Multiomics reveals blood differential metabolites and differential genes in the early onset of ketosis in dairy cows.

Ketosis-a metabolic state characterized by elevated levels of ketone bodies in the blood or urine-reduces the performance and health of dairy cows and causes substantial economic losses for the dairy industry. Currently, beta-hydroxybutyric acid is the gold standard for determining ketosis in cows; however, as this method is only applicable postpartum, it is not conducive to the early intervention of ketosis in dairy cows. In this study, the sera of dry, periparturient, postpartum ketotic, and healthy cows were analyzed by both transcriptomics and metabolomics techniques. Moreover, changes of gene expression and metabolites were observed, and serum physiological and biochemical indexes were detected by ELISA. The purpose was to screen biomarkers that can be used to detect the incidence of dry or periparturient ketosis in cows. The results showed that ketotic cows had increased levels of glycolipid metabolism indexes, oxidizing factors, and inflammatory factors during dry periods and liver damage, which could be used as early biomarkers to predict the onset of ketosis. Transcriptomic results yielded 20 differentially expressed genes (DEGs) between ketotic and healthy cows during dry, peripartum, and postpartum periods. GO and KEGG enrichment analyses indicated that these DEGs were involved in amino acid metabolism, energy metabolism, and disease-related signaling pathways. The metabolomics sequencing results showed that ketotic cows mainly showed enrichment in tricarboxylic acid cycle, butyric acid metabolism, carbon metabolism, lysine degradation, fatty acid degradation, and other signaling pathways. Metabolites differed between ketotic and healthy cows in dry, pre-parturition, and post-parturition periods. Combined transcriptomics and metabolomics analyses identified significant enrichment in the glucagon signaling pathway and the lysine degradation signaling pathway in dry, periparturient, and postpartum ketotic cows. PRKAB2 and SETMAR-key DEGs of the glucagon signaling pathway and lysine degradation signaling pathway, respectively-can be used as key marker genes for determining the early onset of ketosis in dairy cows.

Genomic prediction of blood biomarkers of metabolic disorders in Holstein cattle using parametric and nonparametric models.

BACKGROUND: Metabolic disturbances adversely impact productive and reproductive performance of dairy cattle due to changes in endocrine status and immune function, which increase the risk of disease. This may occur in the post-partum phase, but also throughout lactation, with sub-clinical symptoms. Recently, increased attention has been directed towards improved health and resilience in dairy cattle, and genomic selection (GS) could be a helpful tool for selecting animals that are more resilient to metabolic disturbances throughout lactation. Hence, we evaluated the genomic prediction of serum biomarkers levels for metabolic distress in 1353 Holsteins genotyped with the 100K single nucleotide polymorphism (SNP) chip assay. The GS was evaluated using parametric models best linear unbiased prediction (GBLUP), Bayesian B (BayesB), elastic net (ENET), and nonparametric models, gradient boosting machine (GBM) and stacking ensemble (Stack), which combines ENET and GBM approaches. RESULTS: The results show that the Stack approach outperformed other methods with a relative difference (RD), calculated as an increment in prediction accuracy, of approximately 18.0% compared to GBLUP, 12.6% compared to BayesB, 8.7% compared to ENET, and 4.4% compared to GBM. The highest RD in prediction accuracy between other models with respect to GBLUP was observed for haptoglobin (hapto) from 17.7% for BayesB to 41.2% for Stack; for Zn from 9.8% (BayesB) to 29.3% (Stack); for ceruloplasmin (CuCp) from 9.3% (BayesB) to 27.9% (Stack); for ferric reducing antioxidant power (FRAP) from 8.0% (BayesB) to 40.0% (Stack); and for total protein (PROTt) from 5.7% (BayesB) to 22.9% (Stack). Using a subset of top SNPs (1.5k) selected from the GBM approach improved the accuracy for GBLUP from 1.8 to 76.5%. However, for the other models reductions in prediction accuracy of 4.8% for ENET (average of 10 traits), 5.9% for GBM (average of 21 traits), and 6.6% for Stack (average of 16 traits) were observed. CONCLUSIONS: Our results indicate that the Stack approach was more accurate in predicting metabolic disturbances than GBLUP, BayesB, ENET, and GBM and seemed to be competitive for predicting complex phenotypes with various degrees of mode of inheritance, i.e. additive and non-additive effects. Selecting markers based on GBM improved accuracy of GBLUP.

Determining lactate concentrations in Korean indigenous calves and evaluating its role as a predictor for acidemia in calf diarrhea.

