-21.9; p < 0.01), than cows in group A. There were also moderate and high correlations between the F:P ratio and the BHB for DIM1 (r = 0.57; r2 = 0.33; RSD = 0.09; p < 0.001) and DIM2 (r = 0.78; r2 = 0.60; RSD = 0.07; p < 0.001), respectively. We concluded that animals affected by LDA in the postpartum period have a higher concentration of BHB in milk in DIM1 and all treated animals quickly recover BHB levels up to DIM3. The F:P ratio is a viable and economic indicator, mainly in DIM1 and DIM2, to estimate BHB concentration and energy balance in cows with LDA and other postpartum diseases.

Different inflammatory responses of bovine oviductal epithelial cells in vitro to bacterial species with distinct pathogenicity characteristics and passage number.

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, -3, -5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

The effect of sire predicted transmitting ability for production traits on fertility, survivability, and health of Holstein dairy cows.

The major objective of this study was to evaluate, using survival analysis and multivariable regression models, the relationship of sire predicted transmitting ability (PTA) for production traits with their daughters' milk production, fat, and protein percentage (PROPCT), reproductive performance, postpartum disease incidence, and survivability. Data were collected from six large commercial dairy farms, and data analysis included 22,205 cows. Information regarding each sire's genetic evaluation included the following: PTA for fat yield (FAT), fat percentage (FATPCT), milk yield (MILK), protein yield, and PROPCT. Sire PTA was categorized into quartiles to facilitate data analysis and interpretation. Retained placenta, metritis, displaced abomasum, and clinical mastitis were diagnosed and treated by farm personnel. The overall average daily milk production, milk fat and PROPCT during the first 10 months of lactation was higher for the cows in the highest quartile of sire PTA, and cows in the lowest quartile had lower averages. There was no significant association between sire PTA for production traits and first test day fat to protein ratio or the incidence of postpartum disease. Sire PTA for MILK, FATPCT, and PROPCT were significantly associated with the hazard of pregnancy. The median days from calving to conception were 159, 155, 170, and 181 days for cows in the sire PTA for MILK quartiles 1, 2, 3, and 4, respectively. Sire PTA for PROPCT and FATPCT were also significantly associated with the hazard of pregnancy. The median days from calving to conception were 175, 189, 152, and 145 for cows in the sire PTA for PROPCT groups 1, 2, 3, and 4, respectively. Additionally, cows in the highest quartile for sire PTA for FATPCT had the lowest median days from calving to conception (144 days) and cows in lowest quartile had the highest median interval (177 days). Sire PTA for FAT was the only sire PTA significantly associated with the hazard of death/culling. When compared with the cows in the highest sire PTA for FAT quartile cows in the first, second, and third quartiles were at 1.51, 1.30, and 1.13 times higher hazard of death/culling, respectively. In conclusion, this study shows that high sire PTA for MILK and low sire PTA for milk fat and PROPCT are associated with decreased daughters' reproductive performance. Sire PTA for production traits were not found to be associated with postpartum disease incidence.