BACKGROUND: Calf diarrhea leads to high mortality rates and decreases in growth and productivity, causing negative effects on the livestock industry. Lactate is closely associated with metabolic acidosis in diarrheic calves. However, there have been no reports on lactate concentrations in Korean indigenous (Hanwoo) calves, especially those with diarrhea. This study aimed to determine the reference range of L-lactate and D-lactate concentrations in Hanwoo calves and to better understand the utility of lactate as predictive factors for acidemia in diarrheic calves. RESULTS: L-lactate and D-lactate concentrations were measured in healthy (n = 44) and diarrheic (n = 93) calves, and blood gas analysis was performed on diarrheic calves. The reference range in healthy calves was 0.2-2.25 mmol/L for L-lactate and 0.42-1.38 mmol/L for D-lactate. Diarrheic calves had higher concentrations of L-lactate and D-lactate than healthy calves. In diarrheic calves, L-lactate and D-lactate each had weak negative correlation with pH (r = - 0.31 and r = - 0.35). In diarrheic calves with hyper-L-lactatemia, the combined concentrations of L-lactate and D-lactate had moderate correlation with pH (r = - 0.51) and anion gap (r = 0.55). Receiver operating characteristic analysis showed D-lactate had fair predictive performance (AUC = 0.74) for severe acidemia, with an optimal cut-off value of > 1.43 mmol/L. The combined concentrations of L-lactate and D-lactate showed fair predictive performance for predicting acidemia (AUC = 0.74) and severe acidemia (AUC = 0.72), with cut-off values of > 6.05 mmol/L and > 5.95 mmol/L. CONCLUSIONS: The determined reference ranges for L-lactate and D-lactate in Hanwoo calves enable the identification of hyper-L-lactatemia and hyper-D-lactatemia. Diarrheic calves exhibited increased lactate concentrations correlated with acid-base parameters. While the concentrations of L-lactate and D-lactate have limitations as single diagnostic biomarkers for predicting acidemia or severe acidemia, their measurement remains important, and L-lactate has the advantage of being measurable at the point-of-care. Assessing lactate concentrations should be considered by clinicians, especially when used alongside other clinical indicators and diagnostic tests. This approach can improve calf diarrhea management, contributing positively to animal welfare and providing economic benefits to farms.

Influence of an iron dextran injection in various diseases on hematological blood parameters, including serum ferritin, neonatal dairy calves.

BACKGROUND: Feeding milk substitutes with low iron content or whole milk without iron supplementation is considered a major factor in developing iron-deficiency anemia in neonatal dairy calves. Young calves are often supplemented with iron dextran injections on the first day of life to prevent anemia. However, the effects of preventive treatment and the presence of disease on serum iron (Fe) concentrations, serum ferritin levels, and hematological blood parameters during the early neonatal stages have not been examined in detail. Therefore, we examined and evaluated the effects of iron dextran injections and health status on the development of hematocrit (Ht), red blood cells (RBC), hemoglobin concentration (Hb), erythrocyte indices (mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration), Fe, and serum ferritin concentrations in dairy calves within the first 10 days of life. The suitability of serum ferritin as a reliable indicator of anemia in very young calves was evaluated by correlating ferritin concentrations with known laboratory diagnostic parameters of anemia. RESULTS: Iron supplementation significantly increased Fe levels (P = 0.048) but did not affect serum ferritin levels in neonatal calves. Fe concentrations were significantly lower in diseased than healthy calves (P = 0.0417). Iron supplementation significantly affected the health status, as observed in Ht (Ptreat=0.0057; Phealth=0.0097), RBC (Ptreat=0.0342; Phealth=0.0243), and Hb (Ptreat=0.0170; Phealth=0.0168). Serum ferritin levels did not significantly correlate with Fe levels. Both groups showed marked differences in ferritin levels, with the highest levels measured on day 2. Fe concentrations showed weak negative correlations with Hb and Ht levels on day 3 (ρ=-0.45; P = 0.0034 and ρ=-0.045; P = 0.0032, respectively). RBC count showed strong positive correlations with Hb and Ht levels (ρ = 0.91 and ρ = 0.93; P < 0.001). CONCLUSION: Iron dextran injections increased Fe concentrations but reduced Ht level, RBC count, and Hb level. The presence of diseases led to a reduction in Fe and higher values of Ht, RBC, and Hb in moderate disease than in severe disease. Due to physiological fluctuations during the first 3 days of life, serum ferritin level seems unuseful for evaluating iron storage before day 4 of life.

Serum macroelements and microelements levels in periparturient dairy cows in relation to fatty liver diseases.

BACKGROUND: Fatty liver in dairy cows is a common metabolic disease defined by triglyceride (TG) buildup in the hepatocyte. Clinical diagnosis of fatty liver is usually done by liver biopsy, causing considerable economic losses in the dairy industry owing to the lack of more effective diagnostic methods. Therefore, this study aimed to investigate the potential utility of blood biomarkers for the diagnosis and early warning of fatty liver in dairy cows. RESULTS: A total of twenty-four lactating cows within 28 days after parturition were randomly selected as experimental animals and divided into healthy cows (liver biopsy tested, n = 12) and cows with fatty liver (liver biopsy tested, n = 12). Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the macroelements and microelements in the serum of two groups of cows. Compared to healthy cows (C), concentrations of calcium (Ca), potassium (K), magnesium (Mg), strontium (Sr), selenium (Se), manganese (Mn), boron (B) and molybdenum (Mo) were lower and copper (Cu) was higher in fatty liver cows (F). Meanwhile, the observed differences in macroelements and microelements were related to delivery time, with the greatest major disparity between C and F occurring 7 days after delivery. Multivariable analysis was used to test the correlation between nine serum macroelements, microelements and fatty liver. Based on variable importance projection and receiver operating characteristic (ROC) curve analysis, minerals Ca, Se, K, B and Mo were screened as the best diagnostic indicators of fatty liver in postpartum cows. CONCLUSIONS: Our data suggested that serum levels of Ca, K, Mg, Se, B, Mo, Mn, and Sr were lower in F than in C. The most suitable period for an early-warning identification of fatty liver in cows was 7 days after delivery, and Ca, Se, K, B and Mo were the best diagnostic indicators of fatty liver in postpartum cows.

Cardiac biomarkers as tools in the prediction and diagnosis of traumatic pericarditis and traumatic reticuloperitonitis in cattle and buffaloes.

BACKGROUND: In the livestock industry, Foreign Body Syndrome is a devastating disease condition. Feeding management, lacking of food discrimination, and eating chopped food increase the risk of swallowing sharp foreign bodies in bovine species. In addition to the honeycomb cells shape of the reticulum, the contractions of the reticular wall, gravid uterine pressure, and parturition efforts, foreign bodies can penetrate the reticular wall, causing cascade of problems including traumatic reticulitis, traumatic reticuloperitonitis, and traumatic pericarditis. The present study was carried out to evaluate the diagnostic significance of cardiac troponin I rapid test cassette and other cardiac biomarkers including serum cardiac troponin I (cTn I), creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), and aspartate aminotransferase enzyme (AST), in confirmed cases of traumatic pericarditis (TP) and/or traumatic reticuleoperitonitis (TRP) in cattle and buffaloes. METHODS: A total number of 30 animals (22 cattle and 8 buffaloes) with different signs such as anorexia, jugular distension, brisket edema, and signs of pain (reluctance to move, arching back, and abduction of the forelimbs) were included in the present study. Based on case history, clinical signs, ferroscopic, pericardiocentesis, radiographic and ultrasonographic examinations, TP were confirmed in cattle (n = 10) and buffaloes (n = 8) while TRP were confirmed only in cattle (n = 12). Additionally, 20 clinically healthy animals (n = 10 cattle and 10 buffaloes) were used as a control group. Blood samples were collected for determination of blood level of Tn-I, and activity of CK-MB, LDH, and AST. RESULTS: The obtained results revealed a highly significant increase in serum cTn I in diseased cattle with TP and TRP (P = 0.00), while buffaloes with TP showed no significant changes in serum cTn I (P = 0.111). Both diseased cattle and buffaloes showed increased serum activities of CK-MB, AST, and LDH enzyme. On the other hand, cardiac troponin I rapid test cassette failed to detect cTn I in diseased animals. CONCLUSION: The study concluded that the cardiac troponin I rapid test cassette did not have a diagnostic significance and could not be used as a point-of-care under field condition for diagnosis of TP and TRP in large ruminants. However, the serum troponin I level is helpful in diagnosis of TP and TRP in cattle. Although cardiac biomarkers have some diagnostic values in TP and TRP, the traditional diagnostic methods (clinical, radiography and ultrasonography examinations) are crucial for thorough evaluation of TP/TRP cases in bovine.

Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Progesterone profiles in postpartum dairy cows with inflammatory disorders.

The objective of this prospective cohort study was to determine if progesterone (P4) profiles differed between dairy cows with or without inflammatory disorders early postpartum. A total of 708 cows from 2 commercial herds were enrolled 3 wk before parturition and examined for clinical health disorders (difficult calving, retained placenta, metritis, displaced abomasum, mastitis, or lameness) until 5 wk postpartum. Serum haptoglobin (Hp) was measured in blood at 2 and 6 DIM (range ±2 d); metritis was assessed at 4, 8, 11, and 15 DIM; and purulent vaginal discharge and endometritis (≥6% PMN in endometrial cytology sampled by cytobrush) were assessed at 35 ± 3 DIM. As Hp ≥0.8 g/L or endometritis were associated with ovarian dysfunction in previous studies, cows with serum Hp ≥0.8 g/L at either time point and endometritis, regardless of clinical disease, were classified as the cohort with inflammatory disorders (INFLAM; n = 139). Clinically healthy cows without difficult calving, with singleton birth, with Hp <0.8 g/L at both sampling times, without endometritis or purulent vaginal discharge, and BCS ≥3.00 (1 to 5 scale) were classified as healthy (n = 133). Cows with only one of the 2 conditions (high Hp or endometritis) were excluded. Cohorts had serum P4 measured twice weekly from 35 to 70 DIM (±3 d), and the first detected luteal phase (LP) during the sampling period was defined as the period from onset of luteal activity (P4 increase to ≥1 ng/mL) until decline of P4 to <1 ng/mL. The odds of prolonged LP (≥21 d long), average LP length, peak P4, and time to P4 decline (hazard rate) were analyzed using multivariable mixed logistic, linear, or Cox proportional hazard regression models including INFLAM status, parity, sampling day (when applicable), and herd as a random effect considering the covariates of season, milk yield at first DHIA test, and DIM at onset of cyclicity or LP length (when applicable). Cows with INFLAM had greater odds of prolonged LP (LSM ± SEM; 67% vs. 37% ± 7%), greater average LP length (17 vs. 15 ± 2 d), lesser P4 at d 4 (4.6 vs. 5.5 ± 0.3 ng/mL) and d 7 (6.0 vs. 7.7 ± 0.3 ng/mL) of the LP, and lesser peak P4 (6.9 vs. 8.2 ± 0.3 ng/mL) during the LP than healthy cows. Status of INFLAM was associated with time to P4 decline in multiparous but not primiparous cows; the LP of INFLAM multiparous cows was less likely to have luteolysis (P4 decline) by d 14 (adjusted hazard ratio [AHR] and 95% CI: 0.54; 0.31 to 0.94) or by d 21 (AHR: 0.32; 0.12 to 0.84) than in healthy multiparous cows. In conclusion, postpartum cows with markers of systemic inflammation at wk 1 and uterine inflammation at wk 5 had altered luteal function (prolonged LP and lower P4 concentrations) before first breeding, which is a possible pathway linking postpartum health disorders and reduced fertility.

Plasma and milk metabolomics profiles in dairy cows with subclinical and clinical ketosis.

Ketosis, a commonly observed energy metabolism disorder in dairy cows during the peripartal period, is distinguished by increased concentrations of BHB in the blood. This condition has a negative impact on milk production and quality, causing financial losses. An untargeted metabolomics approach was performed on plasma samples from cows between 5 and 7 DIM diagnosed as controls (CON; BHB <1.2 mM, n = 30), subclinically ketotic (SCK; 1.2 < BHB <3.0 mM, n = 30), or clinically ketotic (CK; BHB >3.0 mM, n = 30). Cows were selected from a commercial farm of 214 Holstein cows (average 305-d yield in the previous lactation of 35.42 ± 7.23 kg/d; parity, 2.41 ± 1.12; BCS, 3.1 ± 0.45). All plasma and milk samples (n = 90) were subjected to liquid chromatography-MS-based metabolomic analysis. Statistical analyses were performed using GraphPad Prism 8.0, MetaboAnalyst 4.0, and R version 4.1.3. Compared with the CON group, both SCK and CK groups had greater milk fat, freezing point, and fat-to-protein ratio, as well as lower milk protein, lactose, solids-not-fat, and milk density. Within 21 d after calving, compared with CON, the SCK group experienced a reduction of 2.65 kg/d in milk yield, while the CK group experienced a decrease of 7.7 kg/d. Untargeted metabolomics analysis facilitated the annotation of a total of 5,259 and 8,423 metabolites in plasma and milk. Differentially affected metabolites were screened in CON versus SCK, CON versus CK, and SCK versus CK (unpaired t-test, false discovery rate <0.05; and absolute value of log(2)-fold change >1.5). A total of 1,544 and 1,888 differentially affected metabolites were detected in plasma and milk. In plasma, glycerophospholipid metabolism, pyrimidine metabolism, tryptophan metabolism, sphingolipid metabolism, amino sugar and nucleotide sugar metabolism, phenylalanine metabolism, and steroid hormone biosynthesis were identified as important pathways. Weighted gene co-expression network analysis (WGCNA) indicated that tryptophan metabolism is a key pathway associated with the occurrence and development of ketosis. Increases in 5-hydroxytryptophan and decreases in kynurenine and 3-indoleacetic acid in SCK and CK were suggestive of an impact at the gut level. The decrease of most glycerophospholipids indicated that ketosis is associated with disordered lipid metabolism. For milk, pyrimidine metabolism, purine metabolism, pantothenate and CoA biosynthesis, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, sphingolipid metabolism, and fatty acid degradation were identified as important pathways. The WGCNA indicated that purine and pyrimidine metabolism in plasma was highly correlated with milk yield during the peripartal period. Alterations in purine and pyrimidine metabolism characterized ketosis, with lower levels of these metabolites in both milk and blood underscoring reduced efficiency in nitrogen metabolism. Our results may help to establish a foundation for future research investigating mechanisms responsible for the occurrence and development of ketosis in peripartal cows.

Oestrogen and progesterone concentrations in intrapartum cows with insufficient cervix dilation.

The cervix is an important organ that has to dilate sufficiently at delivery to allow the foetus to transition to extrauterine life. Insufficient dilatation of the cervix (IDC) is a frequent cause of dystocia in cattle. The mechanisms underlying cervical opening and the pathogenesis of IDC are still widely unclear. Systematic studies on the relationship between IDC and steroid hormones have been limited and have yielded inconsistent findings. This study aimed to measure oestrogen and progesterone (P4) concentrations in intrapartum cows presented with dystocia due to IDC and in a comparison (C) group of cows with eutocic delivery. Before any obstetrical procedures, and right after the initial evaluation, blood samples were taken from IDC and C animals. Concentrations of P4, oestradiol-17ß (E2), free total oestrogens (FTE) and conjugated total oestrogens (CTE) were measured by established radioimmunoassays. Concentrations of P4 (p = .538), FTE (p = .065) and CTE (p = .605) were not statistically different between C and IDC groups. However, E2 levels in group C were significantly lower when compared to those in the IDC group (p = .013), which is inconsistent with the function of oestrogens in cervical dilatation. The correlation analysis demonstrated significant positive correlations between the pairs P4 versus FTE, P4 versus E2 and FTE versus E2 in group C and between the pair FTE versus E2 in group IDC. In conclusion, the results suggest that local activities of steroids relevant to the aetiology of IDC are not reflected by concentrations in the systemic circulation or that other factors are clearly more important.

Identification of genetic markers associated with hyperketonemia patterns in early lactation Holstein cows.

Ketosis, evidenced by hyperketonemia with elevated blood ß-hydroxybutyrate (BHB) levels, is a significant metabolic disorder of dairy cattle, typically diagnosed within the first 6 weeks post-calving when high energy levels are essential to milk production. Our study aimed to identify genetic markers linked to hyperketonemia (HYK) patterns in Holstein cows during early lactation and compare these to HYK-negative cows. We screened 964 cows for HYK using a threshold of BHB ≥1.2 mmol/L during the first 2 weeks postpartum (screening period, SP). Cows that tested negative initially were retested the following week. Cows were deemed HYK-negative (CON group) if BHB levels were below 1.2 mmol/L in both tests, while those with BHB levels exceeding this threshold at any test were treated and classified as HYK-positive (HYK+). Post-treatment, HYK+ cows were monitored for two-week follow-up period (FP) and classified based on their recovery: cured (CUR; consistently low BHB), recurrent (REC; fluctuating BHB levels), severe (SEV; high initial BHB that decreased), or chronic (CHR; persistently high BHB). Using 489 cows that were genotyped, a GWAS was conducted using GCTA software, revealing significant associations of several SNPs across different HYK patterns when compared to the CON group. These SNPs were primarily linked to genes affecting milk traits and were enriched in biological pathways relevant to protein glycosylation, inflammatory response, glucose homeostasis, and fatty acid synthesis. Our findings highlight genomic regions, potential candidate genes, and biological pathways related to ketosis, underscoring potential targets for improving health management in dairy cattle. These insights could lead to better strategies for managing ketosis through genetic selection, ultimately enhancing dairy cattle welfare and productivity. Further research with a larger number of cows is recommended to validate these findings and help confirm the implicated SNPs and genes.

Serum selenium concentrations in neonatal calves: Influence of postnatal selenium injection and health status.

Background: Selenium supply plays a major role in calf rearing, as a deficiency can lead to health problems, economic loss, and even death. Therefore, postnatal selenium injections are often administered as a preventive measure. Objective: In this study, we examined the serum selenium concentrations of healthy and sick calves within the first days of life. Further, serum concentrations after injection with selenium were determined. Animals and procedure: Serum selenium concentrations from 75 calves were measured until the 10th d of life and the differences between sick and healthy calves were investigated. The variations in selenium concentration were analyzed 3 and 6 d after subcutaneous injection of 5.5 mg sodium selenite in 32 calves.To compare serum concentrations between healthy and sick calves, an independent samples t-test was used. For unequal variances, the Satterthwaite method was used; and for equal variances, the pooled sample variance was used. To analyze the statistical differences between the concentrations at different time points, the data were log-transformed and the Bonferroni correction was used. Results: The mean initial selenium concentration was 46 ± 37 µg/L. There was no statistically significant difference (P = 0.60) between sick (46 ± 34 µg/L) and healthy (46 ± 47 µg/L) calves. Serum selenium concentrations 3 and 6 d after injection of calves over 3 samples were 62 ± 19 µg/L and 50 ± 13 µg/L, respectively. Calves with an initial serum concentration of ≥ 72 µg/L showed a decrease of serum selenium concentration despite the injection. Conclusion and clinical relevance: Newborn calves showed a high variation in selenium concentration that was not influenced by health status. A single injection of 5.5 mg of sodium selenite did increase the selenium concentration in calves with selenium undersupply. After injection, none of the calves showed serum concentrations above the reference range for adult cattle. Therefore, the indication for a selenium injection can be interpreted generously if selenium undersupply is suspected.

Concentrations sériques de sélénium chez les veaux nouveau-nés : influence de l'injection postnatale de sélénium et de l'état de santé. Contexte: L'apport en sélénium joue un rôle majeur dans l'élevage des veaux, car une carence peut entraîner des problèmes de santé, des pertes économiques et même la mort. Par conséquent, des injections postnatales de sélénium sont souvent administrées à titre préventif. Objectif: Dans cette étude, nous avons examiné les concentrations sériques de sélénium de veaux sains et malades au cours des premiers jours de vie. De plus, les concentrations sériques après injection de sélénium ont été déterminées. Animaux et procédure: Les concentrations sériques de sélénium de 75 veaux ont été mesurées jusqu'au 10e jour de vie et les différences entre les veaux malades et sains ont été étudiées. Les variations de concentration en sélénium ont été analysées 3 et 6 jours après l'injection sous-cutanée de 5,5 mg de sélénite de sodium chez 32 veaux.Pour comparer les concentrations sériques entre les veaux sains et malades, un test t sur échantillons indépendants a été utilisé. Pour les variances inégales, la méthode de Satterthwaite a été utilisée; et pour des variances égales, la variance de l'échantillon groupé a été utilisée. Pour analyser les différences statistiques entre les concentrations à différents moments, les données ont été transformées par logarithme et la correction de Bonferroni a été utilisée. Résultats: La concentration initiale moyenne en sélénium était de 46 ± 37 µg/L. Il n'y avait pas de différence statistiquement significative (P = 0,60) entre les veaux malades (46 ± 34 µg/L) et sains (46 ± 47 µg/L). Les concentrations sériques de sélénium 3 et 6 jours après l'injection des veaux sur 3 échantillons étaient respectivement de 62 ± 19 µg/L et de 50 ± 13 µg/L. Les veaux avec une concentration sérique initiale ≥ 72 µg/L ont montré une diminution de la concentration sérique en sélénium malgré l'injection. Conclusion et pertinence clinique: Les veaux nouveau-nés ont montré une forte variation de la concentration en sélénium qui n'était pas influencée par l'état de santé. Une injection unique de 5,5 mg de sélénite de sodium a augmenté la concentration de sélénium chez les veaux présentant un apport insuffisant en sélénium. Après l'injection, aucun veau n'a présenté de concentrations sériques supérieures à la plage de référence pour les bovins adultes. Par conséquent, l'indication d'une injection de sélénium peut être interprétée de manière généreuse si un apport insuffisant en sélénium est suspecté.(Traduit par Dr Serge Messier).

Short communication: Association between prepartum subclinical magnesium imbalance and postpartum diseases in grazing dairy cows in Southern Chile.

This study aimed to evaluate the relationship between prepartum subclinical hypomagnesemia (pre-SHMg) and the occurrence of dystocia, metritis, clinical mastitis, lameness, and subclinical hypomagnesemia postpartum (post-SHMg) in pasture-based dairy cows. Also, the difference in means of prepartum magnesium (Mg) concentration by postpartum health events was evaluated. A total of 890 dairy cows from 32 commercial farms located in southern Chile were enrolled. Cows were examined twice, once between 30 and 3 days before and once between 3 and 30 days after calving. Blood samples were collected on both assessments, and cows were considered as having SHMg if serum total Mg < 0.65 mmol/L. On the postpartum visit, cows were evaluated for metritis and lameness. Information about clinical mastitis and dystocia was collected from on-farm records. Data were analyzed using multivariable mixed linear models and multivariable mixed logistic regression models. The overall prevalence of pre-SHMg was 9.9%, and its presence was associated with the occurrence of post-SHMg (odd ratio [OR] = 5.7; P < 0.0001) and metritis (OR = 3.1; P = 0.04). However, we did not detect an association between pre-SHMg and dystocia, clinical mastitis, or lameness after calving. Prepartum serum Mg concentrations were lower in cows that developed post-SHMg than those that did not (LSM ± SE = 0.75 ± 0.02 mmol/L vs. 0.83 ± 0.02 mmol/L; P < 0.0001). In conclusion, pre-SHMg was associated with a higher risk of post-SHMg and metritis in grazing dairy cows but not other postpartum health events.

Methods in isolation and characterization of bovine monocytes and macrophages.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle.

BACKGROUND: DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. RESULTS: The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. CONCLUSIONS: The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

Epidemiology, haematology and molecular characterization of haemoprotozoon and rickettsial organisms causing infections in cattle of Jammu region, North India.

BACKGROUND: The present study was aimed at establishing the prevalence, epidemiology and molecular characterization of major haemoprotozoons (Babesia and Theileria) and rickettsia (Anaplasma) of cattle in Jammu region (North India) using microscopy and Polymerase Chain Reaction (PCR). Hematology, microscopy and PCR based prevalence studies were undertaken with 278 whole blood samples from cattle. Molecular prevalence studies were followed by genetic characterization of the isolates of Babesia, Anaplasma and Theileria spp. based on 18S rRNA, 16S rRNA and Tams1 gene, respectively. The data related to metrology and epidemiological variables like temperature, rainfall, season, age and type of livestock rearing was analyzed and correlated with occurrence of disease by statistical methods. RESULTS: The prevalence based on microscopy was 12.9% (36/278) whereas PCR recorded 30.22% (84/278) animals positive for haemoparasitic infections. All the samples found positive by microscopy were also recorded positive by PCR. Thus the study revealed prevalence of Babesia bigemina, Anaplasma marginale and Theileria annulata to be 9.7, 16.5 and 0.7% respectively. The metrological and epidemiological variables made inroads for the propagation of vector ticks and occurrence of infection. Haematological alterations predominantly related to decrease in haemoglobin, red blood cell count and packed cell volume were evident in diseased animals and collaterally affected the productivity. Further the genetic characterization of Babesia bigemina. (MN566925.1, MN567603, MN566924.1), Anaplasma marginale. (MH733242.1, MN567602.1) and Theileria annulata (MT113479) provided a representative data of the isolates circulating in the region and their proximity with available sequences across the world. CONCLUSIONS: Despite holding much significance to the animal sector, comprehensive disease mapping has yet not been undertaken in several parts of India. The present study provides a blue print of disease mapping, epidemiological correlations and genomic diversity of Babesia bigemina, Anaplasma marginale and Theileria annulata circulating in the region.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle.

BACKGROUND: Serious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle. METHODS: Molecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia. RESULTS: A total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83-81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98-100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle. CONCLUSION: We present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

Effect of hyperketonemia on the diurnal patterns of energy-related blood metabolites in early-lactation dairy cows.

Most dairy cows experience a period of energy deficit in early lactation, resulting in increased plasma concentrations of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). Our objectives were to determine (1) the diurnal variation in plasma BHB and NEFA, (2) the correlation between plasma NEFA and BHB when accounting for diurnal changes, and (3) the effect of hyperketonemia (HYK) on the diurnal pattern of blood metabolites. Jugular catheters were placed in 28 multiparous Holstein cows between 3 and 9 days in milk, and blood samples were collected every 2 h for 96 h. Cows were retrospectively classified as HYK positive (HYK; n = 13) if they had plasma BHB concentrations ≥1.2 mmol/L for ≥3 study days, or HYK negative (non-HYK; n = 15) if they had plasma BHB concentrations ≥1.2 mmol/L for ≤2 study days. Generalized linear mixed models were used to analyze concentrations of analytes over time and differences in metabolites between HYK groups. The correlation between total plasma NEFA and BHB was analyzed by calculating the area under the curve for plasma NEFA and BHB for all cows. Plasma NEFA reached a peak approximately 2 h before morning feed delivery, falling to a nadir in the late evening. Plasma BHB was at a nadir at the time of morning feed delivery, peaking 4 h later. We observed a strong positive correlation between daily plasma NEFA and BHB. Additionally, HYK cows had greater concentrations of plasma NEFA and BHB than non-HYK cows. The HYK cows also experienced a greater magnitude of change in BHB throughout the day than the non-HYK cows. Our results suggest that the time relative to feeding should be considered when analyzing plasma metabolites, as classification of energy status may change throughout a day.

A randomized controlled trial to evaluate propylene glycol alone or in combination with dextrose as a treatment for hyperketonemia in dairy cows.

Our objective was to investigate the effect of i.v. dextrose as an adjunct therapy to oral propylene glycol on the resolution of hyperketonemia (HYK; blood ß-hydroxybutyrate ≥1.2 mmol/L), disease incidence, and early lactation milk yield. Cows (n = 1,249) between 3 and 16 d in milk (DIM) from 4 New York dairy farms were screened once weekly for HYK for 2 wk. Those with HYK and no previous history of retained placenta, metritis, or HYK were randomly assigned to 1 of 3 treatment groups: 300 mL of oral 100% propylene glycol for 3 d (PG3); 300 mL of oral 100% propylene glycol for 3 d plus 500 mL i.v. 50% dextrose on d 1 (PG3D1); or 300 mL of oral 100% propylene glycol for 3 d plus 500 mL i.v. 50% dextrose on all 3 d (PG3D3). Cows with a blood ß-hydroxybutyrate <1.2 mmol/L at initial screening were re-screened the following week and randomly assigned to the above treatment groups if blood ß-hydroxybutyrate was ≥1.2 mmol/L. Cows were assessed for post-treatment HYK resolution 1 and 2 wk after initial HYK diagnosis. We collected farm-diagnosed occurrence of adverse events (sold, died, metritis, displaced abomasum, or ketosis) during the first 60 DIM and milk yield data from the first 10 wk of lactation from herd management software. We used mixed-effects multivariable Poisson regression models to assess the risk of post-treatment HYK resolution at 1 and 2 wk following initial HYK diagnosis and adverse event occurrence among treated cows. We used repeated-measures ANOVA to assess differences in average daily milk yield between treatments. The overall HYK incidence was 30.1% (n = 373). Sixty-four percent of cows (n = 237) were assigned to a treatment group in the first week (3 to 9 DIM), and 36% (n = 136) assigned the second week (10 to 16 DIM). The incidence of 1 or more adverse events during the first 60 DIM was 9.4% (n = 35). We found no effect of treatment on risk of post-treatment HYK resolution at wk 1 (PG3 56.9%, PG3D1 45.0%, PG3D3 50.0%) or wk 2 (PG3 60.0%, PG3D1 52.1%, PG3D3 59.5%) following initial diagnosis, or for risk of adverse event occurrence (PG3 7.4%, PG3D1 8.0%, PG3D3 12.6%). Average daily milk yield (mean ± SE) was similar between treatment groups (PG3: 42.7 ± 0.6 kg/d, PG3D1: 42.4 ± 0.6 kg/d, PG3D3: 42.6 ± 0.6 kg/d). The addition of dextrose for 1 or 3 d provided no improvement in resolution of ketosis assessed once weekly, reduction in adverse events during the first 60 d of lactation, or a difference in average daily milk yield during the first 10 wk of lactation.

Serum retinol, ß-carotene, and α-tocopherol as biomarkers for disease risk and milk production in periparturient dairy cows.

The effectiveness of using serum vitamin concentrations as biomarkers to predict diseases in dairy cows during the periparturient period is not well known. The objective of this study was to evaluate the association between serum ß-carotene, retinol, and α-tocopherol concentrations and periparturient cow diseases in commercial dairies. We measured serum concentrations of these vitamin-active compounds at dry-off and during close-up (approximately 3 wk before calving) and early lactation (approximately 7 d post-calving), and we examined their association with clinical diseases in the first 30 d in milk. Diseases were diagnosed by trained personnel and recorded using database software. Blood samples were taken from 353 cows from 5 different farms over a 3-yr period. Blood samples were analyzed for ß-carotene, retinol, α-tocopherol, and cholesterol. We built separate mixed logistic regression models for each disease outcome: hyperketonuria, lameness, mastitis, uterine diseases (retained placenta or metritis), and an aggregate outcome. For the aggregate outcome, a cow was considered positive if she had one or more of the following: hyperketonuria, lameness, mastitis, uterine disease, pneumonia, milk fever, or displaced abomasum. Concentrations of all 3 fat-soluble vitamins decreased significantly in early lactation relative to the 2 prepartum sampling times. Serum retinol concentrations at close-up and early lactation were negatively associated with odds of developing postpartum hyperketonuria. At early lactation, cows with uterine disease had lower serum retinol concentrations than cows without uterine disease. Similarly, lower serum retinol concentrations were associated with greater odds of having any one disease in the aggregate outcome. First-test 305-d mature-equivalent milk yield was positively correlated with increased serum α-tocopherol and negatively correlated with ß-carotene concentrations. This study demonstrates the potential for serum ß-carotene, retinol, and α-tocopherol to serve as biomarkers for disease risk.